Search results for: in vitro study

Authors: Diky Mudhakir, Satrialdi, Sukmadjaja Asyarie, Yeyet C. Sumirtapura

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Today, cancer remains one of the major diseases that lead to death. The main obstacle in chemotherapy as a main cancer treatment is the toxicity to normal cells due to Multidrug Resistance (MDR) after the use of anticancer drugs. Proposed solution to overcome this problem is the use of MDR efflux inhibitor of cinchona alkaloids which is delivered together with anticancer drugs encapsulated in the form of polymeric nanoparticles. The particles were prepared by the hydration method. The characterization of nanoparticles was particle size, zeta potential, entrapment efficiency and in vitro drug release. Combination nanoparticle size ranged 29-45 nm with a neutral surface charge. Entrapment efficiency was above 87% for the use quinine, quinidine or cinchonidine in combination with etoposide. The release test results exhibited that the cinchona alkaloids release released faster than that of etoposide. Collectively, cinchona alkaloids can be packaged along with etoposide in nanomicelles for better cancer therapy.

Keywords: Cinchona alkaloids, etoposide, MDR efflux inhitor, polymeric nanomicelles.

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Authors: P. I. Bobyleva, E. R. Andreeva, I. V. Andrianova, E. V. Maslova, L.B. Buravkova

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This work studied the ability of adipose tissue-derived mesenchymal stromal cells (MSCs) to form stroma for expansion of cord blood hematopoietic cells. We showed that 72-hour interaction of MSCs with cord blood mononuclear cells (MNCs) in vitro at atmospheric (20%) and low (5%) O2 conditions increased the expression of ICAM-1, HCAM (at the beginning of interaction) on MSCs. Viability of MSCs and MNCs were maintained at high level. Adhesion of MNCs to MSCs was faster at 20% O2. MSCs promoted the proliferation of adhered MNCs to form the suspension containing great number of hematopoietic colony-forming units, and this effect was more pronounced at 5% O2. Thus, adipose-derived MSCs supplied sufficient stromal support to cord blood MNCs both at 20% and 5% О2, providing their adhesion with further expansion of new generation of different hematopoietic lineages.

Keywords: Hematopoietic stem and progenitor cells, mesenchymal stromal cells, tissue-related oxygen.

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Authors: V. G. Sangam, Balasaheb M. Patre

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In this paper we have proposed a methodology to develop an amperometric biosensor for the analysis of glucose concentration using a simple microcontroller based data acquisition system. The work involves the development of Detachable Membrane Unit (enzyme based biomembrane) with immobilized glucose oxidase on the membrane and interfacing the same to the signal conditioning system. The current generated by the biosensor for different glucose concentrations was signal conditioned, then acquired and computed by a simple AT89C51-microcontroller. The optimum operating parameters for the better performance were found and reported. The detailed performance evaluation of the biosensor has been carried out. The proposed microcontroller based biosensor system has the sensitivity of 0.04V/g/dl, with a resolution of 50mg/dl. It has exhibited very good inter day stability observed up to 30 days. Comparing to the reference method such as HPLC, the accuracy of the proposed biosensor system is well within ± 1.5%. The system can be used for real time analysis of glucose concentration in the field such as, food and fermentation and clinical (In-Vitro) applications.

Keywords: Biosensor, DMU, Glucose oxidase andMicrocontroller.

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Authors: Rajan Rajabalaya, Li-Qun Tor, Sheba David

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Transdermal delivery of ondansetron hydrochloride (OdHCl) can prevent the problems encountered with oral ondansetron. In previously conducted studies, effect of amount of polyvinyl pyrrolidone, permeation enhancer and casting solvent on the physicochemical properties on OdHCl were investigated. It is feasible to develop ondansetron transdermal patch by using ethyl cellulose and polyvinyl pyrrolidone with dibutyl pthalate as plasticizer, however, the desired flux is not achieved. The primary aim of this study is to use dimethyl succinate (DMS) and propylene glycol that are not incorporated in previous studies to determine their effect on the physicochemical properties of an OdHCl transdermal patch using ethyl cellulose and polyvinyl pyrrolidone. This study also investigates the effect of permeation enhancer (eugenol and phosphatidylcholine) on the release of OdHCl. The results showed that propylene glycol is a more suitable plasticizer compared to DMS in the fabrication of OdHCl transdermal patch using ethyl cellulose and polyvinyl pyrrolidone as polymers. Propylene glycol containing patch has optimum drug content, thickness, moisture content and water absorption, tensile strength, and a better release profile than DMS. Eugenol and phosphatidylcholine can increase release of OdHCl from the patches. From the physicochemical result and permeation profile, a combination of 350mg of ethyl cellulose, 150mg polyvinyl pyrrolidone, 3% of total polymer weight of eugenol, and 40% of total polymer weight of propylene glycol is the most suitable formulation to develop an OdHCl patch. OdHCl release did not increase with increasing the percentage of plasticiser. DMS 4, PG 4, DMS 9, PG 9, DMS 14, and PG 14 gave better release profiles where using 300mg: 0mg, 300mg: 100mg, and 350mg: 150mg of EC: PVP. Thus, 40% of PG or DMS appeared to be the optimum amount of plasticiser when the above combination where EC: PVP was used. It was concluded from the study that a patch formulation containing 350mg EC, 150mg PVP, 40% PG and 3% eugenol is the best transdermal matrix patch compositions for the uniform and continuous release/permeation of OdHCl over an extended period. This patch design can be used for further pharmacokinetic and pharmacodynamic studies in suitable animal models.

Keywords: Ondansetron hydrochloride, dimethyl succinate, eugenol.

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Authors: Maryam Nejat Dehkordi, Per Lincoln, Hassan Momtaz

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Interaction of Schiff base complexes of Iron and Manganese: Iron [N, N’ Bis (5- (triphenyl phosphonium methyl) salicylidene) -1, 2 ethanediamine) chloride, [Fe Salen]Cl; Manganese [N, N’ Bis (5- (triphenyl phosphonium methyl) salicylidene) -1, 2 ethanediamine) acetate, were investigated by spectroscopic and isothermal titration calorimetry techniques (ITC). The absorbance spectra of complexes have shown hyper and hypochromism in the presence of DNA that is indication of interaction of complexes with DNA. The linear dichroism (LD) measurements confirmed the bending of DNA in the presence of complexes. Furthermore, Isothermal titration calorimetry experiments approved that complexes bound to DNA on the base of both electrostatic and hydrophobic interactions. More, ITC profile exhibits the existence of two binding phases for the complexes. Antibacterial activity of ligand and complexes were tested in vitro to evaluate their activity against the gram positive and negative bacteria.

Keywords: Schiff base complexes, Linear dichroism (LD), Isothermal titration calorimetry (ITC).

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Authors: M. Baryshev, D.Vainauska, S. Kozireva, A.Karpovs

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Liposomal magnetofection is the most powerful nonviral method for the nucleic acid delivery into the cultured cancer cells and widely used for in vitro applications. Use of the static magnetic field condition may result in non-uniform distribution of aggregate complexes on the surface of cultured cells. To prevent this, we developed the new device which allows to concentrate aggregate complexes under dynamic magnetic field, assisting more contact of these complexes with cellular membrane and, possibly, stimulating endocytosis. Newly developed device for magnetofection under dynamic gradient magnetic field, “DynaFECTOR", was used to compare transfection efficiency of human liver hepatocellular carcinoma cell line HepG2 with that obtained by lipofection and magnetofection. The effect of two parameters on transfection efficiency, incubation time under dynamic magnetic field and rotation frequency of magnet, was estimated. Liposomal magnetofection under dynamic gradient magnetic field showed the highest transfection efficiency for HepG2 cells.

Keywords: Dynamic magnetic field, Lipofection, Magnetofection

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Authors: A. N. Gornostaeva, P. I. Bobyleva, E. R. Andreeva, L. B. Buravkova

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Multipotent mesenchymal stromal cells (MSCs) possess immunomodulatory properties. The effect of MSCs on the crucial cellular immunity compartment – T-cells is of a special interest. It is known that MSC tissue niche and expected milieu of their interaction with T- cells are characterized by low oxygen concentration, whereas the in vitro experiments usually are carried out at a much higher ambient oxygen (20%). We firstly evaluated immunomodulatory effects of MSCs on T-cells at tissue-related oxygen (5%) after interaction implied cell-to-cell contacts and paracrine factors only. It turned out that MSCs under reduced oxygen can effectively suppress the activation and proliferation of PHAstimulated T-cells and can provoke decrease in the production of proinflammatory and increase in anti-inflammatory cytokines. In hypoxia some effects were amplified (inhibition of proliferation, antiinflammatory cytokine profile shift). This impact was more evident after direct cell-to-cell interaction; lack of intercellular contacts could revoke the potentiating effect of hypoxia.

Keywords: Cell-to-cell interaction, low oxygen, MSC immunosuppression, T-cells.

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Authors: M Tayari, SZ Moosavi-nejad, A Shabani, M Rezaei Tavirani

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Free Hemoglobin promotes the accumulation of hydroxyl radicals by the heme iron, which can react with endogenous hydrogen peroxide to produce free radicals which may cause severe oxidative cell damage. Haptoglobin binds to Hemoglobin strongly and Haptoglobin-Hemoglobin binding is irreversible. Peroxidase activity of Haptoglobin(2-2)-Hemoglobin complex was assayed by following increase of absorption of produced tetraguaiacol as the second substrate of Haptoglobin-Hemoglobin complex at 470 nm and 42°C by UV-Vis spectrophotometer. The results have shown that peroxidase activity of Haptoglobin(2-2)-Hemoglobin complex is modulated via homotropic effect of hydrogen peroxide as allostric substrate. On the other hand antioxidant property of Haptoglobin(2- 2)-Hemoglobin was increased via heterotropic effect of the two drugs (especially ampicillin) on peroxidase activity of the complex. Both drugs also have mild effect on quality of homotropic property of peroxidase activity of Haptoglobin(2-2)-Hemoglobin complex. Therefore, in vitro studies show that the two drugs may help Hp-Hb complex to remove hydrogen peroxide from serum at pathologic temperature ature (42 C).

Keywords: Haptoglobin, Hemoglobin, Antioxidant, Antibiotics.

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Authors: Adel Dalilottojari, Bahman Delalat, Frances J. Harding, Michaelia P. Cockshell, Claudine S. Bonder, Nicolas H. Voelcker

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Endothelial Progenitor Cell (EPC) based therapies continue to be of interest to treat ischemic events based on their proven role to promote blood vessel formation and thus tissue re-vascularisation. Current strategies for the production of clinical-grade EPCs requires the in vitro isolation of EPCs from peripheral blood followed by cell expansion to provide sufficient quantities EPCs for cell therapy. This study aims to examine the use of different biomolecules to significantly improve the current strategy of EPC capture and expansion on collagen type I (Col I). In this study, four different biomolecules were immobilised on a surface and then investigated for their capacity to support EPC capture and proliferation. First, a cell microarray platform was fabricated by coating a glass surface with epoxy functional allyl glycidyl ether plasma polymer (AGEpp) to mediate biomolecule binding. The four candidate biomolecules tested were Col I, collagen type II (Col II), collagen type IV (Col IV) and vascular endothelial growth factor A (VEGF-A), which were arrayed on the epoxy-functionalised surface using a non-contact printer. The surrounding area between the printed biomolecules was passivated with polyethylene glycol-bisamine (A-PEG) to prevent non-specific cell attachment. EPCs were seeded onto the microarray platform and cell numbers quantified after 1 h (to determine capture) and 72 h (to determine proliferation). All of the extracellular matrix (ECM) biomolecules printed demonstrated an ability to capture EPCs within 1 h of cell seeding with Col II exhibiting the highest level of attachment when compared to the other biomolecules. Interestingly, Col IV exhibited the highest increase in EPC expansion after 72 h when compared to Col I, Col II and VEGF-A. These results provide information for significant improvement in the capture and expansion of human EPC for further application.

Keywords: Cardiovascular disease, cell microarray platform, cell therapy, endothelial progenitor cells, high throughput screening.

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Authors: Irina Spaka, Rita Birkenfelde, Svetlana Kozireva, Jevgenija Osmjana, Madara Upmane, ElenaKashuba, Irina Kholodnyuk Holodnuka

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Epstein-Barr virus (EBV) is implicated in the pathogenesis of the endemic Burkitt-s lymphoma (BL). The EBVpositive BL-derived cell lines initially maintain the original tumor phenotype of EBV infection (latency I, LatI), but most of them drift toward a lymphoblast phenotype of EBV latency III (LatIII) during in vitro culturing. The aim of the present work was to characterize the B-cell subsets in EBV-positive BL cell lines and to verify whether a particular cell subset correlates with the type of EBV infection. The phenotype analysis of two EBV-negative and eleven EBV-positive (three of LatI and eight of LatIII) BL cell lines was performed by polychromatic flow cytomery, based on expression pattern of CD19, CD10, CD38, CD27, and CD5 markers. Two cell subsets, CD19+CD10+ and CD19+CD10-, were defined in LatIII BL cell lines. In both subsets, the CD27 and CD5 cell surface expression was detected in a proportion of the cells.

Keywords: B-cell subsets, Burkitt's lymphoma cell lines, EBV latency, phenotype profiles.

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Authors: T. Qasim, B. El Masoud, D. Ailabouni

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This in vitro study focused on investigating the fatigue resistance of veneered zirconia molar crowns with different veneering ceramic thicknesses, simulating the relative wear depths under simulated cyclic loading. A mandibular first molar was prepared and then scanned using computer-aided design/computer-aided manufacturing (CAD/CAM) technology to fabricate 32 zirconia copings of uniform 0.5 mm thickness. The manufactured copings then veneered with 1.5 mm, 1.0 mm, 0.5 mm, and 0.0 mm representing 0%, 33%, 66%, and 100% relative wear of a normal ceramic thickness of 1.5 mm. All samples were thermally aged to 6000 thermo-cycles for 2 minutes with distilled water between 5 ˚C and 55 ˚C. The samples subjected to cyclic fatigue and fracture testing using SD Mechatronik chewing simulator. These samples are loaded up to 1.25x10⁶ cycles or until they fail. During fatigue, testing, extensive cracks were observed in samples with 0.5 mm veneering layer thickness. Veneering layer thickness 1.5-mm group and 1.0-mm group were not different in terms of resisting loads necessary to cause an initial crack or final failure. All ceramic zirconia-based crown restorations with varying occlusal veneering layer thicknesses appeared to be fatigue resistant. Fracture load measurement for all tested groups before and after fatigue loading exceeded the clinical chewing forces in the posterior region. In general, the fracture loads increased after fatigue loading and with the increase in the thickness of the occlusal layering ceramic.

Keywords: All ceramic, dental crowns, relative wear, chewing simulator, cyclic loading, thermally ageing.

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Authors: Rawia M. Khalil, Ahmed A. Abd El Rahman, Mahfouz A. Kassem, Mohamed S. El Ridi, Mona M. Abou Samra, Ghada E. A. Awad, Soheir S. Mansy

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Solid lipid nanoparticles (SLNs) have gained great attention for the topical treatment of skin associated fungal infection as they facilitate the skin penetration of loaded drugs. Our work deals with the preparation of nystatin loaded solid lipid nanoparticles (NystSLNs) using the hot homogenization and ultrasonication method. The prepared NystSLNs were characterized in terms of entrapment efficiency, particle size, zeta potential, transmission electron microscopy, differential scanning calorimetry, rheological behavior and in vitro drug release. A stability study for 6 months was performed. A microbiological study was conducted in male rats infected with Candida albicans, by counting the colonies and examining the histopathological changes induced on the skin of infected rats. The results showed that SLNs dispersions are spherical in shape with particle size ranging from 83.26±11.33 to 955.04±1.09 nm. The entrapment efficiencies are ranging from 19.73±1.21 to 72.46±0.66% with zeta potential ranging from -18.9 to -38.8 mV and shear-thinning rheological Behavior. The stability studies done for 6 months showed that nystatin (Nyst) is a good candidate for topical SLN formulations. A least number of colony forming unit/ ml (cfu/ml) was recorded for the selected NystSLN compared to the drug solution and the commercial Nystatin® cream present in the market. It can be fulfilled from this work that SLNs provide a good skin targeting effect and may represent promising carrier for topical delivery of Nyst offering the sustained release and maintaining the localized effect, resulting in an effective treatment of cutaneous fungal infection.

Keywords: Candida infections, Hot homogenization, Nystatin, Solid lipid nanoparticles, Stability, Topical delivery.

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Authors: Majid Mirzaei, Saeid Samiezadeh , Abbas Khodadadi, Mohammad R. Ghazavi

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This paper presents a novel method for prediction of the mechanical behavior of proximal femur using the general framework of the quantitative computed tomography (QCT)-based finite element Analysis (FEA). A systematic imaging and modeling procedure was developed for reliable correspondence between the QCT-based FEA and the in-vitro mechanical testing. A speciallydesigned holding frame was used to define and maintain a unique geometrical reference system during the analysis and testing. The QCT images were directly converted into voxel-based 3D finite element models for linear and nonlinear analyses. The equivalent plastic strain and the strain energy density measures were used to identify the critical elements and predict the failure patterns. The samples were destructively tested using a specially-designed gripping fixture (with five degrees of freedom) mounted within a universal mechanical testing machine. Very good agreements were found between the experimental and the predicted failure patterns and the associated load levels.

Keywords: Bone, Osteoporosis, Noninvasive methods, Failure Analysis

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Authors: T. Citarasu, M. Michaelbabu V. N. Vakharia

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The rhizome of Java grass, Cyperus rotundus was extracted different organic polar and non-polar solvents and performed the in vitro antiviral and immunostimulant activities against White Spot Syndrome Virus (WSSV) and Vibrio harveyi respectively. Based on the initial screening the ethyl acetate extract of C. rotundus was strong activities and further it was purified through silica column chromatography and the fractions were screened again for antiviral and immunostimulant activity. Among the different fractions screened against the WSSV and V. harveyi, the fractions, FIII to FV had strong activities. In order to study the in vivo influence of C. rotundus, the fractions (F-III to FV) were pooled and delivered to the F. indicus through artificial feed for 30 days. After the feeding trail the experimental and control diet fed F. indicus were challenged with virulent WSSV and studied the survival, molecular diagnosis, biochemical, haematological, and immunological parameters. Surprisingly, the pooled fractions (F-IV to FVI) incorporated diets helped to significantly (P<0.01) suppressed viral multiplication, showed significant (P<0.01) differences in protein and glucose levels, improved total haemocyte count (THC), coagulase activity, significantly increased (P <= 0.001) prophenol oxidase and intracellular superoxide anion production compared to the control shrimps. Based on the results, C. rotundus extracts effectively suppressed WSSV multiplication and improve the immune system in F. indicus against WSSV infection and this knowledge will helps to develop novel drugs from C. rotundus against WSSV.

Keywords: Antiviral drugs, Cyperus rotundus, Fenneropenaeus indicus, WSSV.

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Authors: May-Ling Tan, Yi Su, Chi-Wan Lim, Liang Zhong, Ru-San Tan

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This paper describes an automatic algorithm to restore the shape of three-dimensional (3D) left ventricle (LV) models created from magnetic resonance imaging (MRI) data using a geometry-driven optimization approach. Our basic premise is to restore the LV shape such that the LV epicardial surface is smooth after the restoration. A geometrical measure known as the Minimum Principle Curvature (κ2) is used to assess the smoothness of the LV. This measure is used to construct the objective function of a two-step optimization process. The objective of the optimization is to achieve a smooth epicardial shape by iterative in-plane translation of the MRI slices. Quantitatively, this yields a minimum sum in terms of the magnitude of κ 2, when κ2 is negative. A limited memory quasi-Newton algorithm, L-BFGS-B, is used to solve the optimization problem. We tested our algorithm on an in vitro theoretical LV model and 10 in vivo patient-specific models which contain significant motion artifacts. The results show that our method is able to automatically restore the shape of LV models back to smoothness without altering the general shape of the model. The magnitudes of in-plane translations are also consistent with existing registration techniques and experimental findings.

Keywords: Magnetic Resonance Imaging, Left Ventricle, ShapeRestoration, Principle Curvature, Optimization

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Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

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Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: Mesenchymal stem cells, Cryopreservation, Stemness, Senescence.

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Authors: YuriyTasinkevych, Ihor Trots, AndrzejNowicki, Marcin Lewandowski

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The paper presents the optimization problem for the multi-element synthetic transmit aperture method (MSTA) in ultrasound imaging applications. The optimal choice of the transmit aperture size is performed as a trade-off between the lateral resolution, penetration depth and the frame rate. Results of the analysis obtained by a developed optimization algorithm are presented. Maximum penetration depth and the best lateral resolution at given depths are chosen as the optimization criteria. The optimization algorithm was tested using synthetic aperture data of point reflectors simulated by Filed II program for Matlab® for the case of 5MHz 128-element linear transducer array with 0.48 mm pitch are presented. The visualization of experimentally obtained synthetic aperture data of a tissue mimicking phantom and in vitro measurements of the beef liver are also shown. The data were obtained using the SonixTOUCH Research systemequipped with a linear 4MHz 128 element transducerwith 0.3 mm element pitch, 0.28 mm element width and 70% fractional bandwidth was excited by one sine cycle pulse burst of transducer's center frequency.

Keywords: synthetic aperture method, ultrasound imaging, beamforming.

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Authors: Brou K., Guéhi T., Konan A. G., Gbakayoro J. B., Gnakri D.

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Two types of crushing were applied to grains of red sorghum: manual crushing using a mortar and pestle of kitchen and mechanical crushing using a hammer mill. The flours obtained at the end of these various crushing were filtered and subdivided in different fractions according to the diameters of the mesh of the sieves (0.16mm; 0.25mm; 0.315mm; 0.4mm, and 0.63mm…). Some physical, chemical and nutritional traits of these flours were evaluated using Association of Official Analytical Chemists (AOAC). In vitro digestibility of these flours was also studied with freezing of flour 1% like substrate and α-amylase from B. licheniformis (E.C.3.2.1.1; Megazyme, Wicklow, Ireland). The results revealed that the batches of flours which have the finest diameters as 0.16mm; 0.25mm are the richest one in nutrients and are also the most digestible. Also mechanical crushing is the best mean to obtain significant amount of flours. In conclusion, the type of crushing and the size of the particles have an impact on the final concentration of some nutrients of the flours obtained. Indeed, the finest particles (0.16mm – 0.25mm 0.315mm) obtained after sifting of the flours are more nutritive and have a better digestibility than others size. So the finest particles could be advised for management of cereals namely the sorghum for the production of the infantile foods.

Keywords: Nutrients, digestibility, crush, flour, milling, granulometry.

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Authors: A. M. Tarola, R. Preti, A. M. Girelli, P. Campana

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Safeguarding, studying and enhancing biodiversity play an important and indispensable role in re-launching agriculture. The ancient local varieties are therefore a precious resource for genetic and health improvement. In order to protect biodiversity through the recovery and valorization of autochthonous varieties, in this study we analyzed 12 samples of four ancient apple cultivars representative of Friuli Venezia Giulia, selected by local farmers who work on a project for the recovery of ancient apple cultivars. The aim of this study is to evaluate the polyphenolic profile and the antioxidant capacity that characterize the organoleptic and functional qualities of this fruit species, besides having beneficial properties for health. In particular, for each variety, the following compounds were analyzed, both in the skins and in the pulp: gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, coumaric acid, ferulic acid, rutin, phlorizin, phloretin and quercetin to highlight any differences in the edible parts of the apple. The analysis of individual phenolic compounds was performed by High Performance Liquid Chromatography (HPLC) coupled with a diode array UV detector (DAD), the antioxidant capacity was estimated using an in vitro essay based on a Free Radical Scavenging Method and the total phenolic compounds was determined using the Folin-Ciocalteau method. From the results, it is evident that the catechins are the most present polyphenols, reaching a value of 140-200 μg/g in the pulp and of 400-500 μg/g in the skin, with the prevalence of epicatechin. Catechins and phlorizin, a dihydrohalcone typical of apples, are always contained in larger quantities in the peel. Total phenolic compounds content was positively correlated with antioxidant activity in apple pulp (r² = 0,850) and peel (r² = 0,820). Comparing the results, differences between the varieties analyzed and between the edible parts (pulp and peel) of the apple were highlighted. In particular, apple peel is richer in polyphenolic compounds than pulp and flavonols are exclusively present in the peel. In conclusion, polyphenols, being antioxidant substances, have confirmed the benefits of fruit in the diet, especially as a prevention and treatment for degenerative diseases. They demonstrated to be also a good marker for the characterization of different apple cultivars. The importance of protecting biodiversity in agriculture was also highlighted through the exploitation of native products and ancient varieties of apples now forgotten.

Keywords: Apple, biodiversity, polyphenols, antioxidant activity, HPLC-DAD, characterization.

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Authors: Salma H. Abu Hafsa, A. Mendonca, B. Brehm-Stecher, A. A. Hassan, S. A. Ibrahim

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Enterococci are important inhabitants of the animal intestine and are widely used in probiotic products. A probiotic strain is expected to possess several desirable properties in order to exert beneficial effects. Therefore, the objective of this study was to isolate, characterize and identify Enterococcus sp. from chicken cecal and fecal samples to determine potential probiotic properties. Enterococci were isolated from chicken ceca and feces of thirty three clinically healthy chickens from a local farm. In vitro studies were performed to assess antibacterial activity of the isolated LAB (using agar well diffusion and cell free supernatant broth technique against Salmonella enterica serotype Enteritidis), survival in acidic conditions, resistance to bile salts, and their survival during simulated gastric juice conditions at pH 2.5. Isolates were identified by biochemical carbohydrate fermentation patterns using an API 50 CHL kit and API ZYM kits and by sequenced 16S rDNA. An isolate belonging to E. faecium species exhibited inhibitory effect against S. enteritidis. This isolate producing a clear zone as large as 10.30 mm or greater and was able to grow in the coculture medium and at the same time, inhibited the growth S. enteritidis. In addition, E. faecium exhibited significant resistance under highly acidic conditions at pH 2.5 for 8 h and survived well in bile salt at 0.2% for 24 h and showing ability to survive in the presence of simulated gastric juice at pH 2.5. Based on these results, E. faecium isolate fulfills some of the criteria to be considered as a probiotic strain and therefore, could be used as a feed additive with good potential for controlling S. Enteritidis in chickens. However, in vivo studies are needed to determine the safety of the strain.

Keywords: Acid tolerance, antimicrobial activity, Enterococcus faecium, probiotic.

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Authors: Kittiwann Nimkerdphol, Masahiro Nakagawa

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Biological reactions of individuals of a testing animal to toxic substance are unique and can be used as an indication of the existing of toxic substance. However, to distinguish such phenomenon need a very complicate system and even more complicate to analyze data in 3 dimensional. In this paper, a system to evaluate in vitro biological activities to acute toxicity of stochastic self-affine non-stationary signal of 3D goldfish swimming by using fractal analysis is introduced. Regular digital camcorders are utilized by proposed algorithm 3DCCPC to effectively capture and construct 3D movements of the fish. A Critical Exponent Method (CEM) has been adopted as a fractal estimator. The hypothesis was that the swimming of goldfish to acute toxic would show the fractal property which related to the toxic concentration. The experimental results supported the hypothesis by showing that the swimming of goldfish under the different toxic concentration has fractal properties. It also shows that the fractal dimension of the swimming related to the pH value of FD Ôëê 0.26pH + 0.05. With the proposed system, the fish is allowed to swim freely in all direction to react to the toxic. In addition, the trajectories are precisely evaluated by fractal analysis with critical exponent method and hence the results exhibit with much higher degree of confidence.

Keywords: 3D locomotion, bioassay, critical exponent method, CEM, fractal analysis, goldfish.

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Authors: Seyed Mohammad Hosseini, Amirhossein Moghanian

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The main purpose of this research was to design a biomimetic system by freeze-drying method for evaluating the effect of adding 5 and 10 mol. % of zinc (Zn) in 58S bioactive glass and gelatin (5ZnBG/G and 10ZnBG/G) in terms of structural and biological changes. The structural analyses of samples were performed by X-Ray Diffraction (XRD), scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) spectroscopy. Also, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alkaline phosphatase (ALP) activity tests were carried out for investigation of MC3T3-E1 cell behaviors. The SEM results demonstrated the spherical shape of the formed hydroxyapatite (HA) phases and also HA characteristic peaks were detected by XRD spectroscopy after 3 days of immersion in the simulated body fluid (SBF) solution. Meanwhile, FTIR spectra proved that the intensity of P–O peaks for 5ZnBG/G was more than 10ZnBG/G and control samples. Moreover, the results of ALP activity test illustrated that the optimal amount of Zn (5ZnBG/G) caused a considerable enhancement in bone cell growth. Taken together, the scaffold with 5 mol.% Zn was introduced as an optimal sample because of its higher biocompatibility, in vitro bioactivity and growth of MC3T3-E1 cells in comparison with other samples in bone tissue engineering.

Keywords: Scaffold, gelatin, modified bioactive glass, ALP, bone tissue engineering.

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Authors: Iman A. Elkiweri, Ph.D, Martha C. Tissot van Patot, Ph.D., Yan Ling Zhang, Ph.D., Uwe Christians, Ph.D., Thomas K. Henthorn, M.D.,

Abstract:

Verapamil has been shown to inhibit fentanyl uptake in vitro and is a potent P-glycoprotein inhibitor. Tissue partitioning of loperamide, a commercially available opioid, is closely controlled by the P-gp efflux transporter. The following studies were designed to evaluate the effect of opioids on verapamil partitioning in the lung and brain, in vivo. Opioid (fentanyl or loperamide) was administered by intravenous infusion to Sprague Dawley rats alone or in combination with verapamil and plasma, with lung and brain tissues were collected at 1, 5, 6, 8, 10 and 60 minutes. Drug dispositions were modeled by recirculatory pharmacokinetic models. Fentanyl slightly increased the verapamil lung (PL) partition coefficient yet decreased the brain (PB) partition coefficient. Furthermore, loperamide significantly increased PLand PB. Fentanyl reduced the verapamil volume of distribution (V1) and verapamil elimination clearance (ClE). Fentanyl decreased verapamil brain partitioning, yet increased verapamil lung partitioning. Also, loperamide increased lung and brain partitioning in vivo. These results suggest that verapamil and fentanyl may be substrates of an unidentified inward transporter in brain tissue and confirm that verapamil and loperamide are substrates of the efflux transporter P-gp.

Keywords: Efflux transporter, elimination clearance, partition coefficient, verapamil

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Authors: S. Techaoei, K. Jarmkom, P. Eakwaropas, W. Khobjai

Abstract:

The objective of this research was focused on investigating in vitro antimicrobial activity of Phellinus linteus fruiting body extracts on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. Phellinus linteus fruiting body was extracted with ethanol and ethyl acetate and was vaporized. The disc diffusion assay was used to assess antimicrobial activity against tested bacterial strains. Primary screening of chemical profile of crude extract was determined by using thin layer chromatography. The positive control and the negative control were used as erythromycin and dimethyl sulfoxide, respectively. Initial screening of Phellinus linteus crude extract with the disc diffusion assay demonstrated that only ethanol had greater antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. The MIC assay showed that the lower MIC was observed with 0.5 mg/ml of Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus and 0.25 mg/ml. of Escherichia coli and Staphylococcus aureus, respectively. TLC chemical profile of extract was represented at R_f ≈ 0.71-0.76.

Keywords: Staphylococcus aureus, Phellinus linteus, methicillin-resistant Staphylococcus aureus, antimicrobial activity, Escherichia coli.

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Authors: P. Thongmuang, Y. Sudjaroen

Abstract:

The methanolic extracts from seeds of tamarind (Tamarindus indica) was prepared by Soxhlet apparatus extraction and evaluated for total phenolic content by Folin-Ciocalteu method. Then, methanolic extract was screened biological activities (In vitro) for anti-melanogenic activity by tyrosinase inhibition test, antiinflammation activity by cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) inhibition test, and cytotoxic screening test with Vero cells. The results showed that total phenolic content, which contained in extract, was contained 27.72 mg of gallic acid equivalent per g of dry weight. The ability to inhibit tyrosinase enzyme, which exerted by Tamarind seed extracts (1 mg/ml) was 52.13 ± 0.42 %. The extract was not possessed inhibitory effect to COX-1 and COX-2 enzymes and cytotoxic effect to Vero cells. The finding is concludes that tested seed extract was possessed antimelanogenic activity with non-toxic effects. However, there was not exhibited anti-inflammatory activity. Further studies include the use of advance biological models to confirm this biological activity, as well as, the isolation and characterization of the purified compounds that it was contained.

Keywords: Tamarindus indica, anti-melanogenic, antiinflammatotion, cytotoxicity, seed, phenolic compounds.

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Authors: Mithilesh Singh, Rakhi Chaturvedi

Abstract:

Neem is a highly heterozygous and commercially important perennial plant. Conventionally, it is propagated by seeds which loose viability within two weeks. Strictly cross pollinating nature of the plant causes serious barrier to the genetic improvement by conventional methods. Alternative methods of tree improvement such as somatic hybridization, mutagenesis and genetic transformation require an efficient in vitro plant regeneration system. In this regard, somatic embryogenesis particularly secondary somatic embryogenesis may offer an effective system for large scale plant propagation without affecting the clonal fidelity of the regenerants. It can be used for synthetic seed production, which further bolsters conservation of this tree species which is otherwise very difficult The present report describes the culture conditions necessary to induce and maintain repetitive somatic embryogenesis, for the first time, in neem. Out of various treatments tested, the somatic embryos were induced directly from immature zygotic embryos of neem on MS + TDZ (0.1 μM) + ABA (4 μM), in more than 76 % cultures. Direct secondary somatic embryogenesis occurred from primary somatic embryos on MS + IAA (5 μM) + GA3 (5 μM) in 12.5 % cultures. Embryogenic competence of the explant as well as of the primary embryos was maintained for a long period by repeated subcultures at frequent intervals. A maximum of 10 % of these somatic embryos were converted into plantlets.

Keywords: Azadirachta indica A. Juss., Cytokinin, Somatic embryogenesis, zygotic embryo culture.

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

Abstract:

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.

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Authors: Ibtisam A. Abbas Al-Darkazly

Abstract:

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.

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Authors: M. Saravanan

Abstract:

Antimicrobial resistant is becoming a major factor in virtually all hospital acquired infection may soon untreatable is a serious public health problem. These concerns have led to major research effort to discover alternative strategies for the treatment of bacterial infection. Nanobiotehnology is an upcoming and fast developing field with potential application for human welfare. An important area of nanotechnology for development of reliable and environmental friendly process for synthesis of nanoscale particles through biological systems In the present studies are reported on the use of fungal strain Aspergillus species for the extracellular synthesis of bionanoparticles from 1 mM silver nitrate (AgNO3) solution. The report would be focused on the synthesis of metallic bionanoparticles of silver using a reduction of aqueous Ag+ ion with the culture supernatants of Microorganisms. The bio-reduction of the Ag+ ions in the solution would be monitored in the aqueous component and the spectrum of the solution would measure through UV-visible spectrophotometer The bionanoscale particles were further characterized by Atomic Force Microscopy (AFM), Fourier Transform Infrared Spectroscopy (FTIR) and Thin layer chromatography. The synthesized bionanoscale particle showed a maximum absorption at 385 nm in the visible region. Atomic Force Microscopy investigation of silver bionanoparticles identified that they ranged in the size of 250 nm - 680 nm; the work analyzed the antimicrobial efficacy of the silver bionanoparticles against various multi drug resistant clinical isolates. The present Study would be emphasizing on the applicability to synthesize the metallic nanostructures and to understand the biochemical and molecular mechanism of nanoparticles formation by the cell filtrate in order to achieve better control over size and polydispersity of the nanoparticles. This would help to develop nanomedicine against various multi drug resistant human pathogens.

Keywords: Bionanoparticles, UV-visible spectroscopy, AtomicForce Microscopy, Extracellular synthesis, Multi drug resistant, antimicrobial activity, Nanomedicine

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Authors: Omima E. Fadlallah, Abdullahi H. El Tinay, Elfadil E. Babiker

Abstract:

Sorghum flour was supplemented with 15 and 30% chickpea flour. Sorghum flour and the supplement were fermented at 35 oC for 0, 8, 16, and 24 h. Changes in pH, titrable acidity, total soluble solids, protein content, in vitro protein digestibility and amino acid composition were investigated during fermentation and/or after supplementation of sorghum flour with chickpea. The pH of the fermenting material decreased sharply with a concomitant increase in the titrable acidity. The total soluble solids remained unchanged with progressive fermentation time. The protein content of sorghum cultivar was found to be 9.27 and that of chickpea was 22.47%. The protein content of sorghum cultivar after supplementation with15 and 30% chickpea was significantly (P ≤ 0.05) increased to 11.78 and 14.55%, respectively. The protein digestibility also increased after fermentation from 13.35 to 30.59 and 40.56% for the supplements, respectively. Further increment in protein content and digestibility was observed when supplemented and unsupplemented samples were fermented for different periods of time. Cooking of fermented samples was found to increase the protein content slightly and decreased digestibility for both supplements. Amino acid content of fermented and fermented and cooked supplements was determined. Supplementation was found to increase the lysine and therionine content. Cooking following fermentation decreased lysine, isoleucine, valine and sulfur containg amino acids.

Keywords: Amino acid, Chickpea, Cooking, Fermentation, protein, Sorghum.

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