Search results for: in vitro assay

Authors: Assel E. Molzhigitova, Amankeldi K. Sadanov, Elvira T. Ismailova, Kulyash A. Iskandarova, Olga N. Shemshura, Ainur I. Seitbattalova

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Fire blight is a very harmful for commercial apple and pear production quarantine bacterial disease. To date, several different methods have been proposed for disease control, including the use of copperbased preparations and antibiotics, which are not always reliable or effective. The use of bacteria as biocontrol agents is one of the most promising and eco-friendly alternative methods. Bacteria with protective activity against the causal agent of fire blight are often present among the epiphytic microorganisms of the phyllosphere of host plants. Therefore, the main objective of our study was screening of local epiphytic bacteria as possible antagonists against Erwinia amylovora, the causal agent of fire blight. Samples of infected organs of apple and pear trees (shoots, leaves, fruits) were collected from the industrial horticulture areas in various agro-ecological zones of Kazakhstan. Epiphytic microorganisms were isolated by standard and modified methods on specific nutrient media. The primary screening of selected microorganisms under laboratory conditions to determine the ability to suppress the growth of Erwinia amylovora was performed by agar-diffusion-test. Among 142 bacteria isolated from the fire blight host plants, 5 isolates, belonging to the genera Bacillus, Lactobacillus, Pseudomonas, Paenibacillus and Pantoea showed higher antagonistic activity against the pathogen. The diameters of inhibition zone have been depended on the species and ranged from 10 mm to 48 mm. The maximum diameter of inhibition zone (48 mm) was exhibited by B. amyloliquefaciens. Less inhibitory effect was showed by Pantoea agglomerans PA1 (19 mm). The study of inhibitory effect of Lactobacillus species against E. amylovora showed that among 7 isolates tested only one (Lactobacillus plantarum 17M) demonstrated inhibitory zone (30 mm). In summary, this study was devoted to detect the beneficial epiphytic bacteria from plants organs of pear and apple trees due to fire blight control in Kazakhstan. Results obtained from the in vitro experiments showed that the most efficient bacterial isolates are Lactobacillus plantarum 17M, Bacillus amyloliquefaciens MB40, and Pantoea agglomerans PA1. These antagonists are suitable for development as biocontrol agents for fire blight control. Their efficacies will be evaluated additionally, in biological tests under in vitro and field conditions during our further study.

Keywords: antagonists, epiphytic bacteria, Erwinia amylovora, fire blight

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

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

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

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Authors: Sonu Gandhi, Neena Capalash, Prince Sharma, C. Raman Suri

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A sensitive chemiluminescence immunoassay (CIA) for heroin and its major metabolites is reported. The method is based on the competitive reaction of horseradish peroxidase (HRP)-labeled anti-MAM antibody and free drug in spiked urine samples. A hapten-protein conjugate was synthesized by using acidic derivative of monoacetyl morphine (MAM) coupled to carrier protein BSA and was used as an immunogen for the generation of anti-MAM (monoacetyl morphine) antibody. A high titer of antibody (1:64,0000) was obtained and the relative affinity constant (Kaff) of antibody was 3.1×107 l/mol. Under the optimal conditions, linear range and reactivity for heroin, mono acetyl morphine (MAM), morphine and codeine were 0.08, 0.09, 0.095 and 0.092 ng/mL respectively. The developed chemiluminescence inhibition assay could detect heroin and its metabolites in standard and urine samples up to 0.01 ng/ml.

Keywords: heroin, metabolites, chemiluminescence immunoassay, horse radish peroxidase

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Authors: Seok Woo Chang

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Aim: Capseal I and Capseal II are calcium silicate and calcium phosphate based experimental root canal sealer. The aim of this study was to evaluate the biocompatibility and mineralization potential of Capseal I and Capseal II. Materials and Methods: The biocompatibility and mineralization-related gene expression (alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN)) of Capseal I and Capseal II were compared using methylthiazol tetrazolium assay and reverse transcription-polymerization chain reaction analysis, respectively. The results were analyzed by Kruskal-Wallis test. P-value of < 0.05 was considered significant. Result: Both Capseal I and Capseal II were favorable in biocompatibility and influenced the messenger RNA expression of ALP and BSP. Conclusion: Within the limitation of this study, Capseal is biocompatible and have mineralization promoting potential, and thus could be a promising root canal sealer.

Keywords: biocompatibility, mineralization-related gene expression, Capseal I, Capseal II

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Authors: Ibrahim Djelloul Berkane

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A survey was conducted to assess the presence of the virus in Citrus tristeza one of the main citrus regions of Algeria, namely the Chlef region, using the technique of Direct Tissue Print Immunoprinting Assay (DTBIA) and the Double Sandwich ELISA antibodies. A nursery citrus, lumber yards, and commercial orchards, which are the main varieties cultivated citrus were subjected to samples collected samples for laboratory analysis. 0.91% of the plants tested orchards were infected with CTV, while no positive case was detected at the nursery the yard, however, it is reported that an alarming rate of 10,5% of orchards tested at the common Chettia were infected with tristeza virus. The investigation was launched to identify the vector species tristeza revealed the presence of a vector is important Aphis gossypii.

Keywords: aphis, chlef, citrus, DAS-ELISA, DTBIA, tristeza

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Authors: Abdol-Hassan Doulah

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In this research, some of the inorganic complexes of uranyl with N- donor ligands were synthesized. Complexes were characteriezed by FT-IR and UV spectra, ¹HNMR, ¹³CNMR and some physical properties. The uranyl unit (UO2) is composed of a center of uranium atom with the charge (+6) and two oxygen atom by forming two U=O double bonds. The structure is linear (O=U=O, 180) and usually stable. So other ligands often coordinate to the U atom in the plane perpendicularly to the O=U=O axis. The antitumor activity of some of ligand and their complexes against a panel of human tumor cell lines (HT29: Haman colon adenocarcinoma cell line T47D: human breast adenocarcinoma cell line) were determined by MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. These data suggest that some of these compounds provide good models for the further design of potent antitumor compounds.

Keywords: inorganic, uranyl complex-donor ligands, Schiff bases, anticancer activity

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Authors: Kakali Bhadra

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Harmalol administration caused remarkable reduction in proliferation of HepG₂ cells with GI₅₀ of 14.2 mM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism and differential scanning calorimetric analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 ^oC. Binding constant and stoichiometry was also calculated using the above biophysical techniques. Further, dose dependent apoptotic induction ability of harmalol was studied in HepG₂ cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub G_o/G₁ population made the cancer cell, HepG₂, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

Keywords: apoptosis, beta carboline alkaloid, comet assay, cytotoxicity, ROS

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Authors: Amir Sasan Mozaffari Nejad

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This study was carried out to detect the presence of aflatoxin B1 (AFB1) in 36 samples of spices from Iran and India that was included of chilli powder (n=12), black pepper powder (n=12) and whole black pepper (n=12). Enzyme-linked immunosorbent assay (ELISA) method was used for analysing the samples. Aflatoxin B1 was found in all the spices samples, the concentration of AFB1 in Iranian samples was ranged from 63.16 to 626.81 ng/kg and in Indian samples was ranged from 31.15 to 245.94 ng/kg. The mean of AFB1 concentration in the chilli powder was significantly higher (P < 0.05) than the whole and powdered black pepper. However, none of the samples exceeded the maximum prescribed limit i.e. 5 µg/kg of European Union regulations for aflatoxin B1. The occurrence of AFB1 in spices samples could be a potential hazard for public health.

Keywords: Aflatoxin B1, chilli, black pepper, ELISA, Iran, India

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Authors: Gurleen Kour, Mowkshi Khullar, B. K. Chandan, Parvinder Pal Singh, Kushalava Reddy Yumpalla, Gurunadham Munagala, Ram A. Vishwakarma, Zabeer Ahmed

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New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). Apart from in-vitro potency against the target, physiochemical properties and pharmacokinetic properties play an imperative role in the process of drug discovery. We have identified novel nitroimidazole derivatives with potential activity against mycobacterium tuberculosis. One lead candidates, MCD-017, which showed potent activity against H37Rv strain (MIC=0.5µg/ml) and was further evaluated in the process of drug development. Methods: Basic physicochemical parameters like solubility and lipophilicity (LogP) were evaluated. Thermodynamic solubility was determined in PBS buffer (pH 7.4) using LC/MS-MS. The partition coefficient (Log P) of the compound was determined between octanol and phosphate buffered saline (PBS at pH 7.4) at 25°C by the microscale shake flask method. The compound followed Lipinski’s rule of five, which is predictive of good oral bioavailability and was further evaluated for metabolic stability. In-vitro metabolic stability was determined in rat liver microsomes. The hepatotoxicity of the compound was also determined in HepG2 cell line. In vivo pharmacokinetic profile of the compound after oral dosing was also obtained using balb/c mice. Results: The compound exhibited favorable solubility and lipophilicity. The physical and chemical properties of the compound were made use of as the first determination of drug-like properties. The compound obeyed Lipinski’s rule of five, with molecular weight < 500, number of hydrogen bond donors (HBD) < 5 and number of hydrogen bond acceptors(HBA) not more then 10. The log P of the compound was less than 5 and therefore the compound is predictive of exhibiting good absorption and permeation. Pooled rat liver microsomes were prepared from rat liver homogenate for measuring the metabolic stability. 99% of the compound was not metabolized and remained intact. The compound did not exhibit cytoxicity in hepG2 cells upto 40 µg/ml. The compound revealed good pharmacokinetic profile at a dose of 5mg/kg administered orally with a half life (t1/2) of 1.15 hours, Cmax of 642ng/ml, clearance of 4.84 ml/min/kg and a volume of distribution of 8.05 l/kg. Conclusion : The emergence of multi drug resistance (MDR) and extensively drug resistant (XDR) Tuberculosis emphasize the requirement of novel drugs active against tuberculosis. Thus, the need to evaluate physicochemical and pharmacokinetic properties in the early stages of drug discovery is required to reduce the attrition associated with poor drug exposure. In summary, it can be concluded that MCD-017 may be considered a good candidate for further preclinical and clinical evaluations.

Keywords: mycobacterium tuberculosis, pharmacokinetics, physicochemical properties, hepatotoxicity

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Authors: Marcela Parra-Vargas, Ana Sandoval-Rodriguez, Roberto Rodriguez-Echevarria, Jose Dominguez-Rosales, Juan Armendariz-Borunda

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Non-alcoholic fatty liver disease (NAFLD) is characterized by an excess of hepatic lipids, and it is to author’s best knowledge, the most prevalent chronic liver disorder. Anthocyanin-rich food consumption is linked to health benefits in metabolic disorders associated with obesity and NAFLD, although the precise functional role of anthocyanidin delphinidin (Dp) has yet to be established. The aim of this study was to investigate the effect of the Dp in NAFLD metabolic alterations by evaluating prevention or amelioration of hepatic lipid accumulation, as well as molecular mechanisms in two experimental obesity-related models of NALFD. In vitro: HepG2 cells were incubated with sodium palmitate (PA, 1 mM) to induce lipotoxic damage, and concomitantly treated with Dp (180 uM) for 24 h. Subsequently, total lipid accumulation was measured by colorimetric staining with Oil Red O, and total intrahepatic triglycerides were determined by an enzymatic assay. To assess molecular mechanisms, cells were pre-treated with PA for 24 h and then exposed to Dp for 1 h. In vivo: four-week-old male C57BL/6Nhsd mice were allocated in two main groups. Mice were fed with standard diet (control) or high-fat and high-carbohydrate diet (45% fat, HFD) for 16 wk to induce NAFLD. Then HFD was divided into subgroups: one treated orally with Dp (15 mg/kg bw, HFD-Dp) every day for 4 wk, while HFD group treated with vehicle (DMSO). Weight and fasting glucose were recorded weekly, while dietary ingestion was measured daily. Insulin tolerance test was performed at the end of treatment. Liver histology was evaluated with H&E and Masson’s trichrome stain. RT-PCR was used to evaluate gene expression and Western Blot to determine levels of protein in both experimental models. Parametric data were analyzed with one-way ANOVA and Tukey’s post-hoc test. Kruskal-Wallis and Mann-Whitney U test for non-parametric data, and P < 0.5 were considered significant. Dp prevented hepatic lipid accumulation by PA in HepG2 hepatocytes. Furthermore, Dp down-regulated gene expression of SREBP1c, FAS, and CPT1a without modifying AMPK phosphorylation levels. In vivo, Dp oral administration did not ameliorate lipid metabolic alterations raised by HFD. Adiposity, dietary ingestion, fasting glucose, and insulin sensitivity after Dp treatment remained similar to HFD group. Histological analysis showed hepatic damage in HFD groups and no differences between HFD and HFD-Dp groups were found. Hepatic gene expression of ACC and FAS were not altered by HFD. SREBP1c was similar in both HFD and HFD-Dp groups. No significant changes were observed in SREBP1c, ACC, and FAS adipose tissue gene expression by HFD or Dp treatment. Additionally, immunoblotting analysis revealed no changes in pathway SIRT1-LKB-AMPK and PPAR alpha by both HFD groups compared to control. In conclusion, the antioxidant Dp may provoke beneficial effects in the prevention of hepatic lipid accumulation. Nevertheless, the oral dose administrated in mice that simulated the total intake of anthocyanins consumed daily by humans has no effect as a treatment on hepatic lipid metabolic alterations and histological abnormalities associated with exposure to chronic HFD. A healthy lifestyle with regular intake of antioxidants such as anthocyanins may prevent metabolic alterations in NAFLD.

Keywords: anthocyanins, antioxidants, delphinidin, non-alcoholic fatty liver disease, obesity

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Authors: Bao-Fang Wen, Meng-Na Dai, Gao-Pei Zhu, Chen-Xi Zhang, Jing Sun, Xun-Bao Yin, Yu-Han Zhao, Hong-Wei Sun, Wei-Fen Zhang

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A drug-loaded nanoparticles containing berberine hydrochloride (BH/FA-CTS-NPs) was prepared. The physicochemical characterizations of BH/FA-CTS-NPs and the inhibitory effect on the HeLa cells were investigated. Folic acid-conjugated chitosan (FA-CTS) was prepared by amino reaction of folic acid active ester and chitosan molecules; BH/FA-CTS-NPs were prepared using ionic cross-linking technique with BH as a model drug. The morphology and particle size were determined by Transmission Electron Microscope (TEM). The average diameters and polydispersity index (PDI) were evaluated by Dynamic Light Scattering (DLS). The interaction between various components and the nanocomplex were characterized by Fourier Transform Infrared Spectroscopy (FT-IR). The entrapment efficiency (EE), drug-loading (DL) and in vitro release were studied by UV spectrophotometer. The effect of cell anti-migratory and anti-invasive actions of BH/FA-CTS-NPs were investigated using MTT assays, wound healing assays, Annexin-V-FITC single staining assays, and flow cytometry, respectively. HeLa nude mice subcutaneously transplanted tumor model was established and treated with different drugs to observe the effect of BH/FA-CTS-NPs in vivo on HeLa bearing tumor. The BH/FA-CTS-NPs prepared in this experiment have a regular shape, uniform particle size, and no aggregation phenomenon. The results of DLS showed that mean particle size, PDI and Zeta potential of BH/FA-CTS NPs were (249.2 ± 3.6) nm, 0.129 ± 0.09, 33.6 ± 2.09, respectively, and the average diameter and PDI were stable in 90 days. The results of FT-IR demonstrated that the characteristic peaks of FA-CTS and BH/FA-CTS-NPs confirmed that FA-CTS cross-linked successfully and BH was encapsulated in NPs. The EE and DL amount were (79.3 ± 3.12) % and (7.24 ± 1.41) %, respectively. The results of in vitro release study indicated that the cumulative release of BH/FA-CTS NPs was (89.48±2.81) % in phosphate-buffered saline (PBS, pH 7.4) within 48h; these results by MTT assays and wund healing assays indicated that BH/FA-CTS NPs not only inhibited the proliferation of HeLa cells in a concentration and time-dependent manner but can induce apoptosis as well. The subcutaneous xenograft tumor formation rate of human cervical cancer cell line HeLa in nude mice was 98% after inoculation for 2 weeks. Compared with BH group and BH/CTS-NPs group, the xenograft tumor growth of BH/FA-CTS-NPs group was obviously slower; the result indicated that BH/FA-CTS-NPs could significantly inhibit the growth of HeLa xenograft tumor. BH/FA-CTS NPs with the sustained release effect could be prepared successfully by the ionic crosslinking method. Considering these properties, block proliferation and impairing the migration of the HeLa cell line, BH/FA-CTS NPs could be an important compound for consideration in the treatment of cervical cancer.

Keywords: folic-acid, chitosan, berberine hydrochloride, nanoparticles, cervical cancer

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Authors: Yew Mun Yip, Dawei Zhang

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Proteins are the biological machinery that executes specific vital functions in every cell of the human body by folding into their 3D structures. When a protein misfolds from its native structure, the machinery will malfunction and lead to misfolding diseases. Although in vitro experiments are able to conclude that the mutations of the amino acid sequence lead to incorrectly folded protein structures, these experiments are unable to decipher the folding process. Therefore, molecular dynamic (MD) simulations are employed to simulate the folding process so that our improved understanding of the folding process will enable us to contemplate better treatments for misfolding diseases. MD simulations make use of force fields to simulate the folding process of peptides. Secondary structures are formed via the hydrogen bonds formed between the backbone atoms (C, O, N, H). It is important that the hydrogen bond energy computed during the MD simulation is accurate in order to direct the folding process to the native structure. Since the atoms involved in a hydrogen bond possess very dissimilar electronegativities, the more electronegative atom will attract greater electron density from the less electronegative atom towards itself. This is known as the polarization effect. Since the polarization effect changes the electron density of the two atoms in close proximity, the atomic charges of the two atoms should also vary based on the strength of the polarization effect. However, the fixed atomic charge scheme in force fields does not account for the polarization effect. In this study, we introduce the polarized structure-specific backbone charge (PSBC) model. The PSBC model accounts for the polarization effect in MD simulation by updating the atomic charges of the backbone hydrogen bond atoms according to equations derived between the amount of charge transferred to the atom and the length of the hydrogen bond, which are calculated from quantum-mechanical calculations. Compared to other polarizable models, the PSBC model does not require quantum-mechanical calculations of the peptide simulated at every time-step of the simulation and maintains the dynamic update of atomic charges, thereby reducing the computational cost and time while accounting for the polarization effect dynamically at the same time. The PSBC model is applied to two different β-peptides, namely the Beta3s/GS peptide, a de novo designed three-stranded β-sheet whose structure is folded in vitro and studied by NMR, and the trpzip peptides, a double-stranded β-sheet where a correlation is found between the type of amino acids that constitute the β-turn and the β-propensity.

Keywords: hydrogen bond, polarization effect, protein folding, PSBC

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Authors: Jigar N. Shah, Hiral J. Shah, Praful D. Bharadia

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The major challenge faced by formulation scientists in transdermal drug delivery system is to overcome the inherent barriers related to skin permeation. The stratum corneum layer of the skin is working as the rate limiting step in transdermal transport and reduce drug permeation through skin. Many approaches have been used to enhance the penetration of drugs through this layer of the skin. The purpose of this study is to investigate the development and evaluation of a combined approach of drug carriers and iontophoresis as a vehicle to improve skin permeation of an analgesic drug. Iontophoresis is a non-invasive technique for transporting charged molecules into and through tissues by a mild electric field. It has been shown to effectively deliver a variety of drugs across the skin to the underlying tissue. In addition to the enhanced continuous transport, iontophoresis allows dose titration by adjusting the electric field, which makes personalized dosing feasible. Drug carrier could modify the physicochemical properties of the encapsulated molecule and offer a means to facilitate the percutaneous delivery of difficult-to-uptake substances. Recently, there are some reports about using liposomes, microemulsions and polymeric nanoparticles as vehicles for iontophoretic drug delivery. Niosomes, the nonionic surfactant-based vesicles that are essentially similar in properties to liposomes have been proposed as an alternative to liposomes. Niosomes are more stable and free from other shortcoming of liposomes. Recently, the transdermal delivery of certain drugs using niosomes has been envisaged and niosomes have proved to be superior transdermal nanocarriers. Proniosomes overcome some of the physical stability related problems of niosomes. The proniosomal structure was liquid crystalline-compact niosomes hybrid which could be converted into niosomes upon hydration. The combined use of drug carriers and iontophoresis could offer many additional benefits. The system was evaluated for Encapsulation Efficiency, vesicle size, zeta potential, Transmission Electron Microscopy (TEM), DSC, in-vitro release, ex-vivo permeation across skin and rate of hydration. The use of proniosomal gel as a vehicle for the transdermal iontophoretic delivery was evaluated in-vitro. The characteristics of the applied electric current, such as density, type, frequency, and on/off interval ratio were observed. The study confirms the synergistic effect of proniosomes and iontophoresis in improving the transdermal permeation profile of selected analgesic drug. It is concluded that proniosomal gel can be used as a vehicle for transdermal iontophoretic drug delivery under suitable electric conditions.

Keywords: iontophoresis, niosomes, permeation enhancement, transdermal delivery

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Authors: Narayana Bhat, Majda Khalil, Hamad Al-Mansour, Anitha Manuvel, Vimla Yeddu

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The aim of this study was to assess the antimicrobial and antioxidant potential and phytochemical composition of Peganum harmala L. For this purpose, powdered shoot, root, and seed samples were extracted in an accelerated solvent extractor (ASE) with methanol, ethanol, acetone, and dichloromethane. The residues were reconstituted in the above solvents and 10% dimethyl sulphoxide (DMSO). The antimicrobial activity of these extracts was tested against two bacterial (Escherichia coli E49 and Staphylococcus aureus CCUG 43507) and two fungi Candida albicans ATCC 24433, Candida glabrata ATCC 15545) strains using the well-diffusion method. The minimum inhibitory concentration (MIC) and growth pattern of these test strains were determined using microbroth dilution method, and the phospholipase assay was performed to detect tissue damage in the host cells. Results revealed that ethanolic, methanolic, and dichloromethane extracts of seeds exhibited significant antimicrobial activities against all tested strains, whereas the acetone extract of seeds was effective against E. coli only. Similarly, ethanolic and methanolic extracts of roots were effective against two bacterial strains only. One sixth of percent (0.6%) yield of methanol extract of seeds was found to be the MIC for Escherichia coli E49, Staphylococcus aureus CCUG 43507, and Candida glabrata ATCC 15545. Overall, seed extracts had greater antimicrobial activities compared to roots and shoot extracts. The original plant extract and MIC dilutions prevented phospholipase secretion in Staphylococcus aureus CCUG 43507 and Candida albicans ATCC 24433. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay revealed radical scavenging activities ranging from 71.80 ± 4.36% to 87.75 ± 1.70%. The main compound present in the root extract was 1-methyl-7-methoxy-beta-carboline (RT: 44.171), followed by norlapachol (3.62%), benzopyrazine (2.20%), palmitic acid (2.12%) and vasicinone (1.96%). In contrast, phenol,4-ethenyl-2-methoxy was in abundance in the methonolic extract of the shoot, whereas 1-methyl-7-methoxy-beta-carboline (79.59%), linoleic acid (9.05%), delta-tocopherol (5.02%), 9,12-octadecadienoic acid, methyl ester (2.65%), benzene, 1,1-1,2 ethanediyl bis 3,4dimethyl (1.15%), anthraquinone (0.58%), hexadecanoic acid, methyl ester (0.54%), palmitic acid (0.35%) and methyl stearate (0.18%) were present in the methanol extract of seeds. Major findings of this study, along with their relevance to developing effective, safe drugs, will be discussed in this presentation.

Keywords: medicinal plants, secondary metabolites, phytochemical screening, bioprospecting, radical scavenging

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Authors: Gokhan Zengin, Abdurrahman Aktumsek

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The key enzyme inhibitory theory is one of the most accepted strategies in the treatment of global health problems including Alzheimer’s Disease and Diabetes mellitus. For this reason, the enzyme inhibitory potentials of different solvent extracts from Linaria genistifolia subsp. genistifolia were investigated against cholinesterase, and tyrosinase. The in vitro enzyme inhibitory potentials were measured with a microplate reader. The acetone and methanol extracts exhibited the strongest enzyme inhibitory effects on cholinesterase. However, the water extract was only active on tyrosinase. The results suggested that Linaria genistifolia subsp. genistifolia could be considered as a source of natural enzyme inhibitors for the treatment of major health problems.

Keywords: enzyme inhibitors, cholinesterase, tyrosinase, linaria, Turkey

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Authors: Fadila Mohamed Mahmoud., Derkaoui I., Krimi Z.

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Date palm is a plant which presents a very good adaptation to the difficult conditions of the environment in particular to the drought and saline stress even at high temperatures. This adaptation is related on the biology of the plant and to the presence of a microflora endophyte which live inside its tissues. Fifteen endophytics fungi isolated from date palm were tested in vitro in the presence of various NaCl concentrations to select halotolerantes isolates. These same endophytes were tested for their colonizing capacity by the description of the production of secondary metabolites more particularly the enzymes (pectinases, proteases, and phosphorylases), and the production of antibiotics and growth hormones. Significant difference was observed between the isolates with respect to the tests carried out.

Keywords: Date palm, Halotolerantes, endophyte, Secondary metabolites.

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Authors: Fuad Ameen, Mohamed Moslem, Sarfaraz Hadi

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Twenty-eight fungal isolates belonging to 12 genera were derived from debris, sediment and water samples collected from Avicennia marina stands 25km south of Jeddah city on the Red Sea coast of Saudi Arabia. Eight of these isolates were found to be able to grow in association cellulosic waste materials under in vitro conditions in the absence of any carbon source. Isolates were further tested for their potential to degrade paper and clothes wastes by co-cultivation under aeration on a rotary shaker. These fungi accumulated significantly higher biomass, produced ligninolytic and cellulase enzymes, and liberated larger volumes of CO2. These observations indicated that the selected isolates were able to break down and consume the waste materials.

Keywords: biodegradation, enzyme activity, waste materials, mangrove

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Authors: S. Jayasinghe, W. G. D. Wickramasingha, V. Karunaratne, D. N. Karunaratne, A. Ekanayake

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Multi-drug resistant microbial pathogens are a serious global health problem, and hence, there is an urgent necessity for discovering new drug therapeutics. However, finding alternatives is a one of the biggest challenges faced by the global drug industry due to the spiraling high cost and serious side effects associated with modern medicine. On the other hand, plants and their secondary metabolites can be considered as good sources of scaffolds to provide structurally diverse bioactive compounds as potential therapeutic agents. 6β-hydroxy betunolic acid is a triterpenoid isolated from bark of Schumacheria castaneifolia which is an endemic plant to Sri Lanka which has shown antibacterial activity against both Staphylococcus aureus (ATCC 29213) and methicillin-resistant S. aureus with Minimum Inhibition Concentration (MIC) of 16 µg/ml. The objective of this study was to determine the anti-bacterial activity for the derivatives of 6β- hydroxy betunolic acid against standard strains of Staphylococcus aureus (ATCC 29213 and ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 35218 and ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), carbepenemas produce Kebsiella pneumonia (ATCC BAA 1705) and carbepenemas non produce Kebsiella pneumonia (ATCC BAA 1706) and four stains of clinically isolated methicillin resistance S. aureus and Acinetobacter. Structural analogues of 6β-hydroxy betunolic acid were synthesized by modifying the carbonyl group at C-3 to obtain olefin and oxime, the hydroxyl group at C-6 position to a ketone, the carboxylic acid at C-17 to obtain amide and halo ester and the olefin group at C-20 position to obtain epoxide. Chemical structures of the synthesized analogues were confirmed with spectroscopic data and antibacterial activity was determined through broth micro dilution assay. Results revealed that 6β- hydroxy betunolic acid shows significant antibacterial activity only against the Gram positive strains and it was inactive against all the tested Gram negative strains for the tested concentration range. However, structural modifications into oxime and olefin at C-3, ketone at C-6 and epoxide at C-20 decreased its antibacterial activity against the gram positive organisms and it was totally lost with the both modifications at C-17 into amide and ester. These results concluded that the antibacterial activity of 6β- hydroxy betunolic acid and derivatives is predominantly depending on the cell wall difference of the bacteria and the presence of carboxylic acid at C-17 is highly important for the antibacterial activity against Gram positive organisms.

Keywords: antibacterial activity, 6β- hydroxy betunolic acid, broth micro dilution assay, structure activity relationship

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Authors: Nitin Jadhav, Pradeep R. Vavia

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Poor water soluble drugs are difficult to promote for oral drug delivery as they demonstrate poor and variable bioavailability because of its poor solubility and dissolution in GIT fluid. Nowadays lipid based formulations especially self microemulsifying drug delivery system (SMEDDS) is found as the most effective technique. With all the impressive advantages, the need of high amount of surfactant (50% - 80%) is the major drawback of SMEDDS. High concentration of synthetic surfactant is known for irritation in GIT and also interference with the function of intestinal transporters causes changes in drug absorption. Surfactant may also reduce drug activity and subsequently bioavailability due to the enhanced entrapment of drug in micelles. In chronic treatment these issues are very conspicuous due to the long exposure. In addition the liquid self microemulsifying system also suffers from stability issues. Recently one novel approach of solid stabilized micro and nano emulsion (Pickering emulsion) has very admirable properties such as high stability, absence or very less concentration of surfactant and easily converts into the dry form. So here we are exploring pickering dry emulsion system for dissolution enhancement of anti-lipemic, extremely poorly water soluble drug (Fenofibrate). Oil moiety for emulsion preparation was selected mainly on the basis of higher solubility of drug. Captex 300 was showed higher solubility for fenofibrate, hence selected as oil for emulsion. With Silica (solid stabilizer); Span 20 was selected to improve the wetting property of it. Emulsion formed by Silica and Span20 as stabilizer at the ratio 2.5:1 (silica: span 20) was found very stable at the particle size 410 nm. The prepared emulsion was further preceded for spray drying and formed microcapsule evaluated for in-vitro dissolution study, in-vivo pharmacodynamic study and characterized for DSC, XRD, FTIR, SEM, optical microscopy etc. The in vitro study exhibits significant dissolution enhancement of formulation (85 % in 45 minutes) as compared to plain drug (14 % in 45 minutes). In-vivo study (Triton based hyperlipidaemia model) exhibits significant reduction in triglyceride and cholesterol with formulation as compared to plain drug indicating increasing in fenofibrate bioavailability. DSC and XRD study exhibit loss of crystallinity of drug in microcapsule form. FTIR study exhibit chemical stability of fenofibrate. SEM and optical microscopy study exhibit spherical structure of globule coated with solid particles.

Keywords: captex 300, fenofibrate, pickering dry emulsion, silica, span20, stability, surfactant

Procedia PDF Downloads 492

Authors: Mohammad Ali Aazami Mavaloo, Mohammad Bagher Hassanpouraghdam

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Intact leaves, leaf segments and petiole sections derived from nodal explants in vitro were employed for the optimization of Pelargonium odoratissimum micropropagation. MS and ½ MS media enriched with BAP (1, 1.5, 2 and 4.5 mg/l) and NAA (0.1, 1 and 1.5 mg/l) were the treatment combinations used for. With leaf segments, the lowest browning incidence, the greatest callogenesis and the highest number of shoots were obtained with the media containing 1.5 mg/L BAP and 1 mg/L NAA. Two mg/L BAP + 0.1 mg/L NAA hold the same results for petiole explants. Intact leaves showed the best results for the three before-mentioned traits with 1 mg/L BAP + 1 mg/L NAA. 0.2 mg/L NAA caused the highest rooting percentage and the greatest mean data for the number and length of the roots. Rooted plantlets were transferred to the pots containing 1:1 peat-moss and perlite. Acclimatization of the plantlets was followed by 90 percent of survival rate in the greenhouse.

Keywords: Pelargonium odoratissimum, micropropagation, BAP, NAA

Procedia PDF Downloads 383

Authors: Ginpreet Kaur, Mohammad Yasir Usmani, Mohammed Kamil Khan

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Diabetes mellitus is a disease with a high global burden and results in significant morbidity and mortality. In India, the number of people suffering with diabetes is expected to rise from 19 to 57 million in 2025. At present, interest in herbal remedies is growing to reduce the side effects associated with conventional dosage form like oral hypoglycemic agents and insulin for the treatment of diabetes mellitus. Our aim was to investigate the antidiabetic activities of combinatorial extract of N. sativa & C. cassia in Streptozotocin induced type-I Diabetic Rats. Thus, the present study was undertaken to screen postprandial glucose excursion potential through α- glucosidase inhibitory activity (In Vitro) and effect of combinatorial extract of N. sativa & C. cassia in Streptozotocin induced type-I Diabetic Rats (In Vivo). In addition changes in body weight, plasma glucose, lipid profile and kidney profile were also determined. The IC50 values for both extract and Acarbose was calculated by extrapolation method. Combinatorial extract of N. sativa & C. cassia at different dosages (100 and 200 mg/kg orally) and Metformin (50 mg/kg orally) as the standard drug was administered for 28 days and then biochemical estimation, body weights and OGTT (Oral glucose tolerance test) were determined. Histopathological studies were also performed on kidney and pancreatic tissue. In In-Vitro the combinatorial extract shows much more inhibiting effect than the individual extracts. The results reveals that combinatorial extract of N. sativa & C. cassia has shown significant decrease in plasma glucose (p<0.0001), total cholesterol and LDL levels when compared with the STZ group The decreasing level of BUN and creatinine revealed the protection of N. sativa & C. cassia extracts against nephropathy associated with diabetes. Combination of N. sativa & C. cassia significantly improved glucose tolerance to exogenously administered glucose (2 g/kg) after 60, 90 and 120 min interval on OGTT in high dose streptozotocin induced diabetic rats compared with the untreated control group. Histopathological studies shown that treatment with N. sativa & C. cassia extract alone and in combination restored pancreatic tissue integrity and was able to regenerate the STZ damaged pancreatic β cells. Thus, the present study reveals that combination of N. sativa & C. cassia extract has significant α- glucosidase inhibitory activity and thus has great potential as a new source for diabetes treatment.

Keywords: lipid levels, OGTT, diabetes, herbs, glucosidase

Procedia PDF Downloads 425

Authors: Jayanwita Sarkar, Usha Chakraborty, Bishwanath Chakraborty

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Plants are constantly interacting with various abiotic and biotic stresses. In changing climate scenario plants are continuously modifying physiological processes to adapt to changing environmental conditions which profoundly affect plant-pathogen interactions. Spot blotch in wheat is a fast-rising disease in the warmer plains of South Asia where the rise in minimum average temperature over most of the year already affecting wheat production. Hence, the study was undertaken to explore the role of elevated temperature in spot blotch disease development and modulation of antioxidative responses by plant growth promoting rhizobacteria (PGPR) for biocontrol of spot blotch at high temperature. Elevated temperature significantly increases the susceptibility of wheat plants to spot blotch causing pathogen Bipolaris sorokiniana. Two PGPR Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) isolated from wheat (Triticum aestivum L.) and blady grass (Imperata cylindrical L.) rhizophere respectively, showing in vitro antagonistic activity against Bipolaris sorokiniana were tested for growth promotion and induction of resistance against spot blotch in wheat. GC-MS analysis showed that Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) produced antifungal and antimicrobial compounds in culture. Seed priming with these two bacteria significantly increase growth, modulate antioxidative signaling and induce resistance and eventually reduce disease incidence in wheat plants at optimum as well as elevated temperature which was further confirmed by indirect immunofluorescence assay using polyclonal antibody raised against Bipolaris sorokiniana. Application of the PGPR led to enhancement in activities of plant defense enzymes- phenylalanine ammonia lyase, peroxidase, chitinase and β-1,3 glucanase in infected leaves. Immunolocalization of chitinase and β-1,3 glucanase in PGPR primed and pathogen inoculated leaf tissue was further confirmed by transmission electron microscopy using PAb of chitinase, β-1,3 glucanase and gold labelled conjugates. Activity of ascorbate-glutathione redox cycle related enzymes such as ascorbate peroxidase, superoxide dismutase and glutathione reductase along with antioxidants such as carotenoids, glutathione and ascorbate and osmolytes like proline and glycine betain accumulation were also increased during disease development in PGPR primed plant in comparison to unprimed plants at high temperature. Real-time PCR analysis revealed enhanced expression of defense genes- chalcone synthase and phenyl alanineammonia lyase. Over expression of heat shock proteins like HSP 70, small HSP 26.3 and heat shock factor HsfA3 in PGPR primed plants effectively protect plants against spot blotch infection at elevated temperature as compared with control plants. Our results revealed dynamic biochemical cross talk between elevated temperature and spot blotch disease development and furthermore highlight PGPR mediated array of antioxidative and molecular alterations responsible for induction of resistance against spot blotch disease at elevated temperature which seems to be associated with up-regulation of defense genes, heat shock proteins and heat shock factors, less ROS production, membrane damage, increased expression of redox enzymes and accumulation of osmolytes and antioxidants.

Keywords: antioxidative enzymes, defense enzymes, elevated temperature, heat shock proteins, PGPR, Real-Time PCR, spot blotch, wheat

Procedia PDF Downloads 159

Authors: Jayeon Kim, Eunjung Yu, Taeki Yoon

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Most spontaneous miscarriage is believed to be a consequence of embryo aneuploidies. Transferring eukaryotic embryos selected by PGS is expected to decrease the miscarriage rate. Current PGS indications include advanced maternal age, recurrent pregnancy loss, repeated implantation failure. Recently, use of PGS for healthy women without above indications for the purpose of improving in vitro fertilization (IVF) outcomes is on the rise. However, it is still controversy about the beneficial effect of PGS in this population, especially, in women with a history of no more than 2 miscarriages or miscarriage of eukaryotic abortus. This study aimed to investigate if karyotyping result of abortus is a good indicator of preimplantation genetic screening (PGS) in subsequent IVF cycle in women with a history of spontaneous abortion. A single-center retrospective cohort study was performed. Women who had spontaneous abortion(s) (less than 3) and dilatation and evacuation, and subsequent IVF from January 2016 to November 2016 were included. Their medical information was extracted from the charts. Clinical pregnancy was defined as presence of a gestational sac with fetal heart beat detected on ultrasound in week 7. Statistical analysis was performed using SPSS software. Total 234 women were included. 121 out of 234 (51.7%) underwent karyotyping of the abortus, and 113 did not have the abortus karyotyped. Embryo biopsy was performed on 3 or 5 days after oocyte retrieval, followed by embryo transfer (ET) on a fresh or frozen cycle. The biopsied materials were subjected to microarray comparative genomic hybridization. Clinical pregnancy rate per ET was compared between PGS and non-PGS group in each study group. Patients were grouped by two criteria: karyotype of the abortus from previous miscarriage (unknown fetal karyotype (n=89, Group 1), eukaryotic abortus (n=36, Group 2) or aneuploidy abortus (n=67, Group 3)), and pursuing PGS in subsequent IVF cycle (pursuing PGS (PGS group, n=105) or not pursuing PGS (non-PGS group, n=87)). The PGS group was significantly older and had higher number of retrieved oocytes and prior miscarriages compared to non-PGS group. There were no differences in BMI and AMH level between those two groups. In PGS group, the mean number of transferable embryos (eukaryotic embryo) was 1.3 ± 0.7, 1.5 ± 0.5 and 1.4 ± 0.5, respectively (p = 0.049). In 42 cases, ET was cancelled because all embryos biopsied turned out to be abnormal. In all three groups (group 1, 2, and 3), clinical pregnancy rates were not statistically different between PGS and non-PGS group (Group 1: 48.8% vs. 52.2% (p=0.858), Group 2: 70% vs. 73.1% (p=0.730), Group 3: 42.3% vs. 46.7% (p=0.640), in PGS and non-PGS group, respectively). In both groups who had miscarriage with eukaryotic and aneuploidy abortus, the clinical pregnancy rate between IVF cycles with and without PGS was not different. When we compare miscarriage and ongoing pregnancy rate, there were no significant differences between PGS and non-PGS group in all three groups. Our results show that the routine application of PGS in women who had less than 3 miscarriages would not be beneficial, even in cases that previous miscarriage had been caused by fetal aneuploidy.

Keywords: preimplantation genetic diagnosis, miscarriage, kpryotyping, in vitro fertilization

Procedia PDF Downloads 171

Authors: Majid Forghani, Michael Khachay

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In genetics, the impact of neighboring amino acids on a target site is referred as the nearest-neighbor effect or simply neighbor effect. In this paper, a new method called wavelet particle decomposition representing the one-dimensional neighbor effect using wavelet packet decomposition is proposed. The main idea lies in known dependence of wavelet packet sub-bands on location and order of neighboring samples. The method decomposes the value of a signal sample into small values called particles that represent a part of the neighbor effect information. The results have shown that the information obtained from the particle decomposition can be used to create better model variables or features. As an example, the approach has been applied to improve the correlation of test and reference sequence distance with titer in the hemagglutination inhibition assay.

Keywords: antigenic variants, neighbor effect, wavelet packet, wavelet particle decomposition

Procedia PDF Downloads 143

Authors: E. Obreque-Slier, F. Orellana-Rodríguez, R. López-Solís

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Phenolic compounds are secondary metabolites present in some foods, such as wine. Polyphenols comprise two main groups: flavonoids (anthocyanins, flavanols, and flavonols) and non-flavonoids (stilbenes and phenolic acids). Phenolic acids are low molecular weight non flavonoid compounds that are usually grouped into benzoic (gallic, vanillinic and protocatechuic acids) and cinnamic acids (ferulic, p-coumaric and caffeic acids). Likewise, tannic acid is an important polyphenol constituted mainly by gallic acid. Phenolic compounds are responsible for important properties in foods and drinks, such as color, aroma, bitterness, and astringency. Astringency is a drying, roughing, and sometimes puckering sensation that is experienced on the various oral surfaces during or immediately after tasting foods. Astringency perception has been associated with interactions between flavanols present in some foods and salivary proteins. Despite the quantitative relevance of phenolic acids in food and beverages, there is no information about its effect on salivary proteins and consequently on the sensation of astringency. The objective of this study was assessed the interaction of several phenolic acids (gallic, vanillinic, protocatechuic, ferulic, p-coumaric and caffeic acids) with saliva. Tannic acid was used as control. Thus, solutions of each phenolic acids (5 mg/mL) were mixed with human saliva (1:1 v/v). After incubation for 5 min at room temperature, 15-μL aliquots of the mixtures were dotted on a cellulose membrane and allowed to diffuse. The dry membrane was fixed in 50 g/L trichloroacetic acid, rinsed in 800 mL/L ethanol and stained for protein with Coomassie blue for 20 min, destained with several rinses of 73 g/L acetic acid and dried under a heat lamp. Both diffusion area and stain intensity of the protein spots were semiqualitative estimates for protein-tannin interaction (diffusion test). The rest of the whole saliva-phenol solution mixtures of the diffusion assay were centrifuged and fifteen-μL aliquots of each supernatant were dotted on a cellulose membrane, allowed to diffuse and processed for protein staining, as indicated above. In this latter assay, reduced protein staining was taken as indicative of protein precipitation (precipitation test). The diffusion of the salivary protein was restricted by the presence of each phenolic acids (anti-diffusive effect), while tannic acid did not alter diffusion of the salivary protein. By contrast, phenolic acids did not provoke precipitation of the salivary protein, while tannic acid produced precipitation of salivary proteins. In addition, binary mixtures (mixtures of two components) of various phenolic acids with gallic acid provoked a restriction of saliva. Similar effect was observed by the corresponding individual phenolic acids. Contrary, binary mixtures of phenolic acid with tannic acid, as well tannic acid alone, did not affect the diffusion of the saliva but they provoked an evident precipitation. In summary, phenolic acids showed a relevant interaction with the salivary proteins, thus suggesting that these wine compounds can also contribute to the sensation of astringency.

Keywords: astringency, polyphenols, tannins, tannin-protein interaction

Procedia PDF Downloads 236

Authors: R. Fegas, S. Zerkout, S. Taberkokt, M. Righezza

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The present work demonstrate the potential of Betacyclodextrine (BCD) for the chiral analysis of a drug .Various separation mechanisms were applied and several parameters affecting the separation were studied, including the type and concentration of chiral selector, and pH of buffer. A simple and sensitive high-performance liquid chromatography (HPLC) method was developed as an assay for fexofenadine enantiomers in pharmaceutical preparation. Fexofenadine enantiomers were separated using a mobile phase of 0.25mM NaH2PO4–acetonitrile (65:35, v/v) – Betacyclodextrine on achiral phenyl-urea column at a flow rate of 1ml/min and measurement at 220nm. The chiral mechanism of separation was mainly based on specific interaction between the solute and the stationary phase. The retention was directly controlled by mobile phase composition but not the selectivity which results of the two mechanisms, electrostatic interactions and partition mechanism.

Keywords: fexofenadine enantiomer, HPLC, achiral phenyl-urea column

Procedia PDF Downloads 451

Authors: Swati Srivastava, Mattia Lauriola, Yuval Gilad, Adi Kimchi, Yosef Yarden

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The glucocorticoid receptor (GR) has been proposed to play important, but incompletely understood roles in cancer. Glucocorticoids (GCs) are widely used as co-medication of various carcinomas, due to their ability to reduce the toxicity of chemotherapy. Furthermore, GR antagonism has proven to be a strategy to treat triple negative breast cancer and castration-resistant prostate cancer. These observations suggest differential GR involvement in cancer subtypes. The goal of our study has been to elaborate the current understanding of GR signaling in tumor progression and metastasis. Our study involves two cellular models, non-tumorigenic breast epithelial cells (MCF10A) and Ewing sarcoma cells (CHLA9). In our breast cell model, the results indicated that the GR agonist dexamethasone inhibits EGF-induced mammary cell migration, and this effect was blocked when cells were stimulated with a GR antagonist, namely RU486. Microarray analysis for gene expression revealed that the mechanism underlying inhibition involves dexamenthasone-mediated repression of well-known activators of EGFR signaling, alongside with enhancement of several EGFR’s negative feedback loops. Because GR mainly acts primarily through composite response elements (GREs), or via a tethering mechanism, our next aim has been to find the transcription factors (TFs) which can interact with GR in MCF10A cells.The TF-binding motif overrepresented at the promoter of dexamethasone-regulated genes was predicted by using bioinformatics. To validate the prediction, we performed high-throughput Protein Complementation Assays (PCA). For this, we utilized the Gaussia Luciferase PCA strategy, which enabled analysis of protein-protein interactions between GR and predicted TFs of mammary cells. A library comprising both nuclear receptors (estrogen receptor, mineralocorticoid receptor, GR) and TFs was fused to fragments of GLuc, namely GLuc(1)-X, X-GLuc(1), and X-GLuc(2), where GLuc(1) and GLuc(2) correspond to the N-terminal and C-terminal fragments of the luciferase gene.The resulting library was screened, in human embryonic kidney 293T (HEK293T) cells, for all possible interactions between nuclear receptors and TFs. By screening all of the combinations between TFs and nuclear receptors, we identified several positive interactions, which were strengthened in response to dexamethasone and abolished in response to RU486. Furthermore, the interactions between GR and the candidate TFs were validated by co-immunoprecipitation in MCF10A and in CHLA9 cells. Currently, the roles played by the uncovered interactions are being evaluated in various cellular processes, such as cellular proliferation, migration, and invasion. In conclusion, our assay provides an unbiased network analysis between nuclear receptors and other TFs, which can lead to important insights into transcriptional regulation by nuclear receptors in various diseases, in this case of cancer.

Keywords: epidermal growth factor, glucocorticoid receptor, protein complementation assay, transcription factor

Procedia PDF Downloads 220

Authors: Amr A. EL Hanafy, Yasir Anwar, Saleh A. Mohamed, Saleh Mohamed Saleh Al-Garni, Jamal S. M. Sabir , Osama A. H. Abu Zinadah, Mohamed Morsi Ahmed

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In the vicinity of the red sea about 15 fungi species were isolated from oil contaminated sites. On the basis of aptitude to degrade the crude oil and DCPIP assay, two fungal isolates were selected amongst 15 oil degrading strains. Analysis of ITS-1, ITS-2 and amplicon pyrosequencing studies of fungal diversity revealed that these strains belong to Penicillium and Aspergillus species. Two strains that proved to be the most efficient in degrading crude oil was Aspergillus niger (54 %) and Penicillium commune (48 %) Subsequent to two weeks of cultivation in BHS medium the degradation rate were recorded by using spectrophotometer and GC-MS. Hence, it is cleared that these fungal strains has the capability of degradation and can be utilized for cleaning the Saudi Arabian environment.

Keywords: fungal strains, hydrocarbon contaminants, molecular identification, biodegradation, GC-MS

Procedia PDF Downloads 514

Authors: R. I. You, C. L. Ho, M. S. Dai, H. M. Hung, S. F. Yen, C. S. Chen, T. Y. Chao

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Neutrophils are the most abundant innate immune cells to against invading microorganisms. Numerous data shown neutrophils have plasticity in response to physiological and pathological conditions. Tumor-associated neutrophils (TAN) exist in distinct types of tumor and play an important role in cancer biology. Different transcriptomic profiles of neutrophils in tumor and non-tumor samples have been identified. Several miRNAs have been recognized as regulators of gene expression in neutrophil, which may have key roles in neutrophil activation. However, the miRNAs expression patterns in TAN are not well known. To address this question, magnetic bead isolated neutrophils from tumor-bearing mice were used in this study. We analyzed production of reactive oxygen species (ROS) by luminol-dependent chemiluminescence assay. The expression of miRNAs targeting NADPH oxidase, ROS generation and autophagy was explored using quantitative real-time polymerase chain reaction. Our data suggest that tumor environment influence neutrophil develop to differential states of activation via miRNAs regulation.

Keywords: tumor-associated neutrophil, miRNAs, neutrophil, ROS

Procedia PDF Downloads 460

Authors: Vandana Gawande, Chandani Satija

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Gnidia glauca is a semi-woody herb of thymelaeaceae family traditionally used as fish poison in India. It is also found in Sri lanka and Africa. In the present study, potential anticancer effect of n-hexane and ethanolic extracts of Gnidia glauca in human breast cancer cells was investigated. Human breast cancer cells (MCF-7) were cultured as monolayers in RPMI 1640 medium. The cells were cultured for 48 hours to allow growth and achieve about 80% confluence in 96-well culture plates. The cells were treated with various concentrations of Gnidia glauca (0.1-100 mg/mL) for 72 hours. Percentage of viable cells after treatment was assessed using a sulforhodamine B colorimetric assay. Both n-hexane and ethanolic extract showed significant cytotoxic activity on MCF-7 cancer cells. This study supports the notion of using Gnidia glauca as a novel anticancer agent for breast cancer.

Keywords: 96 well plate, anticancer activity, Gnidia glauca, MCF-7

Procedia PDF Downloads 281