Search results for: in vitro propagation

Authors: Adebisi Adunola Demehin, Wanlaya Thamnarak, Jaruwan Chatwichien, Chatchakorn Eurtivong, Kiattawee Choowongkomon, Somsak Ruchirawat, Nopporn Thasana

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The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein involved in cellular signalling processes and, its aberrant activity is crucial in the development of many cancers such as lung cancer. Selaginellaceae are fern allies that have long been used in Chinese traditional medicine to treat various cancer types, especially lung cancer. Biflavonoids, the major secondary metabolites in Selaginellaceae, have numerous pharmacological activities, including anti-cancer and anti-inflammatory. For instance, amentoflavone induces a cytotoxic effect in the human NSCLC cell line via the inhibition of PARP-1. However, to the best of our knowledge, there are no studies on biflavonoids as EGFR inhibitors. Thus, this study aims to investigate the EGFR inhibitory activities of biflavonoids isolated from Selaginella siamensis and Selaginella bryopteris. Amentoflavone, tetrahydroamentoflavone, sciadopitysin, robustaflavone, robustaflavone-4-methylether, delicaflavone, and chrysocauloflavone were isolated from the ethyl-acetate extract of the whole plants. The structures were determined using NMR spectroscopy and mass spectrometry. In vitro study was conducted to evaluate their cytotoxicity against A549, HEPG2, and T47D human cancer cell lines using the MTT assay. In addition, a target-based assay was performed to investigate their EGFR inhibitory activity using the kinase inhibition assay. Finally, a molecular docking study was conducted to predict the binding modes of the compounds. Robustaflavone-4-methylether and delicaflavone showed the best cytotoxic activity on all the cell lines with IC50 (µM) values of 18.9 ± 2.1 and 22.7 ± 3.3 on A549, respectively. Of these biflavonoids, delicaflavone showed the most potent EGFR inhibitory activity with an 84% relative inhibition at 0.02 nM using erlotinib as a positive control. Robustaflavone-4-methylether showed a 78% inhibition at 0.15 nM. The docking scores obtained from the molecular docking study correlated with the kinase inhibition assay. Robustaflavone-4-methylether and delicaflavone had a docking score of 72.0 and 86.5, respectively. The inhibitory activity of delicaflavone seemed to be linked with the C2”=C3” and 3-O-4”’ linkage pattern. Thus, this study suggests that the structural features of these compounds could serve as a basis for developing new EGFR-TK inhibitors.

Keywords: anticancer, biflavonoids, EGFR, molecular docking, Selaginellaceae

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Authors: O. Antypenko, I. Vasilieva, S. Kovalenko

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Investigations for new effective and less toxic antimicrobials agents are always up-to-date. The tetrazole derivatives are quite interesting objects as for synthesis as well as for pharmacological screening. Thus, some derivatives of tetrazole demonstrated antimicrobial activity, namely 5-phenyl-tetrazolo[1,5-c]quinazoline was effective one against Staphylococcus aureus and Esherichia faecalis (MIC = 250 mg/L). Besides, investigation of the 9-bromo(chloro)-5-morpholin(piperidine)-4-yl-tetrazolo[1,5-c]quinazoline’s antimicrobial activity against Esherichia coli and Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus revealed that sensitivity of Gram-positive bacteria to the compounds was higher than that of Gram-negative bacteria. So, our previously synthesized, 31 derivatives of 1-(2-(1H-tetrazol-5-yl)-R1-phenyl)-3-R2-phenyl(ethyl)ureas were decided to test for their in vitro antibacterial activity against Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Enterobacter aerogenes, Enterococcus faecalis ATCC 29212), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922, Klebsiella pneumoniae 68) and antifungal properties against Candida albicans ATCC 885653. Agar-diffusion method was used for determination of the preliminary activity compared to well-known reference antimicrobials. All the compounds were dissolved in DMSO at a concentration of 100 μg/disk, using inhibition zone diameter (IZD, mm) as a measure for the antimicrobial activity. The most active turned to be 3 structures, that inhibited several bacterial strains: 1-ethyl-3-(5-fluoro-2-(1H-tetrazol-5-yl)phenyl)urea (1), 1-(4-bromo-2-(1H-tetrazol-5-yl)-phenyl)-3-(4-(trifluoromethyl)phenyl)urea (2) and 1-(4-chloro-2-(1H-tetrazol-5-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (3). IZM (mm) was 40 (Escherichia coli), 25 (Klebsiella pneumonia) for compound 1; 12 (Pseudomonas aeruginosa), 15 (Staphylococcus aureus), 10 (Enterococcus faecalis) for compound 2; 25 (Staphylococcus aureus), 15 (Enterococcus faecalis) for compound 3. The most sensitive to the activity of the substances were Gram-negative bacteria Pseudomonas aeruginosa. While none of compound effected on Candida albicans. Speaking about, reference drugs: Amikacin (30 µg/disk) showed 27 and Ceftazide (30 µg/disk) 25 against Pseudomonas aeruginosa. That is, unfortunately, higher than studied 1-(2-(1H-tetrazol-5-yl)-R1-phenyl)-3-R2-phenyl(ethyl)ureas. Obtained results will be used for further purposeful optimization of the leading compounds in the more effective antimicrobials because of the ever-mounting problem of microorganism’s resistance.

Keywords: antimicrobial, antifungal, compounds, 1-(2-(1H-tetrazol-5-yl)-R1-phenyl)-3-R2-phenyl(ethyl)ureas

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Authors: Aleksandr I. Korobov, Natalia V. Shirgina, Aleksey I. Kokshaiskiy

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The transformation of two types of acoustic waves can occur on a flat interface between two solids at oblique incidence of longitudinal and shear bulk acoustic waves (BAW). This paper presents the results of experimental studies of the properties of reflection and propagation of longitudinal wave and generation of second and third longitudinal and shear harmonics of BAW at oblique incidence of longitudinal BAW on a flat rough boundary between two solids. The experimental sample was a rectangular isosceles pyramid made of D16 aluminum alloy with the plane parallel bases cylinder made of D16 aluminum alloy pressed to the base. The piezoelectric lithium niobate transducer with a resonance frequency of 5 MHz was secured to one face of the pyramid to generate a longitudinal wave. Longitudinal waves emitted by this transducer felt at an angle of 45° to the interface between two solids and reflected at the same angle. On the opposite face of the pyramid, and on the flat side of the cylinder was attached longitudinal transducer with resonance frequency of 10 MHz or the shear transducer with resonance frequency of 15 MHz. These transducers also effectively received signal at a frequency of 5 MHz. In the spectrum of the transmitted and reflected BAW was observed shear and longitudinal waves at a frequency of 5 MHz, as well as longitudinal harmonic at a frequency harmonic of 10 MHz and a shear harmonic at frequency of 15 MHz. The effect of reversing changing of external pressure applied to the rough interface between two solids on the value of the first and higher harmonics of the BAW at oblique incidence on the interface of the longitudinal BAW was experimentally investigated. In the spectrum of the reflected signal from the interface, there was a decrease of amplitudes of the first harmonics of the signal, and non-monotonic dependence of the second and third harmonics of shear wave with an increase of the static pressure applied to the interface. In the spectrum of the transmitted signal growth of the first longitudinal and shear harmonic amplitude and non-monotonic dependence - first increase and then decrease in the amplitude of the second and third longitudinal shear harmonic with increasing external static pressure was observed. These dependencies were hysteresis at reversing changing of external pressure. When pressure applied to the border increased, acoustic contact between the surfaces improves. This increases the energy of the transmitted elastic wave and decreases the energy of the reflected wave. The second longitudinal acoustic harmonics generation was associated with the Hertz nonlinearity on the interface of two pressed rough surfaces, the generation of the third harmonic was caused by shear hysteresis nonlinearity due to dry friction on a rough interface. This study was supported by the Russian Science Foundation (project №14-22-00042).

Keywords: generation of acoustic harmonics, hysteresis nonlinearity, Hertz nonlinearity, transformation of acoustic waves

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Authors: Daphne Meza, Louie Abejar, David A. Rubenstein, Wei Yin

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Endothelial cell (ECs) morphology and function is highly impacted by the mechanical stresses these cells experience in vivo. Any change in the mechanical environment can trigger pathological EC responses. A detailed understanding of EC morphological response and function upon subjection to individual and simultaneous mechanical stimuli is needed for advancement in mechanobiology and preventive medicine. To investigate this, a programmable device capable of simultaneously applying physiological fluid shear stress (FSS) and cyclic strain (CS) has been developed, characterized and validated. Its validation was performed both experimentally, through tracer tracking, and theoretically, through the use of a computational fluid dynamics model. The effectiveness of the device was evaluated through EC morphology changes under mechanical loading conditions. Changes in cell morphology were evaluated through: cell and nucleus elongation, cell alignment and junctional actin production. The results demonstrated that the combined FSS-CS stimulation induced visible changes in EC morphology. Upon simultaneous fluid shear stress and biaxial tensile strain stimulation, cells were elongated and generally aligned with the flow direction, with stress fibers highlighted along the cell junctions. The concurrent stimulation from shear stress and biaxial cyclic stretch led to a significant increase in cell elongation compared to untreated cells. This, however, was significantly lower than that induced by shear stress alone, indicating that the biaxial tensile strain may counteract the elongating effect of shear stress to maintain the shape of ECs. A similar trend was seen in alignment, where the alignment induced by the concurrent application of shear stress and cyclic stretch fell in between that induced by shear stress and tensile stretch alone, indicating the opposite role shear stress and tensile strain may play in cell alignment. Junctional actin accumulation was increased upon shear stress alone or simultaneously with tensile stretch. Tensile stretch alone did not change junctional actin accumulation, indicating the dominant role of shear stress in damaging EC junctions. These results demonstrate that the shearing-stretching device is capable of applying well characterized dynamic shear stress and tensile strain to cultured ECs. Using this device, EC response to altered mechanical environment in vivo can be characterized in vitro.

Keywords: cyclic stretch, endothelial cells, fluid shear stress, vascular biology

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Authors: Aurea C. L. Lacerda, Paulo R. H. Moreno, Bruna M. P. Vianna, Cristina H. R. Serra, Airton Martin, André R. Baby, Vladi O. Consiglieri, Telma M. Kaneko

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The modified Franz cell is the most widely used model for in vitro permeation studies, however it still presents some disadvantages. Thus, some alternative methods have been developed such as Skin PAMPA, which is a bio- artificial membrane that has been applied for skin penetration estimation of xenobiotics based on HT permeability model consisting. Skin PAMPA greatest advantage is to carry out more tests, in a fast and inexpensive way. The membrane system mimics the stratum corneum characteristics, which is the primary skin barrier. The barrier properties are given by corneocytes embedded in a multilamellar lipid matrix. This layer is the main penetration route through the paracellular permeation pathway and it consists of a mixture of cholesterol, ceramides, and fatty acids as the dominant components. However, there is no consensus on the membrane composition. The objective of this work was to compare the performance among different bio-artificial membranes for studying the permeation in skin PAMPA system. Material and methods: In order to mimetize the lipid composition`s present in the human stratum corneum six membranes were developed. The membrane composition was equimolar mixture of cholesterol, ceramides 1-O-C18:1, C22, and C20, plus fatty acids C20 and C24. The membrane integrity assay was based on the transport of Brilliant Cresyl Blue, which has a low permeability; and Lucifer Yellow with very poor permeability and should effectively be completely rejected. The membrane characterization was performed using Confocal Laser Raman Spectroscopy, using stabilized laser at 785 nm with 10 second integration time and 2 accumulations. The membrane behaviour results on the PAMPA system were statistically evaluated and all of the compositions have shown integrity and permeability. The confocal Raman spectra were obtained in the region of 800-1200 cm-1 that is associated with the C-C stretches of the carbon scaffold from the stratum corneum lipids showed similar pattern for all the membranes. The ceramides, long chain fatty acids and cholesterol in equimolar ratio permitted to obtain lipid mixtures with self-organization capability, similar to that occurring into the stratum corneum. Conclusion: The artificial biological membranes studied for Skin PAMPA showed to be similar and with comparable properties to the stratum corneum.

Keywords: bio-artificial membranes, comparison, confocal Raman, skin PAMPA

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Authors: Carolina S. Monteiro, Eugénia Pinto, Miguel A. Faria, Sara C. Cunha

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Rice (Oryza sativa) production plays a vital role in reducing hunger and poverty and assumes particular importance in low-income and developing countries. Rice is a sensitive plant, and production occurs strictly where suitable temperature and water conditions are found. Climatic changes are likely to affect worldwide, and some models have predicted increased temperatures, variations in atmospheric CO₂ concentrations and modification in precipitation patterns. Therefore, the ongoing climatic changes threaten rice production by increasing biotic and abiotic stress factors, and crops will grow in different environmental conditions in the following years. Around the world, the effects will be regional and can be detrimental or advantageous depending on the region. Mediterranean zones have been identified as possible hot spots, where dramatic temperature changes, modifications of CO₂ levels, and rainfall patterns are predicted. The actual estimated atmospheric CO₂ concentration is around 400 ppm, and it is predicted that it can reach up to 1000–1200 ppm, which can lead to a temperature increase of 2–4 °C. Alongside, rainfall patterns are also expected to change, with more extreme wet/dry episodes taking place. As a result, it could increase the migration of pathogens, and a shift in the occurrence of mycotoxins, concerning their types and concentrations, is expected. Mycotoxigenic spoilage fungi can colonize the crops and be present in all rice food chain supplies, especially Penicillium species, mainly resulting in ochratoxin A (OTA) contamination. In this scenario, the objectives of the present study are evaluating the effect of temperature (20 vs. 25 °C), CO₂ (400 vs. 1000 ppm), and water stress (0.93 vs 0.95 water activity) on growth and OTA production by a Penicillium nordicum strain in vitro on rice-based media and when colonizing layers of raw rice. Results demonstrate the effect of temperature, CO₂ and drought on the OTA production in a rice-based environment, thus contributing to the development of mycotoxins predictive models in climate change scenarios. As a result, improving mycotoxins' surveillance and monitoring systems, whose occurrence can be more frequent due to climatic changes, seems relevant and necessary. The development of prediction models for hazard contaminants presents in foods highly sensitive to climatic changes, such as mycotoxins, in the highly probable new agricultural scenarios is of paramount importance.

Keywords: climate changes, ochratoxin A, penicillium, rice

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Authors: Melanie Ostermann, Eivind Birkeland, Ying Xue, Alexander Sauter, Mihaela R. Cimpan

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Background: New reliable and robust techniques to assess biological effects of nanomaterials (NMs) in vitro are needed to speed up safety analysis and to identify key physicochemical parameters of NMs, which are responsible for their acute cytotoxicity. The central aim of this study was to validate and evaluate the applicability and reliability of an impedance-based flow cytometry (IFC) technique for the high-throughput screening of NMs. Methods: Eight inorganic NMs from the European Commission Joint Research Centre Repository were used: NM-302 and NM-300k (Ag: 200 nm rods and 16.7 nm spheres, respectively), NM-200 and NM- 203 (SiO₂: 18.3 nm and 24.7 nm amorphous, respectively), NM-100 and NM-101 (TiO₂: 100 nm and 6 nm anatase, respectively), and NM-110 and NM-111 (ZnO: 147 nm and 141 nm, respectively). The aim was to assess the biological effects of these materials on human monoblastoid (U937) cells. Dispersions of NMs were prepared as described in the NANOGENOTOX dispersion protocol and cells were exposed to NMs at relevant concentrations (2, 10, 20, 50, and 100 µg/mL) for 24 hrs. The change in electrical impedance was measured at 0.5, 2, 6, and 12 MHz using the IFC AmphaZ30 (Amphasys AG, Switzerland). A traditional toxicity assay, Trypan Blue Dye Exclusion assay, and dark-field microscopy were used to validate the IFC method. Results: Spherical Ag particles (NM-300K) showed the highest toxic effect on U937 cells followed by ZnO (NM-111 ≥ NM-110) particles. Silica particles were moderate to non-toxic at all used concentrations under these conditions. A higher toxic effect was seen with smaller sized TiO2 particles (NM-101) compared to their larger analogues (NM-100). No interferences between the IFC and the used NMs were seen. Uptake and internalization of NMs were observed after 24 hours exposure, confirming actual NM-cell interactions. Conclusion: Results collected with the IFC demonstrate the applicability of this method for rapid nanotoxicity assessment, which proved to be less prone to nano-related interference issues compared to some traditional toxicity assays. Furthermore, this label-free and novel technique shows good potential for up-scaling in directions of an automated high-throughput screening and for future NM toxicity assessment. This work was supported by the EC FP7 NANoREG (Grant Agreement NMP4-LA-2013-310584), the Research Council of Norway, project NorNANoREG (239199/O70), the EuroNanoMed II 'GEMN' project (246672), and the UH-Nett Vest project.

Keywords: cytotoxicity, high-throughput, impedance, nanomaterials

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Authors: Adnan Bekhit, Eskedar Lodebo, Ariaya Hymete, Hanan Ragab, Alaa El-Din Bekhit

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Malaria and leishmaniasis have emerged as serious universal health problems throughout history of mankind. According to the WHO 2008 malarial report, half of the world population is at risk of malarial infection with an estimate of 1 million deaths occurring annually mainly in the African region. Furthermore, 12-15 million people are infected with Leishmaniasis worldwide. Despite the continuous introduction of a large number of agents for the treatment of malaria, there is still unmet medical needs due to the emergence of resistance. Resistance has occurred for almost all therapeutic agents approved for the treatment of malaria. Accordingly, it was the aim of this work to design and synthesis a group of antimalarial-antileshmanial agents that would show inhibitory activity against chloroquine-resistant strain of Plasmodium falciparum. The synthesized compounds were designed to contain a pyrazolylpyrazoline moiety having an aromatic group (p-tolyl or p-chlorophenyl) at N1-position of one pyrazoline ring due to the reports of promising activities of such compounds. A formyl or acyl substituent was introduced at the N1-position of the other pyrazoline ring, to investigate the effect of bulkiness of acyl substituents at this position. The synthesized compounds were evaluated for their in-vivo antimalarial activity against Plasmodium berghei infected mice at dose levels of 20 and 30 mg/Kg. the two most active compounds were evaluated for their antimalarial activity against chloroquin-resistant strain (RKL9) of Plasmodium falciparum. In addition, the synthesized compounds were tested for their in-vitro antileshmanial activity against Leishmania aethiopica promastigotes and amastigotes. For both antimalarial and antileishmanial activities, compounds having an N1-p-tolyl group at the first pyrazoline ring did not require bulkiness at the second pyrazoline ring nitrogen where the compound bearing an acetyl group proved to be the most active of the whole series. On the other hand, bulkiness at the N1-position of the second pyazoline ring was necessary in case of compounds carrying the p-chlorophenyl group, where the two derivatives having an N1-butanoyl and an N1-benzoyl moieties at the second pyrazoline showed the best activity. Furthermore, the toxicity of the active compounds were tested and were proved to be non-toxic at 125, 250 and 500 mg/Kg. In addition, docking of the most active compound (having a p-tolyl group at the first pyrazoline-N and an acetyl moiety on the other pyrazoline-N) was performed against dihydrofolate reductase enzyme.

Keywords: pyrazoline derivatives, in-vivo antimalarial activity, docking, dihydrofolate reductase

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

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

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

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Authors: Vivek Kumar Gupta, Kripi Vohra, Monika Gupta

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Pulses have been a vital ingredient of the balanced human diet in India. Lentil (Lens culinaris Medikus or Lens esculenta Moench.) is a common legume known since biblical times. Lentil seeds, with or without hulls, are cooked as dhal and this has been the main dish for millennia in the South Asian region. Oxidative stress can damage lipids, proteins, enzymes, carbohydrates and DNA in cells and tissues, resulting in membrane damage, fragmentation or random cross linking of molecules like DNA, enzymes and structural proteins and even lead to cell death induced by DNA fragmentation and lipid peroxidation. These consequences of oxidative stress construct the molecular basis in the development of cancer, neurodegenerative disorders, cardiovascular diseases, diabetes and autoimmune. The aim of the present work is to assess the antioxidant potential of the peteroleum ether, acetone, methanol and water extract of the Lens esculenta seeds. In vitro antioxidant assessment of the extracts was carried out using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, hydroxyl radical scavenging activity, reducing power assay. The quantitative estimation of total phenolic content, total flavonoid content in extracts and in plant material, total saponin content, total alkaloid content, crude fibre content, total volatile content, fat content and mucilage content in drug material was also carried out. Though all the extracts exhibited dose dependent reducing power activity the acetone extract was found to possess significant hydrogen donating ability in DPPH (45.83%-93.13%) and hydroxyl radical scavenging system (28.7%-46.41%) than the peteroleum ether, methanol and water extracts. Total phenolic content in the acetone and methanol extract was found to be 608 and 188 mg gallic acid equivalent of phenol/g of sample respectively. Total flavonoid content of acetone and methanol extract was found to be 128 and 30.6 mg quercetin equivalent/g of sample respectively. It is evident that acetone extract of Lentil seeds possess high levels of polyphenolics and flavonoids that could be utilized as antioxidants and neutraceuticals.

Keywords: antioxidant, flavanoids, Lens esculenta, polyphenols

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Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

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The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

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Authors: Rema Vazhappilly, Tapas Das

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Oleic acid is a cis unsaturated fatty acid and is known to be a partially essential fatty acid due to its limited endogenous synthesis during pregnancy and lactation. Previous studies have demonstrated the role of oleic acid in neuronal differentiation and brain phospholipid synthesis. These evidences indicate a major role for oleic acid in learning and memory. Interestingly, oleic acid has been shown to enhance hippocampal long term potentiation (LTP), the physiological correlate of long term synaptic plasticity. However the effect of oleic acid on short term synaptic plasticity has not been investigated. Short term potentiation (STP) is the physiological correlate of short term synaptic plasticity which is the key underlying molecular mechanism of short term memory and neuronal information processing. STP in the hippocampal CA1 region has been known to require the activation of N-methyl-D-aspartate receptors (NMDARs). The NMDAR dependent hippocampal STP as a potential mechanism for short term memory has been a subject of intense interest for the past few years. Therefore in the present study the effect of oleic acid on NMDAR dependent hippocampal STP was determined in mouse hippocampal slices (in vitro) using Multi-electrode array system. STP was induced by weak tetanic Stimulation (one train of 100 Hz stimulations for 0.1s) of the Schaffer collaterals of CA1 region of the hippocampus in slices treated with different concentrations of oleic acid in presence or absence of NMDAR antagonist D-AP5 (30 µM) . Oleic acid at 20 (mean increase in fEPSP amplitude = ~135 % Vs. Control = 100%; P<0.001) and 30 µM (mean increase in fEPSP amplitude = ~ 280% Vs. Control = 100%); P<0.001) significantly enhanced the STP following weak tetanic stimulation. Lower oleic acid concentrations at 10 µM did not modify the hippocampal STP induced by weak tetanic stimulation. The hippocampal STP induced by weak tetanic stimulation was completely blocked by the NMDA receptor antagonist D-AP5 (30µM) in both oleic acid and control treated hippocampal slices. This lead to the conclusion that the hippocampal STP elicited by weak tetanic stimulation and enhanced by oleic acid was NMDAR dependent. Together these findings suggest that oleic acid may enhance the short term memory and neuronal information processing through the modulation of NMDAR dependent hippocampal short-term synaptic plasticity. In conclusion this study suggests the possible role of oleic acid to prevent the short term memory loss and impaired neuronal function throughout development.

Keywords: oleic acid, short-term potentiation, memory, field excitatory post synaptic potentials, NMDA receptor

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Authors: Maged El-Sayed Mohamed

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Plant tissue culture practices are presented nowadays as the most promising substitute to a whole plant in the terms of secondary metabolites production. They offer the advantages of high production, tunability and they have less effect on plant ecosystems. Lantana camara is a weed, which is common all over the world as an ornamental plant. Weeds can adapt to any type of soil and climate due to their rich cellular machinery for secondary metabolites’ production. This characteristic is found in Lantana camara as a plant of very rich diversity of secondary metabolites with no dominant class of compounds. Aim: This trait has encouraged the author to develop tissue culture experiments for Lantana camara to be a platform for production and manipulation of secondary metabolites through biotransformation. Methodology: The plant was collected in its flowering stage in September 2014, from which explants were prepared from shoot tip, auxiliary bud and leaf. Different types of culture media were tried as well as four phytohormones and their combinations; NAA, 2,4-D, BAP and kinetin. Explants were grown in dark or in 12 hours dark and light cycles at 25°C. A metabolic profile for the produced callus was made and then compared to the whole plant profile. The metabolic profile was made using GC-MS for volatile constituents (extracted by n-hexane) and by HPLC-MS and capillary electrophoresis-mass spectrometry (CE-MS) for non-volatile constituents (extracted by ethanol and water). Results: The best conditions for the callus induction was achieved using MS media supplied with 30 gm sucrose and NAA/BAP (1:0.2 mg/L). Initiation of callus was favoured by incubation in dark for 20 day. The callus produced under these conditions showed yellow colour, which changed to brownish after 30 days. The rate of callus growth was high, expressed in the callus diameter, which reached to 1.15±0.2 cm in 30 days; however, the induction of callus delayed for 15 days. The metabolic profile for both volatile and non-volatile constituents of callus showed more simple background metabolites than the whole plant with two new (unresolved) peaks in the callus’ nonvolatile constituents’ chromatogram. Conclusion: Lantana camara callus production can be itself a source of new secondary metabolites and could be used for biotransformation studies due to its simple metabolic background, which allow easy identification of newly formed metabolites. The callus production gathered the simple metabolic background with the rich cellular secondary metabolite machinery of the plant, which could be elicited to produce valuable medicinally active products.

Keywords: capillary electrophoresis-mass spectrometry, gas chromatography, metabolic profile, plant tissue culture

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Authors: Benjawan Saengwichian, Alasdair E. Charles, Philip J. Hyde

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Magnetically controlled growing rods (MCGRs) have been used to stabilise and correct spinal curvature in children to support non-invasive scoliosis adjustment. Although the encapsulated driving components are intended to be isolated from body fluid contact, in vivo corrosion was observed on these components due to sealing mechanism damage. Consequently, a corrosion circuit is created with the body fluids, resulting in malfunction of the lengthening mechanism. Particularly, the chloride ions in blood plasma or cerebrospinal fluid (CSF) may corrode the MCGR alloys, possibly resulting in metal ion release in long-term use. However, there is no data available on the corrosion resistance of spinal implant alloys in CSF. In this study, an in vitro immersion configuration was designed to simulate in vivo corrosion of 440C SS-Ti6Al4V couples. The 440C stainless steel (SS) was heat-treated to investigate the effect of tempering temperature on intergranular corrosion (IGC), while crevice and galvanic corrosion were studied by limiting the clearance of dissimilar couples. Tests were carried out in a neutral artificial cerebrospinal fluid (ACSF) and phosphate-buffered saline (PBS) under aeration and deaeration for 2 months. The composition of the passive films and metal ion release were analysed. The effect of galvanic coupling, pH, dissolved oxygen and anion species on corrosion rates and corrosion mechanisms are discussed based on quantitative and qualitative measurements. The results suggest that ACSF is more aggressive than PBS due to the combination of aggressive chlorides and sulphate anions, while phosphate in PBS acts as an inhibitor to delay corrosion. The presence of Vivianite on the SS surface in PBS lowered the corrosion rate (CR) more than 5 times for aeration and nearly 2 times for deaeration, compared with ACSF. The CR of 440C is dependent on passive film properties varied by tempering temperature and anion species. Although the CR of Ti6Al4V is insignificant, it tends to release more Ti ions in deaerated ACSF than under aeration, about 6 µg/L. It seems the crevice-like design has more effect on macroscopic corrosion than combining the dissimilar couple, whereas IGC is dominantly observed on sensitized microstructure.

Keywords: cerebrospinal fluid, crevice corrosion, intergranular corrosion, magnetically controlled growing rods

Procedia PDF Downloads 108

Authors: Pao-Huei Chen, Shu-Mei Lin, Yih-Ming Weng, Zer-Ran Yu, Be-Jen Wang

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Pancreatic α-amylase and intestinal α-glucosidase are the critical enzymes for the breakdown of complex carbohydrates into di- or mono-saccharide, which play an important role in modulating postprandial blood sugars. Angiotensin converting enzyme (ACE) converts inactive angiotensin-I into active angiotensin-II, which subsequently increase blood pressure through triggering vasoconstriction and aldosterone secretion. Thus, inhibition of carbohydrate-digestion enzymes and ACE will help the management of blood glucose and blood pressure, respectively. Studies showed Pleurotus citrinopileatus (PC), an edible mushroom and commonly cultured in oriental countries, exerted anticancer, immune improving, antioxidative, hypoglycemic and hypolipidemic effects. Previous studies also showed various molecular weights (MW) fractioned from extracts may affect biological activities due to varying contents of bioactive components. Thus, the objective of this study is to investigate the in vitro antioxidant, hypoglycemic and hypotenstive effects and distribution of active compounds of various MWs of cold water extract from P. citrinopileatus (CWEPC). CWEPC was fractioned into four various MW fractions, PC-I (＜1 kDa), PC-II (1-3.5 kDa), PC-III (3.5-10 kDa), and PC-IV (＞10 kDa), using an ultrafiltration system. The physiological activities, including antioxidant activities, the inhibition capabilities of pancreatic α-amylase, intestinal α-glucosidase, and hypertension-linked ACE, and the active components, including polysaccharides, protein, and phenolic contents, of CWEPC and four fractions were determined. The results showed that fractions with lower MW exerted a higher antioxidant activity (p<0.05), which was positively correlated to the levels of total phenols. In contrast, the inhibition effects on the activities of α-amylase, α-glucosidase, and ACE of PC-IV fraction were significantly higher than CWEPC and the other three low MW fractions (< 10 kDa), which was more related to protein contents. The inhibition capability of CWEPC and PC-IV on α-amylase activity was 1/13.4 to 1/2.7 relative to that of acarbose (positive control), respectively. However, the inhibitory ability of PC-IV on α-glucosidase (IC50 = 0.5 mg/mL) was significantly higher than acarbose (IC50 = 1.7 mg/mL). Kinetic data revealed that PC-IV fraction followed a non-competitive inhibition on α-glucosidase activity. In conclusion, the distribution of various bioactive components contribute to the functions of different MW fractions on oxidative stress prevention, and blood pressure and glucose modulation.

Keywords: α-Amylase, angiotensin converting enzyme, α-Glucosidase, Pleurotus citrinopileatus

Procedia PDF Downloads 440

Authors: S. M. Mabandla, P. Loveday, C. Gomes, D. T. Maiga, T. T. Phadi

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Lead zirconate titanate (PZT) piezoelectric ceramics are widely used in ultrasonic applications due to their ability to effectively convert electrical energy into mechanical vibrations and vice versa. This paper presents a study on the behaviour of various combinations of pairs of PZT piezoelectric ceramic materials positioned adjacent to each other with an intermediate gap submerged in water, where one piezoelectric ceramic material is excited by a cyclic electric field with constant frequency and amplitude displacement. The transmitted ultrasonic sound propagates through the medium and is received by the PZT ceramic at the other end, the ultrasonic sound signal amplitude displacement experiences attenuation during propagation due to acoustic impedance. The investigation focuses on understanding the causes of extremely high amplitude displacement attenuation that have been observed in various combinations of piezoelectric ceramic pairs that are submerged in water arranged in a manner stipulated earlier. by examining various combinations of pairs of these piezoelectric ceramics, their physical, electrical, and acoustic properties, and behaviour and attributing them to the observed significant signal attenuation. The experimental setup involves exciting one piezoelectric ceramic material at one end with a burst square cyclic electric field signal of constant frequency, which generates a burst of ultrasonic sound that propagates through the water medium to the adjacent piezoelectric ceramic at the other end. Mechanical vibrations of a PZT piezoelectric ceramic are measured using a double-beam laser Doppler vibrometer to mimic the incident ultrasonic waves generated and received ultrasonic waves on the other end due to mechanical vibrations of a PZT. The measured ultrasonic sound wave signals are continuously compared to the applied cyclic electric field at both ends. The impedance matching networks are continuously tuned at both ends to eliminate electromechanical impedance mismatch to improve ultrasonic transmission and reception. The study delves into various physical, electrical, and acoustic properties of the PZT piezoelectric ceramics, such as the electromechanical coupling factor, acoustic coupling, and elasticity, among others. These properties are analyzed to identify potential factors contributing to the unusually high acoustic impedance in the water medium between the ceramics. Additionally, impedance-matching networks are investigated at both ends to offset the high signal attenuation and improve overall system performance. The findings will be reported in this paper.

Keywords: acoustic impedance, impedance mismatch, piezoelectric ceramics, ultrasonic sound

Procedia PDF Downloads 47

Authors: Ieva Bruzauskaite, Jovile Raudoniute, Karina Poliakovaite, Danguole Zabulyte, Daiva Bironaite, Ruta Aldonyte

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Introduction: Air pollution is a growing global problem which has been shown to be responsible for various adverse health outcomes. Immunotoxicity, such as dysregulated inflammation, has been proposed as one of the main mechanisms in air pollution-associated diseases. Chronic obstructive pulmonary disease (COPD) is among major morbidity and mortality causes worldwide and is characterized by persistent airflow limitation caused by the small airways disease (obstructive bronchiolitis) and irreversible parenchymal destruction (emphysema). Exact pathways explaining the air pollution induced and mediated disease states are still not clear. However, modern societies understand dangers of polluted air, seek to mitigate such effects and are in need for reliable biomarkers of air pollution. We hypothesise that post-translational modifications of structural proteins, e.g. citrullination, might be a good candidate biomarker. Thus, we have designed this study, where mice were exposed to diesel exhaust and the ongoing protein modifications and inflammation in lungs and other tissues were assessed. Materials And Methods: To assess the effects of diesel exhaust a in vivo study was designed. Mice (n=10) were subjected to everyday 2-hour exposure to diesel exhaust for 14 days. Control mice were treated the same way without diesel exhaust. The effects within lung and other tissues were assessed by immunohistochemistry of formalin-fixed and paraffin-embedded tissues. Levels of inflammation and citrullination related markers were investigated. Levels of parenchymal damage were also measured. Results: In vivo study corroborates our own data from in vitro and reveals diesel exhaust initiated inflammatory shift and modulation of lung peptidyl arginine deiminase 4 (PAD4), citrullination associated enzyme, levels. In addition, high levels of citrulline were observed in exposed lung tissue sections co-localising with increased parenchymal destruction. Conclusions: Subacute exposure to diesel exhaust renders mice lungs inflammatory and modifies certain structural proteins. Such structural changes of proteins may pave a pathways to lost/gain function of affected molecules and also propagate autoimmune processes within the lung and systemically.

Keywords: air pollution, citrullination, in vivo, lungs

Procedia PDF Downloads 118

Authors: Choupani Andisheh, Temucin Elif Sevval, Bediz Bekir

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Due to their various advantages compared to many other drug delivery systems such as hypodermic injections and oral medications, microneedle arrays (MNAs) are a promising drug delivery system. To achieve enhanced performance of the MN, it is crucial to develop numerical models, optimization methods, and simulations. Accordingly, in this work, the optimized design of dissolvable MNAs, as well as their manufacturing, is investigated. For this purpose, a mechanical model of a single MN, having the geometry of an obelisk, is developed using commercial finite element software. The model considers the condition in which the MN is under pressure at the tip caused by the reaction force when penetrating the skin. Then, a multi-objective optimization based on non-dominated sorting genetic algorithm II (NSGA-II) is performed to obtain geometrical properties such as needle width, tip (apex) angle, and base fillet radius. The objective of the optimization study is to reach a painless and effortless penetration into the skin along with minimizing its mechanical failures caused by the maximum stress occurring throughout the structure. Based on the obtained optimal design parameters, master (male) molds are then fabricated from PMMA using a mechanical micromachining process. This fabrication method is selected mainly due to the geometry capability, production speed, production cost, and the variety of materials that can be used. Then to remove any chip residues, the master molds are cleaned using ultrasonic cleaning. These fabricated master molds can then be used repeatedly to fabricate Polydimethylsiloxane (PDMS) production (female) molds through a micro-molding approach. Finally, Polyvinylpyrrolidone (PVP) as a dissolvable polymer is cast into the production molds under vacuum to produce the dissolvable MNAs. This fabrication methodology can also be used to fabricate MNAs that include bioactive cargo. To characterize and demonstrate the performance of the fabricated needles, (i) scanning electron microscope images are taken to show the accuracy of the fabricated geometries, and (ii) in-vitro piercing tests are performed on artificial skin. It is shown that optimized MN geometries can be precisely fabricated using the presented fabrication methodology and the fabricated MNAs effectively pierce the skin without failure.

Keywords: microneedle, microneedle array fabrication, micro-manufacturing structural optimization, finite element analysis

Procedia PDF Downloads 88

Authors: Nicholas Fletcher, Zachary Houston, Yongmei Zhao, Christopher Howard, Kristofer Thurecht

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One promising approach to enhance nanomedicine therapeutic efficacy is to include a targeting agent, such as an antibody, to increase accumulation at the tumor site. However, the application of such targeted nanomedicines remains limited, in part due to difficulties involved with biomolecule conjugation to synthetic nanomaterials. One approach recently developed to overcome this has been to engineer bispecific antibodies (BsAbs) with dual specificity, whereby one portion binds to methoxy polyethyleneglycol (mPEG) epitopes present on synthetic nanomedicines, while the other binds to molecular disease markers of interest. In this way, noncovalent complexes of nanomedicine core, comprising a hyperbranched polymer (HBP) of primarily mPEG, decorated with targeting ligands are able to be produced by simple mixing. Further work in this area has now demonstrated such complexes targeting the breast cancer marker epidermal growth factor receptor (EGFR) to show enhanced binding to tumor cells both in vitro and in vivo. Indeed the enhanced accumulation at the tumor site resulted in improved therapeutic outcomes compared to untargeted nanomedicines and free chemotherapeutics. The current work on these BsAb-HBP conjugates focuses on further probing antibody-nanomaterial interactions and demonstrating broad applicability to a range of cancer types. Herein are reported BsAb-HBP materials targeted towards prostate-specific membrane antigen (PSMA) and study of their behavior in vivo using ⁸⁹Zr positron emission tomography (PET) in a dual-tumor prostate cancer xenograft model. In this model mice bearing both PSMA+ and PSMA- tumors allow for PET imaging to discriminate between nonspecific and targeted uptake in tumors, and better quantify the increased accumulation following BsAb conjugation. Also examined is the potential for formation of these targeted complexes in situ following injection of individual components? The aim of this approach being to avoid undesirable clearance of proteinaceous complexes upon injection limiting available therapeutic. Ultimately these results demonstrate BsAb functionalized nanomaterials as a powerful and versatile approach for producing targeted nanomedicines for a variety of cancers.

Keywords: bioengineering, cancer, nanomedicine, polymer chemistry

Procedia PDF Downloads 114

Authors: Eisa Younes Alblooshi

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Counterterrorism strategies, to be effective and efficient, require a sound understanding of the dynamics, the interlinked organizational elements of the terrorist outfits being combated, with a view to having cognizance of their strong points to be guarded against, as well as the vulnerable zones that can be targeted for optimal results in a timely fashion by counterterrorism agencies. A unique model regarding the organizational imperatives was evolved in this research through likening the terrorist organizations with the traditional commercial ones, with a view to understanding in detail the dynamics of interconnectivity and dependencies, and the related compulsions facing the leaderships of such outfits that provide counterterrorism agencies with opportunities for forging better strategies. It involved assessing the evolving organizational dynamics and imperatives of different types of terrorist organizations, to enable the researcher to construct a prototype model that defines the progression and linkages of the related organizational elements of such organizations. It required detailed analysis of how the various elements are connected, with sequencing identified, as any outfit positions itself with respect to its external environment and internal dynamics. A case study focusing on a transnational radical religious state-sponsored terrorist organization was conducted to validate the research findings and to further strengthen the specific counterterrorism strategies. Six different variants of the business model of terrorist organizations were identified, categorized based on their outreach, mission, and status of any state sponsorship. The variants represent vast majority of the range of terrorist organizations acting locally or globally. The model shows the progression and dynamics of these organizations through various dimensions including mission, leadership, outreach, state sponsorship status, resulting in the organizational structure, state of autonomy, preference divergence in its fold, recruitment core, propagation avenues, down to their capacity to adapt, resulting critically in their own life cycles. A major advantage of the model is the utility of mapping terrorist organizations according to their fits to the sundry identified variants, allowing for flexibility and differences within, enabling the researchers and counterterrorism agencies to observe a neat blueprint of the organization’s footprint, along with highlighting the areas to be evaluated for focused target zone selection and timing of counterterrorism interventions. Special consideration is given to the dimension of financing, keeping in context the latest developments regarding cryptocurrencies, hawala, and global anti-money laundering initiatives. Specific counterterrorism strategies and intervention points have been identified for each of the respective model variants, with a view to efficient and effective deployment of resources.

Keywords: terrorism, counterterrorism, model, strategy

Procedia PDF Downloads 130

Authors: Lalita Subedi, Jae Kyoung Chae, Yong Un Park, Cho Kyo Hee, Lee Jae Hyuk, Kang Min Cheol, Sun Yeou Kim

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Neuroinflammation may mediate the relationship between low levels of estrogens and neurodegenerative disease. Estrogens are neuroprotective and anti-inflammatory in neurodegenerative disease models. Due to the long term side effects of estrogens, researches have been focused on finding an effective phytoestrogens for biological activities. Daidzein present in soybeans and its active metabolite equol (7-hydroxy-3-(4'-hydroxyphenyl)-chroman) bears strong antioxidant and anticancer showed more potent anti-inflammatory and neuroprotective role in neuroinflammatory model confirmed its in vitro activity with molecular mechanism through NF-κB pathway. Three major CNS cells Microglia (BV-2), Astrocyte (C6), Neuron (N2a) were used to find the effect of equol in inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), MAPKs signaling proteins, apoptosis related proteins by western blot analysis. Nitric oxide (NO) and prostaglandin E2 (PGE2) was measured by the Gries method and ELISA, respectively. Cytokines like tumor necrosis factor-α (TNF-α) and IL-6 were also measured in the conditioned medium of LPS activated cells with or without equol. Equol inhibited the NO production, PGE-2 production and expression of COX-2 and iNOS in LPS-stimulated microglial cells at a dose dependent without any cellular toxicity. At the same time Equol also showed promising effect in modulation of MAPK’s and nuclear factor kappa B (NF-κB) expression with significant inhibition of the production of proinflammatory cytokine like interleukin -6 (IL-6), and tumor necrosis factor -α (TNF-α). Additionally, it inhibited the LPS activated microglia-induced neuronal cell death by downregulating the apoptotic phenomenon in neuronal cells. Furthermore, equol increases the production of neurotrophins like NGF and increase the neurite outgrowth as well. In conclusion the natural daidzein metabolite equol are more active than daidzein, which showed a promising effectiveness as an anti-neuroinflammatory and neuroprotective agent via downregulating the LPS stimulated microglial activation and neuronal apoptosis. This work was supported by Brain Korea 21 Plus project and High Value-added Food Technology Development Program 114006-4, Ministry of Agriculture, Food and Rural Affairs.

Keywords: apoptosis, equol, neuroinflammation, phytoestrogen

Procedia PDF Downloads 341

Authors: A. Zuchowska, A. Buta, M. Mazurkiewicz-Pawlicka, A. Malolepszy, L. Stobinski, Z. Brzozka

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In recent years, graphene-based materials are finding more and more applications in biological science. As a thin, tough, transparent and chemically resistant materials, they appear to be a very good material for the production of implants and biosensors. Interest in graphene derivatives also resulted at the beginning of research about the possibility of their application in cancer therapy. Currently, the analysis of their potential use in photothermal therapy and as a drug carrier is mostly performed. Moreover, the direct anticancer properties of graphene-based materials are also tested. Nowadays, cytotoxic studies are conducted on in vitro cell culture in standard culture vessels (macroscale). However, in this type of cell culture, the cells grow on the synthetic surface in static conditions. For this reason, cell culture in macroscale does not reflect in vivo environment. The microfluidic systems, called Lab-on-a-chip, are proposed as a solution for improvement of cytotoxicity analysis of new compounds. Here, we present the evaluation of cytotoxic properties of graphene oxide (GO) on breast, liver and colon cancer cell line in a microfluidic system in two spatial models (2D and 3D). Before cell introduction, the microchambers surface was modified by the fibronectin (2D, monolayer) and poly(vinyl alcohol) (3D, spheroids) covering. After spheroid creation (3D) and cell attachment (2D, monolayer) the selected concentration of GO was introduced into microsystems. Then monolayer and spheroids viability/proliferation using alamarBlue® assay and standard microplate reader was checked for three days. Moreover, in every day of the culture, the morphological changes of cells were determined using microscopic analysis. Additionally, on the last day of the culture differential staining using Calcein AM and Propidium iodide were performed. We were able to note that the GO has an influence on all tested cell line viability in both monolayer and spheroid arrangement. We showed that GO caused higher viability/proliferation decrease for spheroids than a monolayer (this was observed for all tested cell lines). Higher cytotoxicity of GO on spheroid culture can be caused by different geometry of the microchambers for 2D and 3D cell cultures. Probably, GO was removed from the flat microchambers for 2D culture. Those results were also confirmed by differential staining. Comparing our results with the studies conducted in the macroscale, we also proved that the cytotoxic properties of GO are changed depending on the cell culture conditions (static/ flow).

Keywords: cytotoxicity, graphene oxide, monolayer, spheroid

Procedia PDF Downloads 102

Authors: Ibtisam A. Abbas Al-Darkazly

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In this study, the mechanical properties of alginate hydrogel material for self-standing 3D scaffold architecture with proper shape fidelity are investigated. In-lab built 3D bio-printer extrusion-based technology is utilized to fabricate 3D alginate scaffold constructs. The pressure, needle speed and stage speed are varied using a computer-controlled system. The experimental result indicates that the concentration of alginate solution, calcium chloride (CaCl₂) cross-linking concentration and cross-linking ratios lead to the formation of alginate hydrogel with various gelation states. Besides, the gelling conditions, such as cross-linking reaction time and temperature also have a significant effect on the mechanical properties of alginate hydrogel. Various experimental tests such as the material gelation, the material spreading and the printability test for filament collapse as well as the swelling test were conducted to evaluate the fabricated 3D scaffold constructs. The result indicates that the fabricated 3D scaffold from composition of 3.5% wt alginate solution, that is prepared in DI water and 1% wt CaCl₂ solution with cross-linking ratios of 7:3 show good printability and sustain good shape fidelity for more than 20 days, compared to alginate hydrogel that is prepared in a phosphate buffered saline (PBS). The fabricated self-standing 3D scaffold constructs measured 30 mm × 30 mm and consisted of 4 layers (n = 4) show good pore geometry and clear grid structure after printing. In addition, the percentage change of swelling degree exhibits high swelling capability with respect to time. The swelling test shows that the geometry of 3D alginate-scaffold construct and of the macro-pore are rarely changed, which indicates the capability of holding the shape fidelity during the incubation period. This study demonstrated that the mechanical and physical properties of alginate hydrogel could be tuned for a 3D bio-printing extrusion-based system to fabricate self-standing 3D scaffold soft structures. This 3D bioengineered scaffold provides a natural microenvironment present in the extracellular matrix of the tissue, which could be seeded with the biological cells to generate the desired 3D live tissue model for in vitro and in vivo tissue engineering applications.

Keywords: biomaterial, calcium chloride, 3D bio-printing, extrusion, scaffold, sodium alginate, tissue engineering

Procedia PDF Downloads 89

Authors: Felipe M. de Freitas, Icaro V. Soares, Lucas L. L. Fortes, Sandro T. M. Gonçalves, Úrsula D. C. Resende

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The SPICE-based simulators are quite robust and widely used for simulation of electronic circuits, their algorithms support linear and non-linear lumped components and they can manipulate an expressive amount of encapsulated elements. Despite the great potential of these simulators based on SPICE in the analysis of quasi-static electromagnetic field interaction, that is, at low frequency, these simulators are limited when applied to microwave hybrid circuits in which there are both lumped and distributed elements. Usually the spatial discretization of the FDTD (Finite-Difference Time-Domain) method is done according to the actual size of the element under analysis. After spatial discretization, the Courant Stability Criterion calculates the maximum temporal discretization accepted for such spatial discretization and for the propagation velocity of the wave. This criterion guarantees the stability conditions for the leapfrogging of the Yee algorithm; however, it is known that for the field update, the stability of the complete FDTD procedure depends on factors other than just the stability of the Yee algorithm, because the FDTD program needs other algorithms in order to be useful in engineering problems. Examples of these algorithms are Absorbent Boundary Conditions (ABCs), excitation sources, subcellular techniques, grouped elements, and non-uniform or non-orthogonal meshes. In this work, the influence of the stability of the FDTD method in the modeling of concentrated elements such as resistive sources, resistors, capacitors, inductors and diode will be evaluated. In this paper is proposed, therefore, the electromagnetic modeling of electronic components in order to create models that satisfy the needs for simulations of circuits in ultra-wide frequencies. The models of the resistive source, the resistor, the capacitor, the inductor, and the diode will be evaluated, among the mathematical models for lumped components in the LE-FDTD method (Lumped-Element Finite-Difference Time-Domain), through the parametric analysis of Yee cells size which discretizes the lumped components. In this way, it is sought to find an ideal cell size so that the analysis in FDTD environment is in greater agreement with the expected circuit behavior, maintaining the stability conditions of this method. Based on the mathematical models and the theoretical basis of the required extensions of the FDTD method, the computational implementation of the models in Matlab® environment is carried out. The boundary condition Mur is used as the absorbing boundary of the FDTD method. The validation of the model is done through the comparison between the obtained results by the FDTD method through the electric field values and the currents in the components, and the analytical results using circuit parameters.

Keywords: hybrid circuits, LE-FDTD, lumped element, parametric analysis

Procedia PDF Downloads 124

Authors: A. Mohammad Abdul Motalib Momin, B. Sheikh Mohammad Adil Uddin, C. Md Mamunur Rashid, D. Sheikh Arman Mahbub, E. Mohammad Sazzad Rahman, F. Abdullah Faruque

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The present study was designed to investigate the antioxidant and cytotoxicity potential of methanol and pet ether extracts of the Lagenaria siceraria (LM, LP), Cucumis sativus (CSM, CSP), Cucurbita maxima (CMM, CMP) plants. For the phytochemical screening, crude extract was tested for the presence of different chemical groups. In Lagenaria siceraria the following groups were identified: alkaloids, steroids, glycosides and saponins for methanol extract and alkaloids, steroids, glycosides, tannins and saponins are for pet ether extract. Glycosides, steroids, alkaloids, saponins and tannins are present in the methanol extract of Cucumis sativus; the pet ether extract has the alkaloids, steroids and saponins. Glycosides, steroids, alkaloids, saponins and tannins are present in both the methanolic and pet ether extract of Cucurbita maxima. In vitro antioxidant activity of the extracts were performed using DPPH radical scavenging, nitric oxide (NO) scavenging, total antioxidant capacity, total phenol content, total flavonoid content, and Cupric Reducing Antioxidant Capacity assays. The most prominent antioxidant activity was observed with the CSM in the DPPH free radical scavenging test with an IC50 value of 1667.23±11.00271 μg/ml as opposed to that of standard ascorbic acid (IC50 value of 15.707± 1.181 μg/ml.) In total antioxidant capacity method, CMP showed the highest activity (427.81±11.4 mg ascorbic acid/g). The total phenolic and flavonoids content were determined by Folin-Ciocalteu Reagent and aluminium chloride colorimetric method, respectively. The highest total phenols and total flavonoids content were found in CMM and LP with the value of 79.06±16.06 mg gallic acid/g & 119.0±1.41 mg quercetin/g, respectively. In nitric oxide (NO) scavenging the most prominent antioxidant activity was observed in CMM with an IC50 value of 8.119± 0.0036 μg/ml. The Cupric reducing capacity of the extracts was strong and dose dependent manner and CSM showed lowest reducing capacity. The cytotoxicity was determined by Brine shrimp lethality test and among these extracts most potent cytotoxicity was shown by CMM with LC50 value 16.98 µg/ml. The obtained results indicate that the investigated plants could be potential sources of natural antioxidants and can be used for various types of diseases.

Keywords: antioxidant, cytotoxicity, methanol, petroleum ether

Procedia PDF Downloads 541

Authors: Tatyane Lopes de Freitas, Antonio Diogo S. Vieira, Susana Marta Isay Saad, Maria Ines Genovese

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The fruit processing industries generate a large volume of residues to produce juices, pulps, and jams. These residues, or by-products, consisting of peels, seeds, and pulps, are routinely discarded. Fruits are rich in bioactive compounds, including polyphenols, which have positive effects on health. Dry residues from two fruits, acerola (M. emarginata D. C.) and orange (C. sinensis), were characterized in relation to contents of ascorbic acid, minerals, total dietary fibers, moisture, ash, lipids, proteins, and carbohydrates, and also high performance liquid chromatographic profile of flavonoids, total polyphenols and proanthocyanidins contents, and antioxidant capacity by three different methods (Ferric reducing antioxidant power assay-FRAP, Oxygen Radical Absorbance Capacity-ORAC, 1,1-diphenyl-2-picrylhydrazil (DPPH) radical scavenging activity). Acerola by-products presented the highest acid ascorbic content (605 mg/100 g), and better antioxidant capacity than orange by-products. The dry residues from acerola demonstrated high contents of proanthocyanidins (617 µg CE/g) and total polyphenols (2525 mg gallic acid equivalents - GAE/100 g). Both presented high total dietary fiber (above 60%) and protein contents (acerola: 10.4%; orange: 9.9%), and reduced fat content (acerola: 1.6%; orange: 2.6%). Both residues showed high levels of potassium, calcium, and magnesium, and were considered sources of these minerals. With acerola by-product, four formulations of probiotics fermented milks were produced: F0 (without the addition of acerola residue (AR)), F2 (2% AR), F5 (5% AR) and F10 (10% AR). The physicochemical characteristics of the fermented milks throughout of storage were investigated, as well as the impact of in vitro simulated gastrointestinal conditions on flavonoids and probiotics. The microorganisms analyzed maintained their populations around 8 log CFU/g during storage. After the gastric phase of the simulated digestion, the populations decreased, and after the enteric phase, no colonies were detected. On the other hand, the flavonoids increased after the gastric phase, maintaining or suffering small decrease after enteric phase. Acerola by-products powder is a valuable ingredient to be used in functional foods because is rich in vitamin C, fibers and flavonoids. These flavonoids appear to be highly resistant to the acids and salts of digestion.

Keywords: acerola, orange, by-products, fermented milk

Procedia PDF Downloads 105

Authors: Hossein Havaeji, Tony Wong, Thien-My Dao

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1. Research Problems and Research Objectives: Blockchain Technology-enabled Supply Chain System (BT-enabled SCS) is the system using BT to drive SCS transparency, security, durability, and process integrity as SCS data is not always visible, available, or trusted. The costs of operating BT in the SCS are a common problem in several organizations. The costs must be estimated as they can impact existing cost control strategies. To account for system and deployment costs, it is necessary to overcome the following hurdle. The problem is that the costs of developing and running a BT in SCS are not yet clear in most cases. Many industries aiming to use BT have special attention to the importance of BT installation cost which has a direct impact on the total costs of SCS. Predicting BT installation cost in SCS may help managers decide whether BT is to be an economic advantage. The purpose of the research is to identify some main BT installation cost components in SCS needed for deeper cost analysis. We then identify and categorize the main groups of cost components in more detail to utilize them in the prediction process. The second objective is to determine the suitable Supervised Learning technique in order to predict the costs of developing and running BT in SCS in a particular case study. The last aim is to investigate how the running BT cost can be involved in the total cost of SCS. 2. Work Performed: Applied successfully in various fields, Supervised Learning is a method to set the data frame, treat the data, and train/practice the method sort. It is a learning model directed to make predictions of an outcome measurement based on a set of unforeseen input data. The following steps must be conducted to search for the objectives of our subject. The first step is to make a literature review to identify the different cost components of BT installation in SCS. Based on the literature review, we should choose some Supervised Learning methods which are suitable for BT installation cost prediction in SCS. According to the literature review, some Supervised Learning algorithms which provide us with a powerful tool to classify BT installation components and predict BT installation cost are the Support Vector Regression (SVR) algorithm, Back Propagation (BP) neural network, and Artificial Neural Network (ANN). Choosing a case study to feed data into the models comes into the third step. Finally, we will propose the best predictive performance to find the minimum BT installation costs in SCS. 3. Expected Results and Conclusion: This study tends to propose a cost prediction of BT installation in SCS with the help of Supervised Learning algorithms. At first attempt, we will select a case study in the field of BT-enabled SCS, and then use some Supervised Learning algorithms to predict BT installation cost in SCS. We continue to find the best predictive performance for developing and running BT in SCS. Finally, the paper will be presented at the conference.

Keywords: blockchain technology, blockchain technology-enabled supply chain system, installation cost, supervised learning

Procedia PDF Downloads 97

Authors: Sharat Chandra, Zilong Wang, Ru-Rong Ji, Andrey Bortsov

Abstract:

Chronic pain (CP) therapeutic approaches have limited efficacy. As a result, doctors are prescribing opioids for chronic pain, leading to opioid overuse, abuse, and addiction epidemic. Therefore, the development of effective and safe CP drugs remains an unmet medical need. Voltage-gated sodium (Nav) channels act as cardiovascular and neurological disorder’s molecular targets. Nav channels selective inhibitors are hard to design because there are nine closely-related isoforms (Nav1.1-1.9) that share the protein sequence segments. We are targeting the Nav1.7 found in the peripheral nervous system and engaged in the perception of pain. The objective of this project was to screen a 1.5 million compound library for identification of inhibitors for Nav1.7 with analgesic effect. In this study, we designed a protocol for identification of isoform-selective inhibitors of Nav1.7, by utilizing the prior information on isoform-selective antagonists. First, a similarity search was performed; then the identified hits were docked into a binding site on the fourth voltage-sensor domain (VSD4) of Nav1.7. We used the FTrees tool for similarity searching and library generation; the generated library was docked in the VSD4 domain binding site using FlexX and compounds were shortlisted using a FlexX score and SeeSAR hyde scoring. Finally, the top 25 compounds were tested with molecular dynamics simulation (MDS). We reduced our list to 9 compounds based on the MDS root mean square deviation plot and obtained them from a vendor for in vitro and in vivo validation. Whole-cell patch-clamp recordings in HEK-293 cells and dorsal root ganglion neurons were conducted. We used patch pipettes to record transient Na⁺ currents. One of the compounds reduced the peak sodium currents in Nav1.7-HEK-293 stable cell line in a dose-dependent manner, with IC50 values at 0.74 µM. In summary, our computer-aided analgesic discovery approach allowed us to develop pre-clinical analgesic candidate with significant reduction of time and cost.

Keywords: chronic pain, voltage-gated sodium channel, isoform-selective antagonist, similarity search, virtual screening, analgesics development

Procedia PDF Downloads 97

Authors: Bahaa Eldin A. Fouda, Khaled N. Yossef, Mohamed Elhosseny, Ahmed Lotfy, Mohamed Salama, Mohamed Sobh

Abstract:

Developmental neurotoxicity (DNT) entails the toxic effects imparted by various chemicals on the brain during the early childhood period. As human brains are vulnerable during this period, various chemicals would have their maximum effects on brains during early childhood. Some toxicants have been confirmed to induce developmental toxic effects on CNS e.g. lead, however; most of the agents cannot be identified with certainty due the defective nature of predictive toxicology models used. A novel alternative method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system. This in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects. In the present study, we verified the possible DNT of epoxomicin which is a naturally occurring selective proteasome inhibitor with anti-inflammatory activity. Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. The cortices were aseptically dissected out from the brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was, then, transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neuropsheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0, 4, 5, 11, and 14 days, sphere size was determined by software analyses. The diameter of each neurosphere was measured and exported to excel file further to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability evaluated by the Trypan Blue exclusion test. Epoxomicin was found to affect proliferation and viability of neuropsheres, these effects were positively correlated to doses and progress of time. This study confirms the DNT effects of epoxomicin on 3D neurospheres model. The effects on proliferation suggest possible gross morphologic changes while the decrease in viability propose possible focal lesion on exposure to epoxomicin during early childhood.

Keywords: neural progentor cells, epoxomicin, neurosphere, medical and health sciences

Procedia PDF Downloads 398

Authors: Raghuram Kandimalla, Sanjeeb Kalita, Bhaswati Choudhury, Jibon Kotoky

Abstract:

The quest for developing an ideal suture material prompted our interest to develop a novel suture with advantageous characteristics to market available ones. We developed novel suture biomaterial from muga silk (Antheraea assama) and ramie (Boehmeria nivea) plant fiber. Field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) and thermo gravimetric analysis (TGA) results revealed the physicochemical properties of the fibers which supports the suitability of fibers for suture fabrication. Tensile properties of the prepared sutures were comparable with market available sutures and it found to be biocompatible towards human erythrocytes and nontoxic to mammalian cells. The prepared sutures completely healed the superficial deep wound incisions within seven days in adult male wister rats leaving no rash and scar. Histopathology studies supports the wound healing ability of sutures, as rapid synthesis of collagen, connective tissue and other skin adnexal structures were observed within seven days of surgery. Further muga suture surface modified by exposing the suture to oxygen plasma which resulted in formation of nanotopography on suture surface. Broad spectrum antibiotic amoxicillin was functionalized on the suture surface to prepare an advanced antimicrobial muga suture. Surface hydrophilicity induced by oxygen plasma results in an increase in drug-impregnation efficiency of modified muga suture by 16.7%. In vitro drug release profiles showed continuous and prolonged release of amoxicillin from suture up to 336 hours. The advanced muga suture proves to be effective against growth inhibition of Staphylococcus aureus and Escherichia coli, whereas normal muga suture offers no antibacterial activity against both types of bacteria. In vivo histopathology studies and colony-forming unit count data revealed accelerated wound healing activity of advanced suture over normal one through rapid synthesis and proliferation of collagen, hair follicle and connective tissues.

Keywords: sutures, biomaterials, silk, Ramie

Procedia PDF Downloads 289