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Sample records for 3d in-vitro assay

  1. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

    Science.gov (United States)

    Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

  2. Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays.

    Science.gov (United States)

    Lal-Nag, Madhu; McGee, Lauren; Titus, Steven A; Brimacombe, Kyle; Michael, Sam; Sittampalam, Gurusingham; Ferrer, Marc

    2017-03-01

    Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.

  3. A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

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    Neufeld Gera

    2009-11-01

    Full Text Available Abstract Background The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. Methods We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. Results The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2 is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. Conclusion Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on

  4. 3D in vitro technology for drug discovery.

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    Hosseinkhani, Hossein

    2012-02-01

    Three-dimensional (3D) in vitro systems that can mimic organ and tissue structure and function in vivo, will be of great benefit for a variety of biological applications from basic biology to toxicity testing and drug discovery. There have been several attempts to generate 3D tissue models but most of these models require costly equipment, and the most serious disadvantage in them is that they are too far from the mature human organs in vivo. Because of these problems, research and development in drug discovery, toxicity testing and biotech industries are highly expensive, and involve sacrifice of countless animals and it takes several years to bring a single drug/product to the market or to find the toxicity or otherwise of chemical entities. Our group has been actively working on several alternative models by merging biomaterials science, nanotechnology and biological principles to generate 3D in vitro living organs, to be called "Human Organs-on-Chip", to mimic natural organ/tissues, in order to reduce animal testing and clinical trials. We have fabricated a novel type of mechanically and biologically bio-mimicking collagen-based hydrogel that would provide for interconnected mini-wells in which 3D cell/organ culture of human samples in a manner similar to human organs with extracellular matrix (ECM) molecules would be possible. These products mimic the physical, chemical, and biological properties of natural organs and tissues at different scales. This paper will review the outcome of our several experiments so far in this direction and the future perspectives.

  5. 3D microtumors in vitro supported by perfused vascular networks

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    Sobrino, Agua; Phan, Duc T. T.; Datta, Rupsa; Wang, Xiaolin; Hachey, Stephanie J.; Romero-López, Mónica; Gratton, Enrico; Lee, Abraham P.; George, Steven C.; Hughes, Christopher C. W.

    2016-01-01

    There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This “organs-on-chips” approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This “tumor-on-a-chip” platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging Microscopy and, compared to their surrounding stroma, many show a higher free/bound NADH ratio consistent with their known preference for aerobic glycolysis. The VMT platform provides a unique model for studying vascularized solid tumors in vitro. PMID:27549930

  6. In vitro biological characterization of macroporous 3D Bonelike structures prepared through a 3D machining technique

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    Laranjeira, M.S.; Dias, A.G. [INEB - Instituto de Engenharia Biomedica, Divisao de Biomateriais, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto (Portugal); Santos, J.D. [INEB - Instituto de Engenharia Biomedica, Divisao de Biomateriais, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto (Portugal); Universidade do Porto, Faculdade de Engenharia, Departamento de Engenharia Metalurgica e Materiais, Rua Dr. Roberto Frias, 4200-465 Porto - Portugal (Portugal); Fernandes, M.H., E-mail: mhrf@portugalmail.pt [Universidade do Porto, Faculdade de Medicina Dentaria, Laboratorio de Farmacologia e Biocompatibilidade Celular, Rua Dr. Manuel Pereira da Silva, 4200-392 Porto (Portugal)

    2009-04-30

    3D bioactive macroporous structures were prepared using a 3D machining technique. A virtual 3D structure model was created and a computer numerically controlled (CNC) milling device machined Bonelike samples. The resulting structures showed a reproducible macroporosity and interconnective structure. Macropores size after sintering was approximately 2000 {mu}m. In vitro testing using human bone marrow stroma showed that cells were able to adhere and proliferate on 3D structures surface and migrate into all macropore channels. In addition, these cells were able to differentiate, since mineralized globular structures associated with cell layer were identified. Results obtained showed that 3D structures of Bonelike successfully allow cell migration into all macropores, and allow human bone marrow stromal cells to proliferate and differentiate. This innovative technique may be considered as a step-forward preparation for 3D interconnective macroporous structures that allow bone ingrowth while maintaining mechanical integrity.

  7. Seeding Osteoblasts onto Osteocytes: An In Vitro 3D Study

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    Judith Green

    2006-01-01

    Full Text Available Understanding the mechanisms by which bone cells communicate is vital in exploring diseases characterized by bone degeneration, namely, osteoporosis. Cell seeding has been used in two dimensional (2D cell cultures to study how bone cells interact with one another, specifically, to prove the existence of gap junctions between osteocytes and osteoblasts. However, the natural three dimensional (3D state of bone tissue requires examining it in 3D. Accordingly, the cell seeding procedure was tested on trabecular bone core explants to ascertain whether it is useful in 3D studies as well. When the dye concentrations taken from past 2D experiments were used, Day 1 showed many osteoblasts, but by Day 2 the cells were not visible. The dye concentrations were then doubled to determine if the osteoblasts were still seeded onto the bone cores and viable but not visible, or if they had actually died. With these dye concentrations, the stained osteoblasts were still visible on the second day after seeding, indicating that the cells were seeded and living. According to these results, it is evident that with minor modifications of the 2D procedure, it is possible to seed osteoblasts onto osteocytes in 3D, making this a credible test for the presence of gap junctions in 3D bone tissue.

  8. Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices.

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    Casey, A; Gargotti, M; Bonnier, F; Byrne, H J

    2016-06-01

    The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in "reduced" toxicity or cellular "resistance" to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug

  9. Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

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    Baron, Matthew G.; Mintram, Kate S.; Owen, Stewart F.; Hetheridge, Malcolm J.; Moody, A. John; Purcell, Wendy M.; Jackson, Simon K.; Jha, Awadhesh N.

    2017-01-01

    At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control. PMID:28045944

  10. Engineering an in vitro air-blood barrier by 3D bioprinting

    OpenAIRE

    2015-01-01

    Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual met...

  11. Assessing Drug Efficacy in a Miniaturized Pancreatic Cancer In Vitro 3D Cell Culture Model.

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    Shelper, Todd B; Lovitt, Carrie J; Avery, Vicky M

    2016-09-01

    Pancreatic cancer continues to have one of the poorest prognoses among all cancers. The drug discovery efforts for this disease have largely failed, with no significant improvement in survival outcomes for advanced pancreatic cancer patients over the past 20 years. Traditional in vitro cell culture techniques have been used extensively in both basic and early drug discovery; however, these systems offer poor models to assess emerging therapeutics. More predictive cell-based models, which better capture the cellular heterogeneity and complexities of solid pancreatic tumors, are urgently needed not only to improve drug discovery success but also to provide insight into the tumor biology. Pancreatic tumors are characterized by a unique micro-environment that is surrounded by a dense stroma. A complex network of interactions between extracellular matrix (ECM) components and the effects of cell-to-cell contacts may enhance survival pathways within in vivo tumors. This biological and physical complexity is lost in traditional cell monolayer models. To explore the predictive potential of a more complex cellular system, a three-dimensional (3D) micro-tumor assay was evaluated. Efficacy of six current chemotherapeutics was determined against a panel of primary and metastatic pancreatic tumor cell lines in a miniaturized ECM-based 3D cell culture system. Suitability for potential use in high-throughput screening applications was assessed, including ascertaining the effects that miniaturization and automation had on assay robustness. Cellular health was determined by utilizing an indirect population-based metabolic activity assay and a direct imaging-based cell viability assay.

  12. AlgiMatrix™ based 3D cell culture system as an in-vitro tumor model for anticancer studies.

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    Chandraiah Godugu

    Full Text Available BACKGROUND: Three-dimensional (3D in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. METHODS: Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100-300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin and nanoparticle (NLC were done using spheroids. RESULTS: IC(50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. CONCLUSION: The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

  13. Nanoparticle toxicity assessment using an in vitro 3-D kidney organoid culture model.

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    Astashkina, Anna I; Jones, Clint F; Thiagarajan, Giridhar; Kurtzeborn, Kristen; Ghandehari, Hamid; Brooks, Benjamin D; Grainger, David W

    2014-08-01

    Nanocarriers and nanoparticles remain an intense pharmaceutical and medical imaging technology interest. Their entry into clinical use is hampered by the lack of reliable in vitro models that accurately predict in vivo toxicity. This study evaluates a 3-D kidney organoid proximal tubule culture to assess in vitro toxicity of the hydroxylated generation-5 PAMAM dendrimer (G5-OH) compared to previously published preclinical in vivo rodent nephrotoxicity data. 3-D kidney proximal tubule cultures were created using isolated murine proximal tubule fractions suspended in a biomedical grade hyaluronic acid-based hydrogel. Toxicity in these cultures to neutral G5-OH dendrimer nanoparticles and gold nanoparticles in vitro was assessed using clinical biomarker generation. Neutral PAMAM nanoparticle dendrimers elicit in vivo-relevant kidney biomarkers and cell viability in a 3-D kidney organoid culture that closely reflect toxicity markers reported in vivo in rodent nephrotoxicity models exposed to this same nanoparticle.

  14. 3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

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    Jacob L. Albritton

    2017-01-01

    Full Text Available Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies.

  15. 3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

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    Albritton, Jacob L.

    2017-01-01

    ABSTRACT Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. PMID:28067628

  16. Engineering an in vitro air-blood barrier by 3D bioprinting.

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    Horváth, Lenke; Umehara, Yuki; Jud, Corinne; Blank, Fabian; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

    2015-01-22

    Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.

  17. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

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    Bédouin, Y; Pellen Mussi, P; Tricot-Doleux, S; Chauvel-Lebret, D; Auroy, P; Ravalec, X; Oudadesse, H; Perez, F

    2015-01-01

    This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

  18. 3D In Vitro Model for Breast Cancer Research Using Magnetic Levitation and Bioprinting Method.

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    Leonard, Fransisca; Godin, Biana

    2016-01-01

    Tumor microenvironment composition and architecture are known as a major factor in orchestrating the tumor growth and its response to various therapies. In this context, in vivo studies are necessary to evaluate the responses. However, while tumor cells can be of human origin, tumor microenvironment in the in vivo models is host-based. On the other hand, in vitro studies in a flat monoculture of tumor cells (the most frequently used in vitro tumor model) are unable to recapitulate the complexity of tumor microenvironment. Three-dimensional (3D) in vitro cell cultures of tumor cells have been proven to be an important experimental tool in understanding mechanisms of tumor growth, response to therapeutics, and transport of nutrients/drugs. We have recently described a novel tool to create 3D co-cultures of tumor cells and cells in the tumor microenvironment. Our method utilizes magnetic manipulation/levitation of the specific ratios of tumor cells and cells in the tumor microenvironment (from human or animal origin) aiding in the formation of tumor spheres with defined cellular composition and density, as quickly as within 24 h. This chapter describes the experimental protocols developed to model the 3D structure of the cancer environment using the above method.

  19. Scaffolds for 3D in vitro culture of neural lineage cells.

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    Murphy, Ashley R; Laslett, Andrew; O'Brien, Carmel M; Cameron, Neil R

    2017-03-01

    Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions for this exciting and important field of biomaterials research.

  20. Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro

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    D Widera

    2006-05-01

    Full Text Available The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF- alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF- alpha strongly activates the canonical Nuclear Factor Kappa-B (NF-kappaB pathway. In order to investigate further functions of TNF in neural stem cells (NSCs we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

  1. In vitro vascularization of a combined system based on a 3D printing technique.

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    Zhao, Xinru; Liu, Libiao; Wang, Jiayin; Xu, Yufan; Zhang, Weiming; Khang, Gilson; Wang, Xiaohong

    2016-10-01

    A vital challenge in complex organ manufacturing is to vascularize large combined tissues. The aim of this study is to vascularize in vitro an adipose-derived stem cell (ADSC)/fibrin/collagen incorporated three-dimensional (3D) poly(d,l-lactic-co-glycolic acid) (PLGA) scaffold (10 × 10 × 10 mm(3) ) with interconnected channels. A low-temperature 3D printing technique was employed to build the PLGA scaffold. A step-by-step cocktail procedure was designed to engage or steer the ADSCs in the PLGA channels towards both endothelial and smooth muscle cell lineages. The combined system had sufficient mechanical properties to support the cell/fibrin/collagen hydrogel inside the predefined PLGA channels. The ADSCs encapsulated in the fibrin/collagen hydrogel differentiated to endothelial and smooth muscle cell lineage, respectively, corresponding to their respective locations in the construct and formed vascular-like structures. This technique allows in vitro vascularization of the predefined PLGA channels and provides a choice for complex organ manufacture. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Quantification of blood perfusion using 3D power Doppler: an in-vitro flow phantom study

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    Raine-Fenning, N. J.; Ramnarine, K. V.; Nordin, N. M.; Campbell, B. K.

    2004-01-01

    Three-dimensional (3D) power Doppler data is increasingly used to assess and quantify blood flow and tissue perfusion. The objective of this study was to assess the validity of common 3D power Doppler ‘vascularity’ indices by quantification in well characterised in-vitro flow models. A computer driven gear pump was used to circulate a steady flow of a blood mimicking fluid through various well characterised flow phantoms to investigate the effect of the number of flow channels, flow rate, depth dependent tissue attenuation, blood mimic scatter particle concentration and ultrasound settings. 3D Power Doppler data were acquired with a Voluson 530D scanner and 7.5 MHz transvaginal transducer (GE Kretz). Virtual Organ Computer-aided Analysis software (VOCAL) was used to quantify the vascularisation index (VI), flow index (FI) and vascularisation-flow index (VFI). The vascular indices were affected by many factors, some intuitive and some with more complex or unexpected relationships (e.g. VI increased linearly with an increase in flow rate, blood mimic scatter particle concentration and number of flow channels, and had a complex dependence on pulse repetition frequency). Use of standardised settings and appropriate calibration are required in any attempt at relating ‘vascularity indices’ with flow.

  3. A 3D in vitro bone organ model using human progenitor cells

    Directory of Open Access Journals (Sweden)

    A Papadimitropoulos

    2011-05-01

    Full Text Available Three-dimensional (3D organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0 ±0.3%, pre/osteoclastic (14.0 ±1.4% and mesenchymal/osteoblastic (44.0 ±8.4% phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+ osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides, resorption (by N-terminus collagen-I telopeptides and phosphate levels and osteoclastic activity (by TRAP-5b only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.

  4. In Vitro Biological Evaluation of 3-D Hydroxyapatite/Collagen (50/50 wt. (% Scaffolds

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    Doris Moura Campos

    2012-02-01

    Full Text Available Hydroxyapatite-collagen (HA/Col composites are potential scaffolds for bone tissue engineering. In this work, three-dimensional (3-D HA/Col (50/50 wt. (% scaffolds were synthesized using a self-assembly method and cross-linked with a 0.125% glutaraldehyde solution. Scaffolds were evaluated in vitro by cytotoxicity testing using MC3T3 cells; proliferation and differentiation were studied using STRO-1A human stromal cells for up to 21 days. Morphological and histological examinations showed a fibrous structure with a good distribution and homogeneous HA particles distribution. By thermogravimetric analysis, a ratio of 1.2 between inorganic and organic phase was found. The scaffolds presented no cytotoxicity when evaluated using three different parameters of cell survival and integrity: 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5-carboxanilide (XTT, Neutral Red (NR and Crystal Violet Dye Elution (CVDE. STRO-1A cells were found to adhere, proliferate and differentiate on the 3-D scaffold, but limited cell penetration was observed.

  5. Surface functionalization of 3D glass-ceramic porous scaffolds for enhanced mineralization in vitro

    Science.gov (United States)

    Ferraris, Sara; Vitale-Brovarone, Chiara; Bretcanu, Oana; Cassinelli, Clara; Vernè, Enrica

    2013-04-01

    Bone reconstruction after tissue loosening due to traumatic, pathological or surgical causes is in increasing demand. 3D scaffolds are a widely studied solution for supporting new bone growth. Bioactive glass-ceramic porous materials can offer a three-dimensional structure that is able to chemically bond to bone. The ability to surface modify these devices by grafting biologically active molecules represents a challenge, with the aim of stimulating physiological bone regeneration with both inorganic and organic signals. In this research work glass ceramic scaffolds with very high mechanical properties and moderate bioactivity have been functionalized with the enzyme alkaline phosphatase (ALP). The material surface was activated in order to expose hydroxyl groups. The activated surface was further grafted with ALP both via silanization and also via direct grafting to the surface active hydroxyl groups. Enzymatic activity of grafted samples were measured by means of UV-vis spectroscopy before and after ultrasonic washing in TRIS-HCl buffer solution. In vitro inorganic bioactivity was investigated by soaking the scaffolds after the different steps of functionalization in a simulated body fluid (SBF). SEM observations allowed the monitoring of the scaffold morphology and surface chemical composition after soaking in SBF. The presence of ALP enhanced the in vitro inorganic bioactivity of the tested material.

  6. Comparative endpoint sensitivity of in vitro estrogen agonist assays.

    Science.gov (United States)

    Dreier, David A; Connors, Kristin A; Brooks, Bryan W

    2015-07-01

    Environmental and human health implications of endocrine disrupting chemicals (EDCs), particularly xenoestrogens, have received extensive study. In vitro assays are increasingly employed as diagnostic tools to comparatively evaluate chemicals, whole effluent toxicity and surface water quality, and to identify causative EDCs during toxicity identification evaluations. Recently, the U.S. Environmental Protection Agency (USEPA) initiated ToxCast under the Tox21 program to generate novel bioactivity data through high throughput screening. This information is useful for prioritizing chemicals requiring additional hazard information, including endocrine active chemicals. Though multiple in vitro and in vivo techniques have been developed to assess estrogen agonist activity, the relative endpoint sensitivity of these approaches and agreement of their conclusions remain unclear during environmental diagnostic applications. Probabilistic hazard assessment (PHA) approaches, including chemical toxicity distributions (CTD), are useful for understanding the relative sensitivity of endpoints associated with in vitro and in vivo toxicity assays by predicting the likelihood of chemicals eliciting undesirable outcomes at or above environmentally relevant concentrations. In the present study, PHAs were employed to examine the comparative endpoint sensitivity of 16 in vitro assays for estrogen agonist activity using a diverse group of compounds from the USEPA ToxCast dataset. Reporter gene assays were generally observed to possess greater endpoint sensitivity than other assay types, and the Tox21 ERa LUC BG1 Agonist assay was identified as the most sensitive in vitro endpoint for detecting an estrogenic response. When the sensitivity of this most sensitive ToxCast in vitro endpoint was compared to the human MCF-7 cell proliferation assay, a common in vitro model for biomedical and environmental monitoring applications, the ERa LUC BG1 assay was several orders of magnitude less

  7. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

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    Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y. [Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing (China)

    2013-08-10

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  8. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Directory of Open Access Journals (Sweden)

    Z. Wen

    2013-08-01

    Full Text Available Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  9. 3D bioprinting matrices with controlled pore structure and release function guide in vitro self-organization of sweat gland

    Science.gov (United States)

    Liu, Nanbo; Huang, Sha; Yao, Bin; Xie, Jiangfan; Wu, Xu; Fu, Xiaobing

    2016-01-01

    3D bioprinting matrices are novel platforms for tissue regeneration. Tissue self-organization is a critical process during regeneration that implies the features of organogenesis. However, it is not clear from the current evidences whether 3D printed construct plays a role in guiding tissue self-organization in vitro. Based on our previous study, we bioprinted a 3D matrix as the restrictive niche for direct sweat gland differentiation of epidermal progenitors by different pore structure (300-μm or 400-μm nozzle diameters printed) and reported a long-term gradual transition of differentiated cells into glandular morphogenesis occurs within the 3D construct in vitro. At the initial 14-day culture, an accelerated cell differentiation was achieved with inductive cues released along with gelatin reduction. After protein release completed, the 3D construct guide the self-organized formation of sweat gland tissues, which is similar to that of the natural developmental process. However, glandular morphogenesis was only observed in 300-μm–printed constructs. In the absence of 3D architectural support, glandular morphogenesis was not occurred. This striking finding made us to identify a previously unknown role of the 3D-printed structure in glandular tissue regeneration, and this self-organizing strategy can be applied to forming other tissues in vitro. PMID:27694985

  10. 3D bioprinting matrices with controlled pore structure and release function guide in vitro self-organization of sweat gland.

    Science.gov (United States)

    Liu, Nanbo; Huang, Sha; Yao, Bin; Xie, Jiangfan; Wu, Xu; Fu, Xiaobing

    2016-10-03

    3D bioprinting matrices are novel platforms for tissue regeneration. Tissue self-organization is a critical process during regeneration that implies the features of organogenesis. However, it is not clear from the current evidences whether 3D printed construct plays a role in guiding tissue self-organization in vitro. Based on our previous study, we bioprinted a 3D matrix as the restrictive niche for direct sweat gland differentiation of epidermal progenitors by different pore structure (300-μm or 400-μm nozzle diameters printed) and reported a long-term gradual transition of differentiated cells into glandular morphogenesis occurs within the 3D construct in vitro. At the initial 14-day culture, an accelerated cell differentiation was achieved with inductive cues released along with gelatin reduction. After protein release completed, the 3D construct guide the self-organized formation of sweat gland tissues, which is similar to that of the natural developmental process. However, glandular morphogenesis was only observed in 300-μm-printed constructs. In the absence of 3D architectural support, glandular morphogenesis was not occurred. This striking finding made us to identify a previously unknown role of the 3D-printed structure in glandular tissue regeneration, and this self-organizing strategy can be applied to forming other tissues in vitro.

  11. Development of a 3D matrix for modeling mammalian spinal cord injury in vitro

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    Juan Felipe Diaz Quiroz

    2016-01-01

    Full Text Available Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel therapies, this is in part due to the complexity of the injury and the difficulty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrix in vitro and when injured, the cells respond as they do in vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells after exposure to drugs.

  12. Development of a 3D matrix for modeling mammalian spinal cord injury in vitro

    Science.gov (United States)

    Diaz Quiroz, Juan Felipe; Li, Yuping; Aparicio, Conrado; Echeverri, Karen

    2016-01-01

    Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel therapies, this is in part due to the complexity of the injury and the difficulty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrix in vitro and when injured, the cells respond as they do in vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells after exposure to drugs. PMID:28123426

  13. 3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

    Science.gov (United States)

    Vanderburgh, Joseph; Sterling, Julie A; Guelcher, Scott A

    2017-01-01

    2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

  14. Development of 3D in vitro platform technology to engineer mesenchymal stem cells

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    Hosseinkhani H

    2012-06-01

    Full Text Available Hossein Hosseinkhani,1 Po-Da Hong,1 Dah-Shyong Yu,2 Yi-Ru Chen,3 Diana Ickowicz,4 Ira-Yudovin Farber,4 Abraham J Domb41Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (TAIWANTECH, 2Nanomedicine Research Center, National Defense Medical Center, Taipei, Taiwan, 3Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, IsraelAbstract: This study aims to develop a three-dimensional in vitro culture system to genetically engineer mesenchymal stem cells (MSC to express bone morphogenic protein-2. We employed nanofabrication technologies borrowed from the spinning industry, such as electrospinning, to mass-produce identical building blocks in a variety of shapes and sizes to fabricate electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen. Homogenous nanoparticles of cationic biodegradable natural polymer were formed by simple mixing of an aqueous solution of plasmid DNA encoded bone morphogenic protein-2 with the same volume of cationic polysaccharide, dextran-spermine. Rat bone marrow MSC were cultured on electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen prior to the incorporation of the nanoparticles into the nanofiber sheets. Bone morphogenic protein-2 was significantly detected in MSC cultured on nanofiber sheets incorporated with nanoparticles after 2 days compared with MSC cultured on nanofiber sheets incorporated with naked plasmid DNA. We conclude that the incorporation of nanoparticles into nanofiber sheets is a very promising strategy to genetically engineer MSC and can be used for further applications in regenerative medicine therapy.Keywords: 3D culture, nanoparticles, nanofibers, polycations, tissue engineering

  15. Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.

    Science.gov (United States)

    Frega, Monica; Tedesco, Mariateresa; Massobrio, Paolo; Pesce, Mattia; Martinoia, Sergio

    2014-06-30

    Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems.

  16. Effective inhibition of foot-and-mouth disease virus (FMDV replication in vitro by vector-delivered microRNAs targeting the 3D gene

    Directory of Open Access Journals (Sweden)

    Cai Xuepeng

    2011-06-01

    Full Text Available Abstract Background Foot-and-mouth disease virus (FMDV causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined. Results Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV. Conclusion Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

  17. A Microfabricated 96-Well 3D Assay Enabling High-Throughput Quantification of Cellular Invasion Capabilities

    Science.gov (United States)

    Hao, Rui; Wei, Yuanchen; Li, Chaobo; Chen, Feng; Chen, Deyong; Zhao, Xiaoting; Luan, Shaoliang; Fan, Beiyuan; Guo, Wei; Wang, Junbo; Chen, Jian

    2017-01-01

    This paper presents a 96-well microfabricated assay to study three-dimensional (3D) invasion of tumor cells. A 3D cluster of tumor cells was first generated within each well by seeding cells onto a micro-patterned surface consisting of a central fibronectin-coated area that promotes cellular attachment, surrounded by a poly ethylene glycol (PEG) coated area that is resistant to cellular attachment. Following the formation of the 3D cell clusters, a 3D collagen extracellular matrix was formed in each well by thermal-triggered gelation. Invasion of the tumor cells into the extracellular matrix was subsequently initiated and monitored. Two modes of cellular infiltration were observed: A549 cells invaded into the extracellular matrix following the surfaces previously coated with PEG molecules in a pseudo-2D manner, while H1299 cells invaded into the extracellular matrix in a truly 3D manner including multiple directions. Based on the processing of 2D microscopic images, a key parameter, namely, equivalent invasion distance (the area of invaded cells divided by the circumference of the initial cell cluster) was obtained to quantify migration capabilities of these two cell types. These results validate the feasibility of the proposed platform, which may function as a high-throughput 3D cellular invasion assay. PMID:28240272

  18. Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models.

    Directory of Open Access Journals (Sweden)

    Kyung Eun Sung

    Full Text Available The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com. We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

  19. In vitro activity of ceftazidime-avibactam combination in in vitro checkerboard assays

    NARCIS (Netherlands)

    Berkhout, J.; Melchers, M.J.B.; Mil, A.C. van; Nichols, W.W.; Mouton, J.W.

    2015-01-01

    To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae, 19 Escherichia coli, 1 Citrobacter freundii, and 3 Enterobact

  20. Low-cost, rapidly-developed, 3D printed in vitro corpus callosum model for mucopolysaccharidosis type I

    Science.gov (United States)

    Tabet, Anthony; Gardner, Matthew; Swanson, Sebastian; Crump, Sydney; McMeekin, Austin; Gong, Diana; Tabet, Rebecca; Hacker, Benjamin; Nestrasil, Igor

    2017-01-01

    The rising prevalence of high throughput screening and the general inability of (1) two dimensional (2D) cell culture and (2) in vitro release studies to predict in vivo neurobiological and pharmacokinetic responses in humans has led to greater interest in more realistic three dimensional (3D) benchtop platforms. Advantages of 3D human cell culture over its 2D analogue, or even animal models, include taking the effects of microgeometry and long-range topological features into consideration. In the era of personalized medicine, it has become increasingly valuable to screen candidate molecules and synergistic therapeutics at a patient-specific level, in particular for diseases that manifest in highly variable ways. The lack of established standards and the relatively arbitrary choice of probing conditions has limited in vitro drug release to a largely qualitative assessment as opposed to a predictive, quantitative measure of pharmacokinetics and pharmacodynamics in tissue. Here we report the methods used in the rapid, low-cost development of a 3D model of a mucopolysaccharidosis type I patient’s corpus callosum, which may be used for cell culture and drug release. The CAD model is developed from in vivo brain MRI tracing of the corpus callosum using open-source software, printed with poly (lactic-acid) on a Makerbot Replicator 5X, UV-sterilized, and coated with poly (lysine) for cellular adhesion. Adaptations of material and 3D printer for expanded applications are also discussed. PMID:28357042

  1. Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.

    Science.gov (United States)

    DiMarco, Rebecca L; Dewi, Ruby E; Bernal, Gabriela; Kuo, Calvin; Heilshorn, Sarah C

    2015-10-15

    Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  2. Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications

    Science.gov (United States)

    Stevenson, G.; Rehman, S.; Draper, E.; Hernández‐Nava, E.; Hunt, J.

    2016-01-01

    ABSTRACT In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast‐like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in‐growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre‐clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586–1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26702609

  3. Chemosensitizing acridones: in vitro calmodulin dependent cAMP phosphodiesterase inhibition, docking, pharmacophore modeling and 3D QSAR studies.

    Science.gov (United States)

    Rajendra Prasad, V V S; Deepak Reddy, G; Appaji, D; Peters, G J; Mayur, Y C

    2013-03-01

    Calmodulin inhibitors have proved to play a significant role in sensitizing MDR cancer cells by interfering with cellular drug accumulation. The present investigation focuses on the evaluation of in vitro inhibitory efficacy of chloro acridones against calmodulin dependent cAMP phosphodiesterase (PDE1c). Moreover, molecular docking of acridones was performed with PDE1c in order to identify the possible protein ligand interactions and results thus obtained were compared with in vitro data. In addition an efficient pharmacophore model was developed from a set of 38 chemosensitizing acridones effective against doxorubicin resistant (HL-60/DX) cancer cell lines. Pharmacophoric features such as one hydrogen bond acceptor, one hydrophobic region, a positive ion group and three aromatic rings i.e., AHPRRR have been identified. Ligand based 3D-QSAR was also performed by employing partial least square regression analysis.

  4. 3D super-resolved in vitro multiphoton microscopy by saturation of excitation

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    Nguyen, Anh Dung; Bouwens, Arno; Vanholsbeeck, Frédérique; Egrise, Dominique; Van Simayes, Gaetan; Emplit, Philippe; Goldman, Serge; Gorza, Simon-Pierre

    2015-01-01

    We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples.

  5. High-resolution 3D ultrasound jawbone surface imaging for diagnosis of periodontal bony defects: an in vitro study.

    Science.gov (United States)

    Mahmoud, Ahmed M; Ngan, Peter; Crout, Richard; Mukdadi, Osama M

    2010-11-01

    Although medical specialties have recognized the importance of using ultrasonic imaging, dentistry is only beginning to discover its benefit. This has particularly been important in the field of periodontics which studies infections in the gum and bone tissues that surround the teeth. This study investigates the feasibility of using a custom-designed high-frequency ultrasound imaging system to reconstruct high-resolution (3D) surface images of periodontal defects in human jawbone. The system employs single-element focused ultrasound transducers with center frequencies ranging from 30 to 60 MHz. Continuous acquisition using a 1 GHz data acquisition card is synchronized with a high-precision two-dimensional (2D) positioning system of ±1 μm resolution for acquiring accurate measurements of the mandible, in vitro. Signal and image processing algorithms are applied to reconstruct high-resolution ultrasound images and extract the jawbone surface in each frame. Then, all edges are combined and smoothed in order to render a 3D surface image of the jawbone. In vitro experiments were performed to assess the system performance using mandibles with teeth (dentate) or without (nondentate). The system was able to reconstruct 3D images for the mandible's outer surface with superior spatial resolution down to 24 μm, and to perform the whole scanning in images were confirmed with the anatomical structures on the mandibles. All the anatomical landmarks were detected and fully described as 3D images using this novel ultrasound imaging technique, whereas the 2D X-ray radiographic images suffered from poor contrast. These results indicate the great potential of utilizing high-resolution ultrasound as a noninvasive, nonionizing imaging technique for the early diagnosis of the more severe form of periodontal disease.

  6. The development and characterization of a human mesothelioma in vitro 3D model to investigate immunotoxin therapy.

    Directory of Open Access Journals (Sweden)

    Xinran Xiang

    Full Text Available BACKGROUND: Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma. METHODOLOGY/PRINCIPAL FINDINGS: Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226 and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro. CONCLUSION/SIGNIFICANCE: This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo

  7. Evaluation of immunostimulatory activity of Chyawanprash using in vitro assays.

    Science.gov (United States)

    Madaan, Alka; Kanjilal, Satyajyoti; Gupta, Arun; Sastry, J L N; Verma, Ritu; Singh, Anu T; Jaggi, Manu

    2015-03-01

    Chyawanprash is an ayurvedic formulation used in Indian traditional medicinal system for its beneficial effect on human health. We investigated the immunostimulatory effects of Chyawanprash (CHY) using in vitro assays evaluating the secretion of cytokines such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1beta (IL-1β) and Macrophage Inflammatory Protein-1-alpha (MIP-1-α) from murine bone marrow derived Dendritic Cells (DC) which play pivotal role in immunostimulation. The effects of CHY on phagocytosis in murine macrophages (RAW264.7) and Natural Killer (NK) cell activity were also investigated. At non-cytotoxic concentrations (20-500 μg/ml), CHY enhanced the secretion of all the three cytokines from DC. CHY also stimulated both, macrophage (RAW264.7) as well as NK cell activity, in vitro. In conclusion, the data substantiates the immunoprotective role of CHY at cellular level mediated by immunostimulation in key immune cells viz. dendritic Cells, macrophages and NK cells.

  8. In Vitro Model of the Epidermis: Connecting Protein Function to 3D Structure.

    Science.gov (United States)

    Arnette, Christopher; Koetsier, Jennifer L; Hoover, Paul; Getsios, Spiro; Green, Kathleen J

    2016-01-01

    Much of our understanding of the biological processes that underlie cellular functions in humans, such as cell-cell communication, intracellular signaling, and transcriptional and posttranscriptional control of gene expression, has been acquired from studying cells in a two-dimensional (2D) tissue culture environment. However, it has become increasingly evident that the 2D environment does not support certain cell functions. The need for more physiologically relevant models prompted the development of three-dimensional (3D) cultures of epithelial, endothelial, and neuronal tissues (Shamir & Ewald, 2014). These models afford investigators with powerful tools to study the contribution of spatial organization, often in the context of relevant extracellular matrix and stromal components, to cellular and tissue homeostasis in normal and disease states.

  9. AlgiMatrix™-Based 3D Cell Culture System as an In Vitro Tumor Model: An Important Tool in Cancer Research.

    Science.gov (United States)

    Godugu, Chandraiah; Singh, Mandip

    2016-01-01

    Routinely used two-dimensional cell culture-based models often fail while translating the observations into in vivo models. This setback is more common in cancer research, due to several reasons. The extracellular matrix and cell-to-cell interactions are not present in two-dimensional (2D) cell culture models. Diffusion of drug molecules into cancer cells is hindered by barriers of extracellular components in in vivo conditions, these barriers are absent in 2D cell culture models. To better mimic or simulate the in vivo conditions present in tumors, the current study used the alginate based three-dimensional cell culture (AlgiMatrix™) model, which resembles close to the in vivo tumor models. The current study explains the detailed protocols involved in AlgiMatrix™ based in vitro non-small-cell lung cancer (NSCLC) models. The suitability of this model was studied by evaluating, cytotoxicity, apoptosis, and penetration of nanoparticles into the in vitro tumor spheroids. This study also demonstrated the effect of EphA2 receptor targeted docetaxel-loaded nanoparticles on MDA-MB-468 TNBC cell lines. The methods section is subdivided into three subsections such as (1) preparation of AlgiMatrix™-based 3D in vitro tumor models and cytotoxicity assays, (2) free drug and nanoparticle uptake into spheroid studies, and (3) western blot, IHC, and RT-PCR studies.

  10. In vitro 3-D model based on extending time of culture for studying chronological epidermis aging.

    Science.gov (United States)

    Dos Santos, Morgan; Metral, Elodie; Boher, Aurélie; Rousselle, Patricia; Thepot, Amélie; Damour, Odile

    2015-09-01

    Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the

  11. Development of a 3D matrix for modeling mammalian spinal cord injury in vitro

    Institute of Scientific and Technical Information of China (English)

    Juan Felipe Diaz Quiroz; Yuping Li; Conrado Aparicio; Karen Echeverri

    2016-01-01

    Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel ther-apies, this is in part due to the complexity of the injury and the diffculty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrixin vitro and when injured, the cells respond as they doin vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells atfer exposure to drugs.

  12. In vitro bioactivity of 3D Ti-mesh with bioceramic coatings in simulated body fluid

    Directory of Open Access Journals (Sweden)

    Wei Yi

    2014-09-01

    Full Text Available 3D Ti-mesh has been coated with bioceramics under different coating conditions, such as material compositions and micro-porosity, using a dip casting method. Hydroxyapatite (HA, micro-HA particles (HAp, a bioglass (BG and their different mixtures together with polymer additives were used to control HA-coating microstructures. Layered composites with the following coating-to-substrate designs, such as BG/Ti, HA + BG/BG/Ti and HAp + BG/BG/Ti, were fabricated. The bioactivity of these coated composites and the uncoated Ti-mesh substrate was then investigated in a simulated body fluid (SBF. The Ti-mesh substrate and BG/Ti composite did not induce biomimetic apatite deposition when they were immersed in SBF for the selected BG, a pressable dental ceramic, used in this study. After seven days in SBF, an apatite layer was formed on both HA + BG/BG/Ti and HAp + BG/BG/Ti composites. The difference is the apatite layer on the HAp + BG/BG/Ti composite was rougher and contained more micro-pores, while the apatite layer on the HA + BG/BG/Ti composite was dense and smooth. The formation of biomimetic apatite, being more bioresorbable, is favored for bone regeneration.

  13. Development of 3D in vitro platform technology to engineer mesenchymal stem cells.

    Science.gov (United States)

    Hosseinkhani, Hossein; Hong, Po-Da; Yu, Dah-Shyong; Chen, Yi-Ru; Ickowicz, Diana; Farber, Ira-Yudovin; Domb, Abraham J

    2012-01-01

    This study aims to develop a three-dimensional in vitro culture system to genetically engineer mesenchymal stem cells (MSC) to express bone morphogenic protein-2. We employed nanofabrication technologies borrowed from the spinning industry, such as electrospinning, to mass-produce identical building blocks in a variety of shapes and sizes to fabricate electrospun nanofiber sheets comprised of composites of poly (glycolic acid) and collagen. Homogenous nanoparticles of cationic biodegradable natural polymer were formed by simple mixing of an aqueous solution of plasmid DNA encoded bone morphogenic protein-2 with the same volume of cationic polysaccharide, dextran-spermine. Rat bone marrow MSC were cultured on electrospun nanofiber sheets comprised of composites of poly (glycolic acid) and collagen prior to the incorporation of the nanoparticles into the nanofiber sheets. Bone morphogenic protein-2 was significantly detected in MSC cultured on nanofiber sheets incorporated with nanoparticles after 2 days compared with MSC cultured on nanofiber sheets incorporated with naked plasmid DNA. We conclude that the incorporation of nanoparticles into nanofiber sheets is a very promising strategy to genetically engineer MSC and can be used for further applications in regenerative medicine therapy.

  14. 3D polylactide-based scaffolds for studying human hepatocarcinoma processes in vitro

    Directory of Open Access Journals (Sweden)

    Roberto Scaffaro, Giada Lo Re, Salvatrice Rigogliuso and Giulio Ghersi

    2012-01-01

    Full Text Available We evaluated the combination of leaching techniques and melt blending of polymers and particles for the preparation of highly interconnected three-dimensional polymeric porous scaffolds for in vitro studies of human hepatocarcinoma processes. More specifically, sodium chloride and poly(ethylene glycol (PEG were used as water-soluble porogens to form porous and solvent-free poly(L,D-lactide (PLA-based scaffolds. Several characterization techniques, including porosimetry, image analysis and thermogravimetry, were combined to improve the reliability of measurements and mapping of the size, distribution and microarchitecture of pores. We also investigated the effect of processing, in PLA-based blends, on the simultaneous bulk/surface modifications and pore architectures in the scaffolds, and assessed the effects on human hepatocarcinoma viability and cell adhesion. The influence of PEG molecular weight on the scaffold morphology and cell viability and adhesion were also investigated. Morphological studies indicated that it was possible to obtain scaffolds with well-interconnected pores of assorted sizes. The analysis confirmed that SK-Hep1 cells adhered well to the polymeric support and emitted surface protrusions necessary to grow and differentiate three-dimensional systems. PEGs with higher molecular weight showed the best results in terms of cell adhesion and viability.

  15. Characterization of Silk Fibroin/Chitosan 3D Porous Scaffold and In Vitro Cytology.

    Directory of Open Access Journals (Sweden)

    Shuguang Zeng

    Full Text Available Bone tissue engineering is a powerful tool to treat bone defects caused by trauma, infection, tumors and other factors. Both silk fibroin (SF and chitosan (CS are non-toxic and have good biocompatibility, but are poor biological scaffolds when used alone. In this study, the microscopic structure and related properties of SF/CS composite scaffolds with different component ratios were examined. The scaffold material most suitable for osteoblast growth was determined, and these results offer an experimental basis for the future reconstruction of bone defects. First, via freeze-drying and chemical crosslinking methods, SF/CS composites with different component ratios were prepared and their structure was characterized. Changes in the internal structure of the SF and CS mixture were observed, confirming that the mutual modification between the two components was complete and stable. The internal structure of the composite material was porous and three-dimensional with a porosity above 90%. We next studied the pore size, swelling ratio, water absorption ratio, degradation and in vitro cell proliferation. For the 40% SF-60% CS group, the pore size of the scaffold was suitable for the growth of osteoblasts, and the rate of degradation was steady. This favors the early adhesion, growth and proliferation of MG-63 cells. In addition to good biocompatibility and satisfactory cell affinity, this material promotes the secretion of extracellular matrix materials by osteoblasts. Thus, 40% SF-60% CS is a good material for bone tissue engineering.

  16. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

    Science.gov (United States)

    Knight, Eleanor; Przyborski, Stefan

    2015-12-01

    Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.

  17. A Novel Qualitative and Quantitative Biofilm Assay Based on 3D Soft Tissue

    Directory of Open Access Journals (Sweden)

    Bodil Hakonen

    2014-01-01

    Full Text Available The lack of predictable in vitro methods to analyze antimicrobial activity could play a role in the development of resistance to antibiotics. Current used methods analyze planktonic cells but for the method to be clinically relevant, biofilm in in vivo like conditions ought to be studied. Hence, our group has developed a qualitative and quantitative method with in vivo like 3D tissue for prediction of antimicrobial activity in reality. Devices (wound dressings were applied on top of Pseudomonas aeruginosa inoculated Muller-Hinton (MH agar or 3D synthetic soft tissues (SST and incubated for 24 hours. The antibacterial activity was then analyzed visually and by viable counts. On MH agar two out of three silver containing devices showed zone of inhibitions (ZOI and on SST, ZOI were detected for all three. Corroborating results were found upon evaluating the bacterial load in SST and shown to be silver concentration dependent. In conclusion, a novel method was developed combining visual rapid screening and quantitative evaluation of the antimicrobial activity in both tissue and devices. It uses tissue allowing biofilm formation thus mimicking reality closely. These conditions are essential in order to predict antimicrobial activity of medical devices in the task to prevent device related infections.

  18. Generation of orientation tools for automated zebrafish screening assays using desktop 3D printing

    Science.gov (United States)

    2014-01-01

    Background The zebrafish has been established as the main vertebrate model system for whole organism screening applications. However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. While technical solutions to the orientation problem exist, dissemination is often hindered by the lack of simple and inexpensive ways of distributing and duplicating tools. Results Here, we provide a cost effective method for the production of 96-well plate compatible zebrafish orientation tools using a desktop 3D printer. The printed tools enable the positioning and orientation of zebrafish embryos within cavities formed in agarose. Their applicability is demonstrated by acquiring lateral and dorsal views of zebrafish embryos arrayed within microtiter plates using an automated screening microscope. This enables the consistent visualization of morphological phenotypes and reporter gene expression patterns. Conclusions The designs are refined versions of previously demonstrated devices with added functionality and strongly reduced production costs. All corresponding 3D models are freely available and digital design can be easily shared electronically. In combination with the increasingly widespread usage of 3D printers, this provides access to the developed tools to a wide range of zebrafish users. Finally, the design files can serve as templates for other additive and subtractive fabrication methods. PMID:24886511

  19. Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correlation with 2D dose responses

    Science.gov (United States)

    Chapman, K. E.; Thomas, A. D.; Jenkins, G. J. S.

    2014-01-01

    Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructed skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay’s fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 µg/ml and methyl methanesulfonate (MMS) at 1750 µg/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 µg/ml, which was very similar to the topical LOGEL when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm™. Our preliminary validation here demonstrates that the RSMN assay may be a valuable follow-up to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in

  20. Repair of Cartilage injuries using in vitro engineered 3D cartilage tissue- Preliminary Results of Our Animal Studies

    Directory of Open Access Journals (Sweden)

    Arumugam S

    2011-01-01

    Full Text Available Introduction: The cartilage injuries demand novel therapeutic approaches as the success rates of the current conventional strategies for the repair of injured articular cartilages are not that encouraging. Earlier we have reported that the Thermoreversible Gelation Polymer (TGP is an ideal scaffold for human chondrocyte expansion in vitro. In this study, we report the preliminary results of the in vitro expansion, characterization and experimental in vivo transplantation of chondrocytes in a rabbit model of cartilage injury Materials & Methods: Nine rabbits were included in this study scheduled for two years, after approval by the ethics committee. In the first animal, Chondrocytes were isolated from the weight bearing area of patellar groove in the left hindlimb and cultured in TGP Scaffold and maintained at 37°C in 5% carbon dioxide incubator for 64 days without growth factors. Then the TGP-Chondrocyte construct was transplanted into an experimental defect created in the knee of the right forelimb of the same rabbit. After a period of 10 weeks, a biopsy was taken from the transplanted region and subjected to morphological analysis, characterization by histopathology (H&E stain and Immunohistochemistry (S-100 staining.Results: The chondrocytes in the 3D TGP culture had round to oval shaped morphology without any de-differentiation which is otherwise observed in Conventional 2D cultures. A macroscopic structure which resembled cartilage was appreciated in the TGP construct in vitro after 64 days which was then transplanted to the rabbit. The H&E and Immunohistochemistry studies confirmed the presence of chondrocytes in the biopsy tissue. Conclusion: Based on the results, we conclude that the TGP significantly supports the in vitro expansion of chondrocytes for a longer period and the 3D culture using TGP preserves the phenotype of the articular chondrocytes. The tissue thus grown when implanted with the TGP has engrafted well without any

  1. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

    Science.gov (United States)

    Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

  2. Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

    Directory of Open Access Journals (Sweden)

    G Bouet

    2015-05-01

    Full Text Available An engineered three dimensional (3D in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a “proof-of-concept” for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

  3. Development of in vitro assay method with radioisotope

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Chang Woon; Lim, S. M.; An, S. H.; Woo, K. S.; Chung, W. S.; Lim, S. J.; Hong, S. W. [Korea Atomic Energy Research Institute. Korea Cancer Center Hospital, Seoul (Korea, Republic of); Oh, O. D. [Yonsei University, Seoul (Korea, Republic of)

    1999-04-01

    Radioimmunoassay (RIA) and related competitive protein-binding methods began a little over 20 years ago as a cumbersome research methodology in a few specialized laboratories. Endocrinology has been greatly enriched by the new knowledge that has come as a direct result of RIA methods. Establishment of the taxol RIA system will be expected to develop RIA for drug monitoring. Scintillation proximity assay was useful since any separation step is not required, it has the advantage of dealing with multiple samples. The increased sensitivity of the new assay in determining HCV RT([{sup 125}I]dUTP) suggests that it would be worth investigating whether the system can be applied to analysis. [{sup 125}I] lodotyramine with 98.5% radiochemical purity. Optimal background counts was certificated using varied radioactivity of radionuclides. Appropriate standard curve was obtained from SPA method successively, and the concentration of hCG from unknown serum was determined by standard curve. The result concentration of hCG from unknown serum was determined by synthesized successively and purified by HPLC system. Hybridoma reducing monoclonal anti thyroglobulin antibodies titer is measured by ELISA. These studies play an important role in development of in vitro assay with radionuclides.

  4. Non-invasive transcranial ultrasound therapy based on a 3D CT scan: protocol validation and in vitro results

    Energy Technology Data Exchange (ETDEWEB)

    Marquet, F; Pernot, M; Aubry, J-F; Montaldo, G; Tanter, M; Fink, M [Laboratoire Ondes et Acoustique, ESPCI, Universite Paris VII, UMR CNRS 7587, 10 rue Vauquelin, 75005 Paris (France); Marsac, L [Supersonic Imagine, Les Jardins de la Duranne, 510 rue Rene Descartes, 13857 Aix-en-Provence (France)], E-mail: fabrice.marquet@espci.org

    2009-05-07

    A non-invasive protocol for transcranial brain tissue ablation with ultrasound is studied and validated in vitro. The skull induces strong aberrations both in phase and in amplitude, resulting in a severe degradation of the beam shape. Adaptive corrections of the distortions induced by the skull bone are performed using a previous 3D computational tomography scan acquisition (CT) of the skull bone structure. These CT scan data are used as entry parameters in a FDTD (finite differences time domain) simulation of the full wave propagation equation. A numerical computation is used to deduce the impulse response relating the targeted location and the ultrasound therapeutic array, thus providing a virtual time-reversal mirror. This impulse response is then time-reversed and transmitted experimentally by a therapeutic array positioned exactly in the same referential frame as the one used during CT scan acquisitions. In vitro experiments are conducted on monkey and human skull specimens using an array of 300 transmit elements working at a central frequency of 1 MHz. These experiments show a precise refocusing of the ultrasonic beam at the targeted location with a positioning error lower than 0.7 mm. The complete validation of this transcranial adaptive focusing procedure paves the way to in vivo animal and human transcranial HIFU investigations.

  5. Non-invasive transcranial ultrasound therapy based on a 3D CT scan: protocol validation and in vitro results

    Science.gov (United States)

    Marquet, F.; Pernot, M.; Aubry, J.-F.; Montaldo, G.; Marsac, L.; Tanter, M.; Fink, M.

    2009-05-01

    A non-invasive protocol for transcranial brain tissue ablation with ultrasound is studied and validated in vitro. The skull induces strong aberrations both in phase and in amplitude, resulting in a severe degradation of the beam shape. Adaptive corrections of the distortions induced by the skull bone are performed using a previous 3D computational tomography scan acquisition (CT) of the skull bone structure. These CT scan data are used as entry parameters in a FDTD (finite differences time domain) simulation of the full wave propagation equation. A numerical computation is used to deduce the impulse response relating the targeted location and the ultrasound therapeutic array, thus providing a virtual time-reversal mirror. This impulse response is then time-reversed and transmitted experimentally by a therapeutic array positioned exactly in the same referential frame as the one used during CT scan acquisitions. In vitro experiments are conducted on monkey and human skull specimens using an array of 300 transmit elements working at a central frequency of 1 MHz. These experiments show a precise refocusing of the ultrasonic beam at the targeted location with a positioning error lower than 0.7 mm. The complete validation of this transcranial adaptive focusing procedure paves the way to in vivo animal and human transcranial HIFU investigations.

  6. Assessment of gastrointestinal motility using three different assays in vitro.

    Science.gov (United States)

    Pozzoli, Cristina; Poli, Enzo

    2010-11-01

    The protocols detailed in this unit are designed to assess the motor activity of different gastric and intestinal muscle preparations in vitro and the effects of drugs that modulate gastrointestinal motility. The preparations described are characterized by different contractile behaviors, consisting of spontaneous (duodenum), neurogenic (ileum), and drug-stimulated (fundus, ileum) motility; these reproduce motility patterns occurring in the gut wall in vivo. These protocols document the variety of factors that can influence the responses of isolated tissues and describe how such tissues can be used for testing substances that affect gut movements. These preparations allow evaluation of direct interactions with the processes that control contractile machinery, as well as indirect effects resulting from the modification of neurotransmitter release from myenteric neurons. These models can be exploited to assay novel compounds undergoing preclinical development or to evaluate the functional toxicity exerted by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins, as well as the mechanisms underlying these effects.

  7. In Vitro Transcription Assays and Their Application in Drug Discovery.

    Science.gov (United States)

    Yang, Xiao; Ma, Cong

    2016-09-20

    In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

  8. Simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay.

    Science.gov (United States)

    Demir, Ihsan Ekin; Tieftrunk, Elke; Schäfer, Karl-Herbert; Friess, Helmut; Ceyhan, Güralp O

    2014-04-14

    Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

  9. High-frequency 3D echodentographic imaging modality for early assessment of periodontal diseases: in vitro study

    Science.gov (United States)

    Mahmoud, Ahmed M.; Ngan, Peter; Crout, Richard; Mukdadi, Osama M.

    2009-02-01

    The use of ultrasound in dentistry is still an open growing area of research. Currently, there is a lack of imaging modalities to accurately predict minute structures and defects in the jawbone. In particular, the inability of 2D radiographic images to detect bony periodontal defects resulted from infection of the periodontium. This study investigates the feasibility of high frequency ultrasound to reconstruct high resolution 3D surface images of human jawbone. Methods: A dentate and non-dentate mandibles were used in this study. The system employs high frequency single-element ultrasound focused transducers (15-30 MHz) for scanning. Continuous acquisition using a 1 GHz data acquisition card is synchronized with a high precision two-dimensional stage positioning system of +/-1 μm resolution for acquiring accurate and quantitative measurements of the mandible in vitro. Radio frequency (RF) signals are acquired laterally 44-45.5 μm apart for each frame. Different frames are reconstructed 500 μm apart for the 3D reconstruction. Signal processing algorithms are applied on the received ultrasound signals for filtering, focusing, and envelope detection before frame reconstruction. Furthermore, an edge detection technique is adopted to detect the bone surface in each frame. Finally, all edges are combined together in order to render a 3D surface image of the jawbone. Major anatomical landmarks on the resultant images were confirmed with the anatomical structures on the mandibles to show the efficacy of the system. Comparison were also made with conventional 2D radiographs to show the superiority of the ultrasound imaging system in diagnosing small defects in the lateral, axial and elevation planes of space. Results: The landmarks on all ultrasound images matched with those on the mandible, indicating the efficacy of the system in detecting small structures in human jaw bones. Comparison with conventional 2D radiographic images of the same mandible showed superiority of

  10. Genotoxicity Assessment of Mixed Oligomers of Chlorotrifluoroethylene using a Battery of In Vitro and In Vivo/In Vitro Assays

    Science.gov (United States)

    1990-11-01

    exchange among more than two chromosomes or fragments which is the result of several breaks. D Dicentric : An exchange between two chromosomes which results...in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF. DF Dicentric with... Chromosome Aberration Assay, the BALB/c- 3T3 Cell Transformation Assay an in In Vivo/lIn Vitro Unscheduled DNA Synthesis Assay and S-Phase Synthesis Assay

  11. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Kuh, Hyo-Jeong, E-mail: hkuh@catholic.ac.kr [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of)

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  12. Strain and rate-dependent neuronal injury in a 3D in vitro compression model of traumatic brain injury

    Science.gov (United States)

    Bar-Kochba, Eyal; Scimone, Mark T.; Estrada, Jonathan B.; Franck, Christian

    2016-08-01

    In the United States over 1.7 million cases of traumatic brain injury are reported yearly, but predictive correlation of cellular injury to impact tissue strain is still lacking, particularly for neuronal injury resulting from compression. Given the prevalence of compressive deformations in most blunt head trauma, this information is critically important for the development of future mitigation and diagnosis strategies. Using a 3D in vitro neuronal compression model, we investigated the role of impact strain and strain rate on neuronal lifetime, viability, and pathomorphology. We find that strain magnitude and rate have profound, yet distinctively different effects on the injury pathology. While strain magnitude affects the time of neuronal death, strain rate influences the pathomorphology and extent of population injury. Cellular injury is not initiated through localized deformation of the cytoskeleton but rather driven by excess strain on the entire cell. Furthermore we find that, mechanoporation, one of the key pathological trigger mechanisms in stretch and shear neuronal injuries, was not observed under compression.

  13. Strain and rate-dependent neuronal injury in a 3D in vitro compression model of traumatic brain injury.

    Science.gov (United States)

    Bar-Kochba, Eyal; Scimone, Mark T; Estrada, Jonathan B; Franck, Christian

    2016-08-02

    In the United States over 1.7 million cases of traumatic brain injury are reported yearly, but predictive correlation of cellular injury to impact tissue strain is still lacking, particularly for neuronal injury resulting from compression. Given the prevalence of compressive deformations in most blunt head trauma, this information is critically important for the development of future mitigation and diagnosis strategies. Using a 3D in vitro neuronal compression model, we investigated the role of impact strain and strain rate on neuronal lifetime, viability, and pathomorphology. We find that strain magnitude and rate have profound, yet distinctively different effects on the injury pathology. While strain magnitude affects the time of neuronal death, strain rate influences the pathomorphology and extent of population injury. Cellular injury is not initiated through localized deformation of the cytoskeleton but rather driven by excess strain on the entire cell. Furthermore we find that, mechanoporation, one of the key pathological trigger mechanisms in stretch and shear neuronal injuries, was not observed under compression.

  14. Assay of mitochondrial functions by resazurin in vitro

    Institute of Scientific and Technical Information of China (English)

    Hai-xia ZHANG; Guan-hua DU; Jun-tian ZHANG

    2004-01-01

    AIM: To study the mechanism of resazurin as indicator of mitochondrial function and to develop a rapid and sensitive assay for measuring metabolic activity of isolated mitochondria from rat liver in vitro. METHODS: The screening was carried out on 96-well microtitre plates by monitoring fluorescence intensity of resazurin reduced by mitochondria. Experimental conditions were optimized and influences of several inhibitors on mitochondrial function were observed. RESULTS: Fluorescence intensity increased in a linear manner when the mitochondrial protein concentration from 5 to 50 μg protein per well was incubated with resazurin (5 μmol/L) during 230 min period at 37 ℃. Edetic acid could promote the reduction of resazurin in mitochondria. The fluorescence intensity decreased greatly after pretreatment with NaN3, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),and oligomycin compared with the control. However, the typical complex I inhibitor, rotenone enhanced the fluorescence intensity without mitochondria. CONCLUSION: Using resazurin to determine mitochondrial function is sensitive, inexpensive and could be easily automated for high throughput screening.

  15. Structure, Properties, and In Vitro Behavior of Heat-Treated Calcium Sulfate Scaffolds Fabricated by 3D Printing.

    Directory of Open Access Journals (Sweden)

    Mitra Asadi-Eydivand

    Full Text Available The ability of inkjet-based 3D printing (3DP to fabricate biocompatible ceramics has made it one of the most favorable techniques to generate bone tissue engineering (BTE scaffolds. Calcium sulfates exhibit various beneficial characteristics, and they can be used as a promising biomaterial in BTE. However, low mechanical performance caused by the brittle character of ceramic materials is the main weakness of 3DP calcium sulfate scaffolds. Moreover, the presence of certain organic matters in the starting powder and binder solution causes products to have high toxicity levels. A post-processing treatment is usually employed to improve the physical, chemical, and biological behaviors of the printed scaffolds. In this study, the effects of heat treatment on the structural, mechanical, and physical characteristics of 3DP calcium sulfate prototypes were investigated. Different microscopy and spectroscopy methods were employed to characterize the printed prototypes. The in vitro cytotoxicity of the specimens was also evaluated before and after heat treatment. Results showed that the as-printed scaffolds and specimens heat treated at 300°C exhibited severe toxicity in vitro but had almost adequate strength. By contrast, the specimens heat treated in the 500°C-1000°C temperature range, although non-toxic, had insufficient mechanical strength, which was mainly attributed to the exit of the organic binder before 500°C and the absence of sufficient densification below 1000°C. The sintering process was accelerated at temperatures higher than 1000°C, resulting in higher compressive strength and less cytotoxicity. An anhydrous form of calcium sulfate was the only crystalline phase existing in the samples heated at 500°C-1150°C. The formation of calcium oxide caused by partial decomposition of calcium sulfate was observed in the specimens heat treated at temperatures higher than 1200°C. Although considerable improvements in cell viability of heat

  16. Structure, Properties, and In Vitro Behavior of Heat-Treated Calcium Sulfate Scaffolds Fabricated by 3D Printing.

    Science.gov (United States)

    Asadi-Eydivand, Mitra; Solati-Hashjin, Mehran; Shafiei, Seyedeh Sara; Mohammadi, Sepideh; Hafezi, Masoud; Abu Osman, Noor Azuan

    2016-01-01

    The ability of inkjet-based 3D printing (3DP) to fabricate biocompatible ceramics has made it one of the most favorable techniques to generate bone tissue engineering (BTE) scaffolds. Calcium sulfates exhibit various beneficial characteristics, and they can be used as a promising biomaterial in BTE. However, low mechanical performance caused by the brittle character of ceramic materials is the main weakness of 3DP calcium sulfate scaffolds. Moreover, the presence of certain organic matters in the starting powder and binder solution causes products to have high toxicity levels. A post-processing treatment is usually employed to improve the physical, chemical, and biological behaviors of the printed scaffolds. In this study, the effects of heat treatment on the structural, mechanical, and physical characteristics of 3DP calcium sulfate prototypes were investigated. Different microscopy and spectroscopy methods were employed to characterize the printed prototypes. The in vitro cytotoxicity of the specimens was also evaluated before and after heat treatment. Results showed that the as-printed scaffolds and specimens heat treated at 300°C exhibited severe toxicity in vitro but had almost adequate strength. By contrast, the specimens heat treated in the 500°C-1000°C temperature range, although non-toxic, had insufficient mechanical strength, which was mainly attributed to the exit of the organic binder before 500°C and the absence of sufficient densification below 1000°C. The sintering process was accelerated at temperatures higher than 1000°C, resulting in higher compressive strength and less cytotoxicity. An anhydrous form of calcium sulfate was the only crystalline phase existing in the samples heated at 500°C-1150°C. The formation of calcium oxide caused by partial decomposition of calcium sulfate was observed in the specimens heat treated at temperatures higher than 1200°C. Although considerable improvements in cell viability of heat-treated scaffolds were

  17. MO-F-CAMPUS-I-04: Magnetic Resonance Imaging of An in Vitro 3D Tumor Model

    Energy Technology Data Exchange (ETDEWEB)

    Veiga, C; Long, T; Siow, B; Loizidou, M; Royle, G; Ricketts, K [University College London, London (United Kingdom)

    2015-06-15

    Purpose: To investigate the use of an in vitro 3D tumor model (tumoroid) as a bio-phantom for repetitive and sequential magnetic resonance imaging (MRI) studies. Methods: The tissue engineered tumoroid comprised an artificial cancer mass (ACM) containing 30 million HT29 cancer cells seeded in a collagen type I matrix, whose density was increased by plastic compression (dry/wet weight=40%). The ACM was embedded in an uncompressed collagen gel that mimicked the tumor stroma, and the tumoroid was incubated for 24h before imaging. Images were acquired using the 1T ICON™ (Bruker Corporation, Billerica, MA) MRI scanner. T1 maps were calculated using an IR-RARE sequence (TE=12ms, TR=10000ms, 7 inversion times), while for T2 maps a MSME technique (TR=6000ms, 16 echoes) was used. T1 and T2 fittings were performed using a pixel-wise approach to produce relaxometric parametric maps. Results: The images acquired and corresponding T1 and T2 maps indicate contrast between the ACM and the stroma. T1 was 2500 and 2800ms, while T2 was 520 and 760ms, for the ACM and stroma respectively. The ACM construct was not homogenous and internal features were visible, which can be explained by local gradients of cell and/or collagen density. The viability of the cells was confirmed via confocal microscopy for several days after the imaging session, demonstrating the suitability of the tumoroid for sequential imaging studies. Conclusions: We have engineered a tumor model compatible with repetitive and sequential MRI. We found T1 and T2 contrast between the ACM and stroma using a pre-clinical MRI scanner. The model, which enables controllable cell and matrix densities, has potential for a wide range of applications in radiotherapy, such as to study tumor progression and to validate imaging biomarkers. Further work is necessary to understand the mechanisms behind the contrast achieved, and to correlate findings with biology and histology data.

  18. Toxicity assessment of aggregated/agglomerated cerium oxide nanoparticles in an in vitro 3D airway model: The influence of mucociliary clearance

    NARCIS (Netherlands)

    Kuper, C.F.; Gröllers-Mulderij, M.; Maarschalkerweerd, T.; Meulendijks, N.M.M.; Reus, A.; Acker, F. van; Zondervan-van den Beuken, E.K.; Wouters, M.E.L.; Bijlsma, S.; Kooter, I.M.

    2014-01-01

    We investigated the toxicity of aggregated nanoparticles of cerium oxide (CeO2) using an in vitro 3D human bronchial epithelial model that included a mucociliary apparatus (MucilAir™). CeO2 was dispersed in saline and applied to the apical surface of the model. CeO2 did not induce distinct effects i

  19. A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging.

    Science.gov (United States)

    Tseng, Hubert; Gage, Jacob A; Shen, Tsaiwei; Haisler, William L; Neeley, Shane K; Shiao, Sue; Chen, Jianbo; Desai, Pujan K; Liao, Angela; Hebel, Chris; Raphael, Robert M; Becker, Jeanne L; Souza, Glauco R

    2015-09-14

    An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5'-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (-control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z' = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

  20. Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

    Directory of Open Access Journals (Sweden)

    Debora B. Petropolis

    2014-04-01

    Full Text Available Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D environment mainly composed of Collagen I (COL I. This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.

  1. A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

    Directory of Open Access Journals (Sweden)

    Robert J Polackwich

    Full Text Available New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1 increased the strength of cell-induced forces on the ECM, 2 did not significantly change migration speed, and 3 increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

  2. Noninvasive quantification of in vitro osteoblastic differentiation in 3D engineered tissue constructs using spectral ultrasound imaging.

    Science.gov (United States)

    Gudur, Madhu Sudhan Reddy; Rao, Rameshwar R; Peterson, Alexis W; Caldwell, David J; Stegemann, Jan P; Deng, Cheri X

    2014-01-01

    Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5-15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×10(4) at day 1 to 0.9±0.2×10(4) at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.

  3. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    Science.gov (United States)

    Langer, Gernot

    2016-01-01

    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  4. Polymer-based mesh as supports for multi-layered 3D cell culture and assays.

    Science.gov (United States)

    Simon, Karen A; Park, Kyeng Min; Mosadegh, Bobak; Subramaniam, Anand Bala; Mazzeo, Aaron D; Ngo, Philip M; Whitesides, George M

    2014-01-01

    Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system-Cells-in-Gels-in-Mesh (CiGiM)-that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells-layer-by-layer-within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis-(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.

  5. Experiences with the in vivo and in vitro comet assay in regulatory testing.

    Science.gov (United States)

    Frötschl, Roland

    2015-01-01

    The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages.

  6. A Functional High-Throughput Assay of Myelination in Vitro

    Science.gov (United States)

    2014-07-01

    Myelination in Vitro PRINCIPAL INVESTIGATOR: Moore, Michael J. CONTRACTING ORGANIZATION: Tulane University...information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and...maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect

  7. Influence of In Vitro Assay pH and Extractant Composition on As Bioaccessibility in Contaminated Soils

    Science.gov (United States)

    In vitro bioaccessibility assays are often utilised to determine the potential human exposure to soil contaminants through soil ingestion. Comparative studies have identified inconsistencies in the results obtained with different in vitro assays. In this study we investigated the...

  8. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins.

    Science.gov (United States)

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran

    2016-09-24

    Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

  9. In vitro study of 3D PLGA/n-HAp/β-TCP composite scaffolds with etched oxygen plasma surface modification in bone tissue engineering

    Science.gov (United States)

    Roh, Hee-Sang; Jung, Sang-Chul; Kook, Min-Suk; Kim, Byung-Hoon

    2016-12-01

    Three-dimensional (3D) scaffolds have many advantageous properties for bone tissue engineering application, due to its controllable properties such as pore size, structural shape and interconnectivity. In this study, effects on oxygen plasma surface modification and adding of nano-hydroxyapatite (n-HAp) and β-tricalcium phosphate (β-TCP) on the 3D PLGA/n-HAp/β-TCP scaffolds for improving preosteoblast cell (MC3T3-E1) adhesion, proliferation and differentiation were investigated. The 3D PLGA/n-HAp/β-TCP scaffolds were fabricated by 3D Bio-Extruder equipment. The 3D scaffolds were prepared with 0°/90° architecture and pore size of approximately 300 μm. In addition 3D scaffolds surface were etched by oxygen plasma to enhance the hydrophilic property and surface roughness. After oxygen plasma treatment, the surface chemistry and morphology were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. And also hydrophilic property was measured by contact angle. The MC3T3-E1 cell proliferation and differentiation were investigated by MTT assay and ALP activity. In present work, the 3D PLGA/HAp/beta-TCP composite scaffold with suitable structure for the growth of osteoblast cells was successfully fabricated by 3D rapid prototyping technique. The surface hydrophilicity and roughness of 3D scaffold increased by oxygen plasma treatment had a positive effect on cell adhesion, proliferation, and differentiation. Furthermore, the differentiation of MC3T3-E1 cell was significantly enhanced by adding of n-HAp and β-TCP on 3D PLGA scaffold. As a result, combination of bioceramics and oxygen plasma treatment showed a synergistic effect on biocompatibility of 3D scaffolds. This result confirms that this technique was useful tool for improving the biocompatibility in bone tissue engineering application.

  10. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false In vitro human immunodeficiency virus (HIV) drug... OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  11. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin A.; Orosz Coghlan, Patricia A.; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza; Nickerson, Cheryl A.

    2007-01-30

    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D on porous collagen I-coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in-situ hybridization were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts using differentiated monolayer cultures failed.

  12. An in vitro clonogenic assay to assess radiation damage in rat CNS glial progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Maazen, R.W.M. van der; Verhagen, I.; Kogel, A.J. van der (Katholieke Univ., Nijmegen (Netherlands). Inst. of Radiotherapy)

    1990-11-01

    Normal glial progenitor cells can be isolated from the rat central nervous system (CNS) and cultured in vitro on a monolayer of type-1 astrocytes. These monolayers are able to support and stimulate explanted glial progenitor cells to proliferate. Employing these in vitro interactions of specific glial cell types, an in vivo-in vitro clonogenic assay has been developed. This method offers the possibility to study the intrinsic radiosensitivity, repair and regeneration of glial progenitor cells after in vitro or in vivo irradiation. (author).

  13. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    Science.gov (United States)

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.

  14. Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology

    NARCIS (Netherlands)

    Frega, M.; Tedesco, M.; Massobrio, P.; Pesce, M.; Martinoia, S.

    2014-01-01

    Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These

  15. The EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Historical control data and proof of principle studies for mechanistic assay adaptations.

    Science.gov (United States)

    Roy, Shambhu; Kulkarni, Rohan; Hewitt, Nicola J; Aardema, Marilyn J

    2016-07-01

    The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising novel animal alternative for evaluating genotoxicity of topically applied chemicals. It is particularly useful for assessing cosmetic ingredients that can no longer be tested using in vivo assays. To advance the use of this test especially for regulatory decision-making, we have established the RSMN assay in our laboratory according to Good Laboratory Practice and following the principles of the OECD test guideline 487 in vitro mammalian cell micronucleus test. Proficiency with the assay was established by correctly identifying direct-acting genotoxins and genotoxins requiring metabolism, as well as non-genotoxic/non-carcinogenic chemicals. We also report the analysis of our historical control data that demonstrate vehicle control and positive control values for %micronuclei in binucleated cells are in the ranges reported previously. Technical issues including evaluating various solvents with both 48h and 72h treatment regimens were investigated. For the first time, mechanistic studies using CREST analysis revealed that the RSMN assay is suitable for distinguishing aneugens and clastogens. Moreover, the assay is also suitable for measuring cytokines as markers for proliferative and toxic effects of chemicals.

  16. Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays.

    NARCIS (Netherlands)

    Tanneberger, K.; Rico Rico, A.; Kramer, N.I.; Busser, F.J.M.; Hermens, J.L.M.; Schirmer, K.

    2010-01-01

    Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly underst

  17. The comet assay: assessment of in vitro and in vivo DNA damage.

    Science.gov (United States)

    Bajpayee, Mahima; Kumar, Ashutosh; Dhawan, Alok

    2013-01-01

    Rapid industrialization and pursuance of a better life have led to an increase in the amount of chemicals in the environment, which are deleterious to human health. Pesticides, automobile exhausts, and new chemical entities all add to air pollution and have an adverse effect on all living organisms including humans. Sensitive test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cells from both in vitro and in vivo sources as well as in humans. Already, the in vivo comet assay has gained importance as the preferred test for assessing DNA damage in animals for some international regulatory guidelines. The advantages of the in vivo comet assay are its ability to detect DNA damage in any tissue, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the comet assay have resulted in establishment of guidelines. The in vitro comet assay conducted in cultured cells and cell lines can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high-throughput screening method for new chemical entities, as well as environmental samples. This chapter details the in vitro comet assay using the 96-well plate and in vivo comet assay in multiple organs of the mouse.

  18. Synthesis, Spectral Analysis and Preliminary in Vitro Evaluation of Some Tetrapyrrolic Complexes with 3d Metal Ions

    Directory of Open Access Journals (Sweden)

    Radu Socoteanu

    2015-08-01

    Full Text Available In this paper, two tetrapyrrolic complexes, Zn(II-5-(3-hydroxyphenyl-10,15,20-tris-(4-acetoxy-3-methoxyphenylporphyrin and Cu(II-5-(3-hydroxyphenyl-10,15,20-tris-(4-acetoxy-3-methoxyphenylporphyrin were synthesized, and characterized from a spectral and biological point of view. The study provided data concerning the behavior of identical external substituents vs. two different core insertions. Some of the properties of the proposed tetrapyrrolic structures were highlighted, having photodynamic therapy of cancer as a targeted biomedical application. Elemental analysis, NMR, FTIR and UV-Vis data in various solvents were provided. A preliminary in vitro study on normal and cancer cultured cells was carried out for biocompatibility assessment in dark conditions. The preliminary in vitro study performed on human peripheral mononuclear cells exposed to tetrapyrrolic compounds (2 µM showed that the proposed compounds had a convenient cytotoxic profile on human normal peripheral blood mononuclear cells under dark conditions. Meanwhile, the investigated compounds reduced the number of metabolically active breast tumor MCF-7 cells, with the exception of Zn(II complex-containing a symmetrical ligand. Accordingly, preliminary in vitro data suggest that the proposed tetrapyrrolic compounds are good candidates for PDT, as they limit tumor expansion even under dark conditions, whilst sparing normal cells.

  19. 3D bone mineral density distribution and shape reconstruction of the proximal femur from a single simulated DXA image: an in vitro study

    Science.gov (United States)

    Whitmarsh, Tristan; Humbert, Ludovic; De Craene, Mathieu; del Río Barquero, Luis M.; Fritscher, Karl; Schubert, Rainer; Eckstein, Felix; Link, Thomas; Frangi, Alejandro F.

    2010-03-01

    Area Bone Mineral Density (aBMD) measured by Dual-energy X-ray Absorptiometry (DXA) is an established criterion in the evaluation of hip fracture risk. The evaluation from these planar images, however, is limited to 2D while it has been shown that proper 3D assessment of both the shape and the Bone Mineral Density (BMD) distribution improves the fracture risk estimation. In this work we present a method to reconstruct both the 3D bone shape and 3D BMD distribution of the proximal femur from a single DXA image. A statistical model of shape and a separate statistical model of the BMD distribution were automatically constructed from a set of Quantitative Computed Tomography (QCT) scans. The reconstruction method incorporates a fully automatic intensity based 3D-2D registration process, maximizing the similarity between the DXA and a digitally reconstructed radiograph of the combined model. For the construction of the models, an in vitro dataset of QCT scans of 60 anatomical specimens was used. To evaluate the reconstruction accuracy, experiments were performed on simulated DXA images from the QCT scans of 30 anatomical specimens. Comparisons between the reconstructions and the same subject QCT scans showed a mean shape accuracy of 1.2mm, and a mean density error of 81mg/cm3. The results show that this method is capable of accurately reconstructing both the 3D shape and 3D BMD distribution of the proximal femur from DXA images used in clinical routine, potentially improving the diagnosis of osteoporosis and fracture risk assessments at a low radiation dose and low cost.

  20. 3D kinematic in-vitro comparison of posterolateral corner reconstruction techniques in a combined injury model.

    Science.gov (United States)

    Nau, Thomas; Chevalier, Yan; Hagemeister, Nicola; Duval, Nicolas; deGuise, Jacques A

    2005-10-01

    With the variable injury pattern to the posterolateral structures (PLS) of the knee, a number of reconstructive procedures have been introduced. It was the aim of the present study to evaluate the resulting 3D kinematics following three different surgical techniques of reconstruction in a combined posterior cruciate ligament (PCL)/PLS injury model. In nine human cadaveric knees, 3D kinematics were recorded during the path of flexion-extension using a computer based custom made 6-degree-of-freedom (DOF) testing apparatus. Additional laxity tests were conducted at 30 and 90 degrees of flexion. Testing was performed before and after cutting the PLS and PCL, followed by PCL reconstruction alone. Reconstructing the posterolateral corner, three surgical techniques were compared: (a) the posterolateral corner sling procedure (PLCS), (b) the biceps tenodesis (BT), and (c) a bone patellar-tendon bone (BTB) allograft reconstruction. Posterior as well as rotational laxity were significantly increased after PCL/PLS transection at 30 and 90 degrees of flexion. Isolated PCL reconstruction resulted in a remaining external rotational deficiency for both tested flexion angles. Additional PLS reconstruction closely restored external rotation as well as posterior translation to intact values by all tested procedures. Compared to the intact knee, dynamic testing revealed a significant internal tibial rotation for (b) BT (mean=3.9 degrees, p=0.043) and for (c) BTB allograft (mean=4.3 degrees, p=0.012). (a) The PLCS demonstrated a tendency to internal tibial rotation between 0 and 60 degrees of flexion (mean=2.2 degrees, p=0.079). Varus/valgus rotation as well as anterior/posterior translation did not show significant differences for any of the tested techniques. The present study shows that despite satisfying results in static laxity testing, pathological 3D knee kinematics were not restored to normal, demonstrated by a nonphysiological internal tibial rotation during the path of

  1. In vitro antileukemia, antibacterial and antifungal activities of some 3d metal complexes: chemical synthesis and structure - activity relationships.

    Science.gov (United States)

    Gulea, Aurelian; Poirier, Donald; Roy, Jenny; Stavila, Vitalie; Bulimestru, Ion; Tapcov, Victor; Birca, Maria; Popovschi, Lilia

    2008-12-01

    The present paper describes the synthesis, characterization and in vitro biological evaluation screening of different classes (ammoniacates, dioximates, carboxylates, semi- and thiosemicarbazidates) of Co(II), Co(III), Cu(II), Ni(II), Mn(II), Zn(II) and Fe(III) complexes. Schiff bases were obtained from the reaction of some salicyl aldehydes with, respectively, furoylhydrazine, benzoylhydrazine, semicarbazide, thiosemicarbazide and S-methylthiosemicarbazide to give tridentate ligands containing ONO, ONS or ONN as donor atoms. The synthetic metal complexes are of various geometrical and electronic structures, thermodynamic and thermal stabilities, and magnetic and conductance properties. All complexes, except those of Cu, are octahedral. Some Cu, Co and Mn compounds have a dimeric or a polymeric structure. The composition and structure of complexes were analysed by elemental analysis, IR and (1)H NMR and (13)C NMR spectroscopies, and magnetochemical, thermoanalytical and molar conductance measurements. All ligands and metal complexes were tested as inhibitors of human leukemia (HL-60) cells growth, and the most potent, the Cu(II) complexes, have been also tested for their in vitro antibacterial and antifungal activities. Structure-activity relationships were carried out.

  2. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System

    Directory of Open Access Journals (Sweden)

    Fanny Knöspel

    2016-04-01

    Full Text Available Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR, while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro.

  3. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System.

    Science.gov (United States)

    Knöspel, Fanny; Jacobs, Frank; Freyer, Nora; Damm, Georg; De Bondt, An; van den Wyngaert, Ilse; Snoeys, Jan; Monshouwer, Mario; Richter, Marco; Strahl, Nadja; Seehofer, Daniel; Zeilinger, Katrin

    2016-04-16

    Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro.

  4. The development of 3-D, in vitro, endothelial culture models for the study of coronary artery disease

    Directory of Open Access Journals (Sweden)

    Fraser Richard

    2009-10-01

    Full Text Available Abstract The response of the vascular endothelium to wall shear stress plays a central role in the development and progression of atherosclerosis. Current studies have investigated endothelial response using idealized in vitro flow chambers. Such cell culture models are unable to accurately replicate the complex in vivo wall shear stress patterns arising from anatomical geometries. To better understand this implication, we have created both simplified/tubular and anatomically realistic in vitro endothelial flow models of the human right coronary artery. A post-mortem vascular cast of the human left ventricular outflow tract was used to create geometrically accurate silicone elastomer models. Straight, tubular models were created using a custom made mold. Following the culture of human abdominal aortic endothelial cells within the inner lumen, cells were exposed to steady flow (Re = 233 for varying time periods. The resulting cell morphology was analyzed in terms of shape index and angle of orientation relative to the flow direction. In both models a progressive elongation and alignment of the endothelium in the flow direction was observed following 8, 12, and 24 hours. This change, however, was significantly less pronounced in the anatomical model (as observed from morphological variations indicative of localized flow features. Differences were also observed between the inner and outer walls at the disease-prone proximal region. Since morphological adaptation is a visual indication of endothelial shear stress activation, the use of anatomical models in endothelial genetic and biochemical studies may offer better insight into the disease process.

  5. Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro

    Science.gov (United States)

    Funk, Juergen; Robbins, Justin B.; Crogan-Grundy, Candace; Presnell, Sharon C.; Singer, Thomas; Roth, Adrian B.

    2016-01-01

    Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level. PMID:27387377

  6. Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro.

    Directory of Open Access Journals (Sweden)

    Deborah G Nguyen

    Full Text Available Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI. This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM. Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

  7. Gain in cellular organization of inflammatory breast cancer: A 3D in vitro model that mimics the in vivo metastasis

    Directory of Open Access Journals (Sweden)

    Alpaugh Mary L

    2009-12-01

    Full Text Available Abstract Background The initial step of metastasis in carcinomas, often referred to as the epithelial-mesenchymal transition (EMT, occurs via the loss of adherens junctions (e.g. cadherins by the tumor embolus. This leads to a subsequent loss of cell polarity and cellular differentiation and organization, enabling cells of the embolus to become motile and invasive. However highly malignant inflammatory breast cancer (IBC over-expresses E-cadherin. The human xenograft model of IBC (MARY-X, like IBC, displays the signature phenotype of an exaggerated degree of lymphovascular invasion (LVI in situ by tumor emboli. An intact E-cadherin/α, β-catenin axis mediates the tight, compact clump of cells found both in vitro and in vivo as spheroids and tumor emboli, respectively. Methods Using electron microscopy and focused ion beam milling to acquire in situ sections, we performed ultrastructural analysis of both an IBC and non-IBC, E-cadherin positive cell line to determine if retention of this adhesion molecule contributed to cellular organization. Results Here we report through ultrastructural analysis that IBC exhibits a high degree of cellular organization with polar elements such as apical/lateral positioning of E-cadherin, apical surface microvilli, and tortuous lumen-like (canalis structures. In contrast, agarose-induced spheroids of MCF-7, a weakly invasive E-cadherin positive breast carcinoma cell line, do not exhibit ultrastructural polar features. Conclusions This study has determined that the highly metastatic IBC with an exaggerated malignant phenotype challenges conventional wisdom in that instead of displaying a loss of cellular organization, IBC acquires a highly structured architecture. These findings suggest that the metastatic efficiency might be linked to the formation and maintenance of these architectural features. The comparative architectural features of both the spheroid and embolus of MARY-X provide an in vitro model with

  8. Synthesis and in vitro Antibacterial Activities of 5-(2,3,4,5- Tetrahydro-1 H-chromeno[2,3-dJpyrimidin-5-yl)pyrimidione Derivatives%Synthesis and in vitro Antibacterial Activities of 5-(2,3,4,5- Tetrahydro-1 H-chromeno[2,3-dJpyrimidin-5-yl)pyrimidione Derivatives

    Institute of Scientific and Technical Information of China (English)

    Cheng, Qingfang; Wang, Qifa; Tan, Ting; Wang, Mingxiao; Chen, Na

    2012-01-01

    A series of novel 5-(2,3,4,5-tetrahydro-lH-chromeno[2,3-d]pyrimidin-5-yl)pyrimidione derivatives have been synthesized from substituted salicylaldehydes and barbituric acid or 2-thiobarbituric acid in water catalyzed by phase transfer catalysis of triethylbenzyl ammonium chloride (TEBA). Elemental analysis, IR, 1HNMR, and 13C NMR elucidated the structures of all the newly synthesized compounds. In vitro antimicrobial activities of synthesized compounds have been investigated against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudornonas aeruginosa. These newly synthesized derivatives exhibited significant in vitro antibacterial activity.

  9. Production and in vitro characterization of 3D porous scaffolds made of magnesium carbonate apatite (MCA)/anionic collagen using a biomimetic approach

    Energy Technology Data Exchange (ETDEWEB)

    Sader, Marcia S., E-mail: msader@metalmat.ufrj.br [Prog. Engenharia Metalúrgica e Materiais, COPPE/UFRJ, RJ (Brazil); Martins, Virginia C.A. [Depto. de Química e Física Molecular, IQSC/USP, SP (Brazil); Gomez, Santiago [Dept. Anatomía Patológica, Universidad de Cádiz, Cadiz (Spain); LeGeros, Racquel Z. [Department of Biomaterials and Biomimetics, New York University College of Dentistry, NY (United States); Soares, Gloria A. [Prog. Engenharia Metalúrgica e Materiais, COPPE/UFRJ, RJ (Brazil)

    2013-10-15

    3D porous scaffolds are relevant biomaterials to bone engineering as they can be used as templates to tissue reconstruction. The aim of the present study was to produce and characterize in vitro 3D magnesium-carbonate apatite/collagen (MCA/col) scaffolds. They were prepared by using biomimetic approach, followed by cross-linking with 0.25% glutaraldehyde solution (GA) and liofilization. Results obtained with Fourier-transform infrared spectroscopy (FT-IR) confirmed the type-B carbonate substitution, while by X-ray diffraction (XRD), a crystallite size of ∼ 10 nm was obtained. Optical and electron microscopy showed that the cylindrical samples exhibited an open-porous morphology, with apatite nanocrystals precipitated on collagen fibrils. The cross-linked 3D scaffolds showed integrity when immersed in culture medium up to 14 days. Also, the immersion of such samples into an acid buffer solution, to mimic the osteoclastic resorption environment, promotes the release of important ions for bone repair, such as calcium, phosphorus and magnesium. Bone cells (SaOs2) adhered, and proliferated on the 3D composite scaffolds, showing that synthesis and the cross-linking processes did not induce cytotoxicity. Highlights: • 3D scaffolds of Mg-carbonate–apatite and anionic-collagen were produced. • The biomimetic approach and the cross-linking with 0.25% GA solution were employed. • The scaffolds showed open-porous structure and apatite crystals on collagen fibrils. • The cross-linked scaffolds exhibited integrity when immersed in culture medium. • SaOs2 cells adhered and proliferated on the cross-linked scaffolds confirming no cytotoxicity.

  10. In vitro assays for cobblestone area-forming cells, LTC-IC, and CFU-C.

    Science.gov (United States)

    van Os, Ronald P; Dethmers-Ausema, Bertien; de Haan, Gerald

    2008-01-01

    Various assays exist that measure the function of hematopoietic stemcells (HSCs). In this chapter, in vitro assays are described that measure the frequency of progenitors (colony-forming unit in culture; CFU-C), stem cells (long-term culture-initiating cell; LTC-IC), or both (cobblestone area-forming cell assay; CAFC). These assays measure the potential of a test cell population retrospectively, i.e., at the time its activity is evident when the stem cell itself is often not detectable anymore. Although the in vitro LTC-IC and CAFC assays have been shown to correlate with in vivo activity, in vivo transplantation assays, where it can be shown that cells possess the ability to indefinitely repopulate all blood lineages, are the ultimate proof for HSC activity. Nevertheless, these in vitro assays provide an excellent method to screen for stem cell activity of a putative stem cell population or for screening the effect of a certain treatment on HSCs.

  11. In vitro comparison of Doppler and catheter-measured pressure gradients in 3D models of mitral valve calcification.

    Science.gov (United States)

    Herrmann, Tarrah A; Siefert, Andrew W; Pressman, Gregg S; Gollin, Hannah R; Touchton, Steven A; Saikrishnan, Neelakantan; Yoganathan, Ajit P

    2013-09-01

    Mitral annular calcification (MAC) involves calcium deposition in the fibrous annulus supporting the mitral valve (MV). When calcification extends onto the leaflets, valve opening can be restricted. The influence of MAC MV geometry on Doppler gradients is unknown. This study describes a novel methodology to rapid-prototype subject-specific MAC MVs. Replicated valves were used to assess the effects of distorted annular-leaflet geometry on Doppler-derived, transmitral gradients in comparison to direct pressure measurements and to determine if transmitral gradients vary according to measurement location. Three-dimensional echocardiography data sets were selected for two MAC MVs and one healthy MV. These MVs were segmented and rapid prototyped in their middiastolic configuration for in vitro testing. The effects of MV geometry, measurement modality, and measurement location on transmitral pressure gradient were assessed by Doppler and catheter at three locations along the MV's intercommissural axis. When comparing dimensions of the rapid-prototyped valves to the subject echocardiography data sets, mean relative errors ranged from 6.2% to 35%. For the evaluated MVs, Doppler pressure gradients exhibited good agreement with catheter-measured gradients at a variety of flow rates, though with slight systematic overestimation in the recreated MAC valves. For all of the tested MVs, measuring the transmitral pressure gradient at differing valve orifice positions had minimal impact on observed gradients. Upon the testing of additional normal and calcific MVs, these data may contribute to an improved clinical understanding of MAC-related mitral stenosis. Moreover, they provide the ability to statistically evaluate between measurement locations, flow rates, and valve geometries for Doppler-derived pressure gradients. Determining these end points will contribute to greater clinical understanding for the diagnosis MAC patients and understanding the use and application of Doppler

  12. Innovative mode of action based in vitro assays for detection of marine neurotoxins

    NARCIS (Netherlands)

    Nicolas, J.A.Y.

    2015-01-01

    Innovative mode of action based in vitro assays for detection of marine neurotoxins J. Nicolas, P.J.M. Hendriksen, T.F.H. Bovee, I.M.C.M. Rietjens Marine biotoxins are naturally occurring compounds produced by particular phytoplankton species. These toxins often accumulate in seafood and thereby rep

  13. A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds.

    Science.gov (United States)

    Göttlich, Claudia; Müller, Lena C; Kunz, Meik; Schmitt, Franziska; Walles, Heike; Walles, Thorsten; Dandekar, Thomas; Dandekar, Gudrun; Nietzer, Sarah L

    2016-01-01

    In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining's, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its

  14. In vitro screening assay for teratogens using growth inhibition of human embryonic cells.

    Science.gov (United States)

    Pratt, R M; Willis, W D

    1985-01-01

    We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo. Images PMID:3862095

  15. In vitro screening assay for teratogens using growth inhibition of human embryonic cells

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, R.M.; Willis, W.D.

    1985-09-01

    The authors have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. The authors suggest that this assay, along with the complementary tumor cell-attachment assay of Braun may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

  16. Thyroid endocrine system disruption by pentachlorophenol: an in vitro and in vivo assay.

    Science.gov (United States)

    Guo, Yongyong; Zhou, Bingsheng

    2013-10-15

    The present study aimed to evaluate the disruption caused to the thyroid endocrine system by pentachlorophenol (PCP) using in vitro and in vivo assays. In the in vitro assay, rat pituitary GH3 cells were exposed to 0, 0.1, 0.3, and 1.0 μM PCP. PCP exposure significantly downregulated basal and triiodothyronine (T3)-induced Dio 1 transcription, indicating the antagonistic activity of PCP in vitro. In the in vivo assay, zebrafish embryos were exposed to 0, 1, 3, and 10 μg/L of PCP until 14 days post-fertilization. PCP exposure resulted in decreased thyroxine (T4) levels, but elevated contents of whole-body T3. PCP exposure significantly upregulated the mRNA expression of genes along hypothalamic-pituitary-thyroid (HPT) axis, including those encoding thyroid-stimulating hormone, sodium/iodide symporter, thyroglobulin, Dio 1 and Dio 2, alpha and beta thyroid hormone receptor, and uridinediphosphate-glucuronosyl-transferase. PCP exposure did not influence the transcription of the transthyretin (TTR) gene. The results indicate that PCP potentially disrupts the thyroid endocrine system both in vitro and in vivo.

  17. Synthesis of Some Polysubstituted Nicotinonitriles and Derived Pyrido[2,3-d]pyrimidines as In Vitro Cytotoxic and Antimicrobial Candidates

    Directory of Open Access Journals (Sweden)

    Hassan M. Faidallah

    2016-01-01

    Full Text Available The synthesis of polysubstituted pyridines, in addition to some derived pyrido[2,3-d]pyrimidine ring systems supported with chemotherapeutically active functionalities, is described. They were evaluated for their in vitro cytotoxic effects against three different human tumor cell lines (human colon carcinoma HT29, hepatocellular carcinoma Hep-G2, and Caucasian breast adenocarcinoma MCF7. Nine compounds displayed variable cytotoxic potential, among which alkylthio analogs 33, 34, and 37 emerged as the most active members, being almost twice as active as doxorubicin against the colon carcinoma HT29 cell line. In addition, the same three analogs showed a clear differential cytotoxic profile as they exhibited a marginal inhibitory effect on the growth of the normal nontransformed human foreskin fibroblast Hs27 cell line. Meanwhile, nineteen compounds were able to exhibit significant antibacterial activity against both Gram-positive and Gram-negative bacteria, together with moderate antifungal activities. The pyrido[2,3-d]pyrimidine-2(1H-thione 30 together with its alkylthio derivatives 33 and 34 stemmed as the most active antimicrobial members being equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a noticeable antifungal activity against C. albicans. Compounds 33 and 34 could be considered as a promising template for possible dual antimicrobial-anticancer candidates.

  18. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

    NARCIS (Netherlands)

    Legler, J.; Zeinstra, L.M.; Schuitemaker, F.; Lanser, P.H.; Bogerd, J.; Brouwer, A.; Vethaak, A.D.; Voogt, de P.; Murk, A.J.; Burg, van der B.

    2002-01-01

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably trans

  19. Human low density lipoprotein as a substrate for in vitro steroidogenesis assays with fathead minnow ovary explants

    Science.gov (United States)

    Gonad explant in vitro steroidogenesis assays are used as part of a multifaceted strategy to detect endocrine active chemicals capable of altering steroid hormone synthesis. An in vitro steroidogenesis assay used in our laboratory involves exposing fathead minnow (FHM) gonad exp...

  20. Comparison of the unlabeled and labeled pre-mRNA splicing assays in vitro

    Institute of Scientific and Technical Information of China (English)

    TIAN XU BU; JING XIN HONG; ZHI YAO; JIE YANG

    2006-01-01

    Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro, 32P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB)staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.

  1. Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

    Science.gov (United States)

    Lorge, Elisabeth; Lambert, Carine; Gervais, Véronique; Becourt-Lhote, Nathalie; Delongeas, Jean-Luc; Claude, Nancy

    2007-04-01

    The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.

  2. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  3. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs

    Science.gov (United States)

    Sewing, Sabine; Boess, Franziska; Moisan, Annie; Bertinetti-Lapatki, Cristina; Minz, Tanja; Hedtjaern, Maj; Tessier, Yann; Schuler, Franz; Singer, Thomas; Roth, Adrian B.

    2016-01-01

    Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development. PMID:27442522

  4. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

    Directory of Open Access Journals (Sweden)

    Sabine Sewing

    Full Text Available Single stranded oligonucleotides (SSO represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.

  5. Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Holmstrøm, Kim; Jørgensen, Flemming;

    2015-01-01

    sorting (FACS) sorted CD4 + CD25(high)CD127(dim/-)CD45RA+ naïve Treg cells followed by in vitro expansion. We report on the use of these cells in a short-term assay based on Treg mediated inhibition of the early effector T cell activation markers CD69 and CD154. Additionally, we investigate the use...

  6. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

    Science.gov (United States)

    Kimura, Aoi; Miyata, Atsuro; Honma, Masamitsu

    2013-09-01

    The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

  7. Transcription in Archaea: in vitro transcription assays for mjRNAP.

    Science.gov (United States)

    Smollett, Katherine; Blombach, Fabian; Werner, Finn

    2015-01-01

    The fully recombinant Methanocaldococcus jannaschii RNA polymerase allows for a detailed dissection of the different stages of the transcription. In the previous chapter, we discussed how to purify the different components of the M. jannaschii transcription system, the RNA polymerase subunits, and general transcription factors and how to assemble a functional M. jannaschii enzyme. Standard in vitro transcription assays can be used to examine the different stages of transcription. In this chapter, we describe how some of these assays have been optimized for M. jannaschii RNA polymerase, which transcribes at much higher temperatures than many other transcription complexes.

  8. In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites.

    Science.gov (United States)

    Heredero-Bermejo, I; Copa-Patiño, J L; Soliveri, J; Gómez, R; de la Mata, F J; Pérez-Serrano, J

    2013-12-01

    The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

  9. Antioxidant activities of Indigofera cassioides Rottl. Ex. DC. using various in vitro assay models

    Institute of Scientific and Technical Information of China (English)

    R Senthil Kumar; B Rajkapoor; P Perumal

    2012-01-01

    Objective: To evaluate the antioxidant potential of methanolic leaf extract of Indigoferacassioides (MEIC) using various in vitro antioxidant assay systems. Methods: Antioxidant and free radical scavenging activity of MEIC was assayed by using different in vitro models like ABTS, DPPH, nitric oxide, superoxide, hydrogen peroxide and hydroxyl radical. Reductive ability of the extract was tested by the complex formation with potassium ferricyanide. Further total phenol and flavonoid contents of the crude extract were also determined. Rutin and ascorbic acid were used as standards. Results: MEIC exhibited potent and concentration dependent free radical scavenging activity in all the tested models. Reductive ability was also found to increase with increase in MEIC concentration. Total phenol and flavonoid content determination showed that the extract is rich in phenols and flavonoids. Conclusions: All the results of the in vitro antioxidant assays reveal potent antioxidant and free radical scavenging activity of the leaves of Indigofera cassioides, equivalent to that of standard ascorbic acid and rutin. This potent antioxidant activity may be attributed to its high phenolic and flavonoid contents

  10. Evaluation of wetland and tertiary wastewater treatments for estrogenicity using in vivo and in vitro assays.

    Science.gov (United States)

    Xie, L; Sapozhnikova, Y; Bawardi, O; Schlenk, D

    2005-01-01

    The effects of wetland wastewater treatment on estrogenic activity and estrogenic activity of water after tertiary treatment were evaluated using in vivo (rainbow trout vitellogenin [VTG] expression) and in vitro (yeast estrogen screening) assays. Juvenile rainbow trout exposed to tertiary-treated wastewater from the Green Acres Treatment Plant in Orange County Water District had increased plasma VTG levels compared with control fish. When trout were exposed to wastewater-dominated water before it entered into Prado Wetland (Riverside County, CA), VTG concentrations were increased above those of controls and were not significantly different from fish exposed to water exiting the wetland. VTG E2-equivalent concentrations (EEQs) of the water samples from the Green Acres Plant were 16.92 +/- 16.49 ng/L. Activity of water entering Prado Wetland was 29.80 +/- 28.41 ng/L EEQ, and water exiting was 24.34 +/- 23.17 ng/L EEQ. In vitro assays estimated that estrogenic activity of water from the Green Acres Plant was estrogen screening EEQs of 2.57 and estrogens that are not potent estrogen-receptor ligands exist in wastewaters from the Green Acres Plant as well as water entering and exiting Prado Wetland. Wetland treatment did not remove environmental estrogens in the water. Our results also suggest that in vitro assays may underestimate estrogenic activity of sampled water.

  11. Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay.

    Science.gov (United States)

    Li, Yan; Chen, David H; Yan, Jian; Chen, Ying; Mittelstaedt, Roberta A; Zhang, Yongbin; Biris, Alexandru S; Heflich, Robert H; Chen, Tao

    2012-06-14

    Silver nanoparticles (AgNPs) have antimicrobial properties, which have contributed to their widespread use in consumer products. A current issue regarding nanomaterials is the extent to which existing genotoxicity assays are useful for evaluating the risks associated with their use. In this study, the genotoxicity of 5 nm AgNPs was assessed using two standard genotoxicity assays, the Salmonella reverse mutation assay (Ames test) and the in vitro micronucleus assay. Using the preincubation version of the Ames assay, Salmonella strains TA102, TA100, TA1537, TA98, and TA1535 were treated with 0.15-76.8 μg/plate of the AgNPs. Toxicity limited the doses that could be assayed to 2.4-38.4 μg/plate; no increases in mutant frequency over the vehicle control were found for the concentrations that could be assayed. Human lymphoblastoid TK6 cells were treated with 10-30 μg/ml AgNPs, and additional cells were treated with water and 0.73 gy X-rays as vehicle and positive controls. Micronucleus frequency was increased by the AgNP treatment in a dose-dependent manner. At a concentration of 30 μg/ml (with 45.4% relative population doubling), AgNPs induced a significant, 3.17-fold increase with a net increase of 1.60% in micronucleus frequency over the vehicle control, a weak positive response by our criteria. These results demonstrate that the 5 nm AgNP are genotoxic in TK6 cells. Also, the data suggest that the in vitro micronucleus assay may be more appropriate than the Ames test for evaluating the genotoxicity of the AgNPs.

  12. Prevalidation of the rat CFU-GM assay for in vitro toxicology applications.

    Science.gov (United States)

    Pessina, Augusto; Bonomi, Arianna; Cavicchini, Loredana; Albella, Beatriz; Cerrato, Laura; Parent-Massin, Dominique; Sibiril, Yann; Parchment, Ralph; Behrsing, Holger; Verderio, Paolo; Pizzamiglio, Sara; Giangreco, Manuela; Baglio, Carolina; Coccè, Valentina; Sisto, Francesca; Gribaldo, Laura

    2010-05-01

    In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies - however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. In previous studies, the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood) and for predicting the in vivo human maximal tolerated dose (MTD) by adjusting in vivo data on mouse toxicity. Recently, a Colony Forming Unit-Megakaryocyte (CFU-MK) assay has also been prevalidated for testing drug toxicity toward megakaryocytes. The rat CFU-GM assay has been used by many researchers for its ability to evaluate in vitro haematotoxicity. Although it is not yet available, a standardised procedure for data comparison could be very important, since the rat is the most widely-used species for the in vivo testing of toxicants. This report presents the results of the prevalidation study developed to analyse the intra-laboratory and inter-laboratory variability of a standardised operating procedure for this assay and its performance for the in vitro determination of the inhibitory concentration (IC) values of drugs on rat myeloid progenitors (CFU-GM). The results demonstrate that the CFU-GM assay can be performed with cryopreserved rat bone-marrow cells (rBMC). The assay represents a useful tool for evaluating the toxicity of a compound, in terms of both relative toxicity (when different molecules are compared) and the prediction of the degree of in vivo toxicity. The use of this assay could greatly reduce the number of rats used in experimental procedures, and

  13. Potencies of estrogenic compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo.

    Science.gov (United States)

    Segner, H; Navas, J M; Schäfers, C; Wenzel, A

    2003-03-01

    The objective of this study was to compare the estrogenic potency of environmental estrogens at two testing tiers: at the initial level of in vitro screening assays, and at the level of definitive fish reproduction tests in vivo. The in vitro tests included a recombinant yeast estrogen receptor (ER) assay, a competitive radioreceptor assay using the hepatic ER of carp (Cyprinus carpio), and assays on vitellogenin induction in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss) and carp. In vivo, full life cycle tests with zebrafish (Danio rerio) were performed, using fertilization success as estrogen-sensitive reproductive endpoint. The test compounds included the natural estrogen 17beta-estradiol (E2) (only applied in the in vitro assays); the synthetic estrogen ethynylestradiol (EE2); and two xenoestrogens, 4-tert-octylphenol (OP) and bisphenol A (BPA). Among the in vitro assays, differences were observed in the relative ranking of the test substances, and in the absolute sensitivity (EC50 values), although the interassay differences of EC50 values were within one order of magnitude. The in vivo activity of the test compounds was not accurately predicted by the in vitro assays, with respect to neither sensitivity nor ranking. The in vitro assays tended to overestimate the relative potency of the xenoestrogens; i.e. the ratio between the activity of the reference compound, EE2, and that of the test compound. The best prediction of the in vivo fish test results was obtained from the recombinant yeast assay.

  14. Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment.

    Directory of Open Access Journals (Sweden)

    Rachel F Cox

    Full Text Available Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

  15. Factors affecting the in vitro micronucleus assay for evaluation of nanomaterials.

    Science.gov (United States)

    Li, Yan; Doak, Shareen H; Yan, Jian; Chen, David H; Zhou, Min; Mittelstaedt, Roberta A; Chen, Ying; Li, Chun; Chen, Tao

    2017-01-01

    A number of in vitro methodologies have been used to assess the genotoxicity of different nanomaterials, including titanium dioxide nanoparticles (TiO2 NPs) and silver nanoparticles (AgNPs). The in vitro micronucleus assay is one of the most commonly used test methods for genotoxicity evaluation of nanomaterials. However, due to the novel features of nanomaterials, such as high adsorption capacity and fluorescence properties, there are unexpected interactions with experimental components and detection systems. In this study, we evaluate the interference by two nanoparticles, AgNPs and TiO2 NPs, with the in vitro micronucleus assay system and possible confounding factors affecting cytotoxicity and genotoxicity assessment of the nanomaterials including cell lines with different p53 status, nanoparticle coatings and fluorescence, cytochalasin B, fetal bovine serum in cell treatment medium and different measurement methodologies for detecting micronuclei. Our results showed that micronucleus induction by AgNPs was similar when evaluated using flow cytometry or microscope, whereas the induction by TiO2 NPs was different using the two methods due to TiO2's fluorescence interference with the cytometry equipment. Cells with the mutated p53 gene were more sensitive to micronucleus induction by AgNPs than the p53 wild-type cells. The presence of serum during treatment increased the toxicity of AgNPs. The coatings of nanoparticles played an important role in the genotoxicity of AgNPs. These collective data highlight the importance of considering the unique properties of nanoparticles in assessing their genotoxicity using the in vitro micronucleus assay.

  16. Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Nair MS

    2015-05-01

    Full Text Available Maya S Nair,1 Ullas Mony,1 Deepthy Menon,1 Manzoor Koyakutty,1 Neeraj Sidharthan,2 Keechilat Pavithran,2 Shantikumar V Nair,1 Krishnakumar N Menon11Amrita Centre for Nanosciences and Molecular Medicine, 2Department of Oncology, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kerala, IndiaAbstract: Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU/poly-L-lactic acid (PLLA in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34+/CD38-/CD33- phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27Kip1leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27

  17. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.

    2003-01-01

    Full Text Available Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method.

  18. Dose assessment of SiC nanoparticle dispersions during in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Mejia, Jorge, E-mail: jorge.mejiamendoza@unamur.be [University of Namur, Research Centre for the Physics of Matter and Radiation (LARN-PMR) NARILIS (Belgium); Piret, Jean-Pascal; Noeel, Florence [University of Namur, Research Unit in Cellular Biology (URBC) NARILIS (Belgium); Masereel, Bernard [University of Namur, Department of Pharmacy NAMEDIC, Namur Thrombosis and Homeostasis Center (NTHC) NARILIS (Belgium); Toussaint, Olivier [University of Namur, Research Unit in Cellular Biology (URBC) NARILIS (Belgium); Lucas, Stephane [University of Namur, Research Centre for the Physics of Matter and Radiation (LARN-PMR) NARILIS (Belgium)

    2013-08-15

    Here, we show that key physicochemical parameters of commercial Silicon Carbide nanoparticles, such as the primary particles of about 53 nm in size, the agglomerates size, and the surface composition, are considerably modified with respect to the pristine conditions, during in vitro assessment. The use of sample conditioning stages, such as the pre-dispersion in aqueous media and the subsequent dispersion in a culture medium specific to the in vitro assay, produce modifications as the absorption of N, C, and O, from the culture medium, in the nanoparticles surface. Our results show that the sedimented dose, fraction of sedimented NPs during incubation and consequently in contact with cells seeded at the bottom, of Silicon Carbide nanoparticles can be measured from the particle size distribution obtained using a centrifugal liquid sedimentation technique. It is underlined that the variations observed in the physicochemical properties are related to the in vitro assay conditions. Culture medium and incubation time are found to influence the most the sedimented dose and consequently the cells dose uptake.

  19. [In vitro evaluation of the chemosensitivity of malignant gastrointestinal tumors by stem cell assay].

    Science.gov (United States)

    Scheithauer, W; Temsch, E M; Schieder, H; Funovics, J; Schiessel, R; Grabner, G

    1984-01-01

    The Human Tumor Stem Cell Assay, originally described by Hamburger and Salmon, was shown to be a useful in-vitro technique for predicting response or lack of response in individual patients' tumors. In the present study 34 GI-tumors were assayed for evaluation of in-vitro growth characteristics and sensitivity-patterns to standard chemotherapeutic drugs as well as to recombinant interferon alpha-2(rIF). Sufficient growth for evaluation of anticancer drug activity (greater than 30 colonies/control plate) was obtained in 56% of specimens: 2/9 colorectal, 0/3 stomach, 0/3 pancreatic tumors and 1/4 hepatomas revealed a 50% (or more) decrease of TCFUs, that was considered the minimum for in-vitro efficacy. Our results suggest a very limited overall activity of rIF in gastrointestinal malignancies. Only 1 pancreatic cancer (of 18 evaluable specimens) showed a significant decrease of colony formation (70%), when 100 U of interferon/ml were added to the culture system.

  20. The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

    Science.gov (United States)

    Tanner, Rachel; O’Shea, Matthew K.; White, Andrew D.; Müller, Julius; Harrington-Kandt, Rachel; Matsumiya, Magali; Dennis, Mike J.; Parizotto, Eneida A.; Harris, Stephanie; Stylianou, Elena; Naranbhai, Vivek; Bettencourt, Paulo; Drakesmith, Hal; Sharpe, Sally; Fletcher, Helen A.; McShane, Helen

    2017-01-01

    The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays. PMID:28256545

  1. Development of a novel in vitro assay for the evaluation of integron DNA integrase activity

    Directory of Open Access Journals (Sweden)

    Fatemeh Tohidi

    2016-05-01

    Full Text Available Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment.

  2. In vitro antioxidant assay of selected aqueous plant extracts and their polyherbal formulation

    Directory of Open Access Journals (Sweden)

    Ganga Raju M.

    2015-04-01

    Full Text Available To support the use of selected plant extracts in Ayurveda, naturopathy, the antioxidant potential of the aqueous extract of Vincarosea (VR, Gymnemasylvestre (GS, Tinosporacordifolia (TC and Emblicaofficinalis (EO and their mixture (PHF of Indian origin was investigated for in vitro antioxidant activity by using in vitro models like superoxide, hydroxyl radical scavenging activity and lipid peroxide inhibition assay. The results were compared with standard (ascorbic acid, a known antioxidant. The various phytoconstituents identified in the above selected plants extracts were poly phenols, flavonoids, terpenoids, tannins, alkaloids. The terpenoids were reported to protect lipids, blood and body fluids against the attack of free radicals, some types of reactive oxygen, hydroxylic groups, peroxides and superoxide radicals. The presence of these phytoconstituents in selected plants might be responsible for antioxidant activity with that of known antioxidant ascorbic acid.

  3. Development and in vitro assay of oxidative stress modifying formulations for wound healing promotion.

    Science.gov (United States)

    Atrux-Tallau, Nicolas; Callejon, Sylvie; Migdal, Camille; Padois, Karine; Bertholle, Valérie; Denis, Alain; Chavagnac-Bonneville, Marlène; Haftek, Marek; Falson, Françoise; Pirot, Fabrice

    2011-05-01

    Often presented as metabolism byproducts, reactive oxygen species are linked to detrimental effects such as chronic wound, mutagenesis, cancer and skin ageing. However, recent in vitro and in vivo observations suggest that ROS, and mainly hydrogen peroxide, interfere with cell signaling acting like second messenger and inducing adaptive responses. This is particularly observed in skin wound healing where cells are exposed to H₂O₂ following injury. In this study, we developed and characterized an innovative formulation producing H₂O₂ at low concentrations, in order to mimic physiological inflammation phase. Then, this pro-oxidative formulation (CAM-GOx) was assayed in vitro on keratinocytes cell culture, compared to the blank formulation (CAM) and the anti-oxidative formulation (CAM-CAT) to assess whether oxidative stress was implied or not in cellular responses.

  4. Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

    Energy Technology Data Exchange (ETDEWEB)

    Vieira, J.M.B.D., E-mail: jmanya@terra.com.br [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil); Seabra, S.H. [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Vallim, D.C. [Instituto Oswaldo Cruz, Rio de Janeiro (Brazil); Americo, M.A.; Fracallanza, S.E.L. [Laboratorio de Bacteriologia Medica, IMPPG, UFRJ, Rio de Janeiro (Brazil); Vommaro, R.C. [Laboratorio de Ultra-estrutura Celular Hertha Meyer, IBCCF, UFRJ (Brazil); Domingues, R.M.C.P. [Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil)

    2009-10-02

    Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

  5. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics

    Science.gov (United States)

    Joubert, Marisa K.; Deshpande, Meghana; Yang, Jane; Reynolds, Helen; Bryson, Christine; Fogg, Mark; Baker, Matthew P.; Herskovitz, Jonathan; Goletz, Theresa J.; Zhou, Lei; Moxness, Michael; Flynn, Gregory C.; Narhi, Linda O.; Jawa, Vibha

    2016-01-01

    An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed. PMID:27494246

  6. In vitro pituitary and thyroid cell proliferation assays and their relevance as alternatives to animal testing.

    Science.gov (United States)

    Jomaa, Barae; Aarts, Jac M M J G; de Haan, Laura H J; Peijnenburg, Ad A C M; Bovee, Toine F H; Murk, Albertinka J; Rietjens, Ivonne M C M

    2013-01-01

    This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

  7. Replacing animal experiments in developmental toxicity testing of phenols by combining in vitro assays with physiologically based kinetic (PBK) modelling

    NARCIS (Netherlands)

    Strikwold, Marije

    2016-01-01

    Many efforts have been undertaken over the past decades to develop in vitro tests for a wide range of toxicological endpoints as an alternative to animal testing. The principle application of in vitro toxicity assays still lies in the hazard assessment and the prioritisation of chemicals for further

  8. Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays

    Directory of Open Access Journals (Sweden)

    Crabb Brendan S

    2010-06-01

    Full Text Available Abstract Background Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. Methods The D10 P. falciparum line was transfected to express green fluorescent protein (GFP. In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Results Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Conclusions Flow cytometry based assays using GFP

  9. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

    Directory of Open Access Journals (Sweden)

    Fukumori Nobutaka

    2009-09-01

    Full Text Available Abstract Background Recently, manufactured nano/microparticles such as fullerenes (C60, carbon black (CB and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

  10. In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

    Science.gov (United States)

    Darne, Christian; Coulais, Catherine; Terzetti, Francine; Fontana, Caroline; Binet, Stéphane; Gaté, Laurent; Guichard, Yves

    2016-01-15

    Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard

  11. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  12. Synthesis, in vitro antimycobacterial evaluation and docking studies of some new 5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one schiff bases.

    Science.gov (United States)

    Malothu, Narender; Bhandaru, Jaswanth S; Kulandaivelu, Umasankar; Jojula, Malathi; Adidala, Raghuram Reddy; K R, Umadevi; A V N, Dusthackeer; Kaki, Venkat Rao; Akkinepally, Raghuram R

    2016-02-01

    Development of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) has been considered as major health burden, globally. In order to develop novel, potential molecules against drug resistant TB, twenty two (22) new 3-substituted-7-benzyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6a-k) and 3-substituted-7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a-k) derivatives were designed and synthesized by using appropriate synthetic protocols. Pantothenate synthetase (PS) was considered as the target for the molecular docking studies and evaluated the binding pattern at active site, as PS plays a significant role in the biosynthesis of pantothenate in Mycobacterium tuberculosis (MTB). The preliminary in vitro antibacterial screening of test compounds was carried out against two strains of Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The antimycobacterial screening was performed against MTB H37Rv and an isoniazid-resistant clinical isolate of MTB. The compounds 6b, 6c, 6d, 6k, 7b, 7c, 7d and 7k exhibited promising antibacterial activity MIC in the range of 15-73 μM against all bacterial strains used and compounds 6d and 7b showed antimycobacterial activity (IC50 <340 μM in LRP assay) and (MIC <9 μM in broth microdilution method).

  13. Fabrication, in vitro Degradation and Cytotoxic Assay of Different Cystalline Phases Calcium Polyphosphate

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Fabrication, in vitro degradation and cytotoxic assay of different crystalline phases calcium polyphosphate (CPP) were reported. The CPP ceramics were fabricated by crystallizing the amorphous frits , and sintered at 550 ℃ ,875 ℃ ,1000 ℃ for 1 h to obtain the γ-CPP, β-CPP anda-CPP respectively. The effects of the different crystalline phases on their weight loss and released PO4 3- were investigated during the degradation.And the surface change was observed by the SEM. The osteoblastic ROS17/2.8 cell line was used to estimate the cytotoxicity of CPP. The effects of CPP on cells' proliferation were evaluated by using MTT assay. The experimental results showed that γ-CPP, β- CPP and α-CPP did not exert cytotoxic effect on the cells. In addition, the proliferation of the growth of ROS17/2.8 cells on β-CPP was optimal.

  14. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

    Science.gov (United States)

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-07-08

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

  15. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA for Infectious Diseases

    Directory of Open Access Journals (Sweden)

    Harpal Singh

    2015-07-01

    Full Text Available Enzyme-linked Immunosorbent Assay (ELISA-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

  16. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases

    Science.gov (United States)

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines. PMID:26184194

  17. Validation of an in vitro contractility assay using canine ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.; Pullen, G.F.; Valentin, J.P.; Pollard, C.E.

    2012-04-15

    Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of accuracy.

  18. Evaluation of Antioxidant Capacity of Solanum sessiliflorum (Cubiu Extract: An In Vitro Assay

    Directory of Open Access Journals (Sweden)

    Diego Rocha de Lucena Herrera Mascato

    2015-01-01

    Full Text Available Cubiu is a vegetable of Solanaceae family, native to the Amazon, which is widely distributed through Brazil, Peru, and Colombia. It is used in food, medicine, and cosmetics by native populations. Research has shown that cubiu extracts have antioxidant activities with great biological relevance. We performed a phytochemical screening to identify the main chemical groups that could confer antioxidant activity to this extract. Several tests and qualitative precipitation specific staining for major classes of secondary metabolites were used. Antioxidant capacity in vitro tests (DPPH and ABTS were also used to assess the extract’s ability to sequester free radicals of 70% hydroethanolic and aqueous extracts of cubiu flour. Alkaloids, organic acids, phenols, flavonoid glycosides, and coumarins were found in the hydroethanolic extract while the aqueous extract presented anthocyanins, gums, tannins and mucilage, amino groups, and volatile and fixed acids. For in vitro tests, the IC50 value obtained in the DPPH assay was 606.3 ± 3.5 μg/mL while that for the ABTS assay was 290.3 ± 10.7 µg/mL. Although cubiu extracts present chemical compounds directly related to antioxidant activity, our results show that it has a low antioxidant activity. Additional studies will be needed to isolate and characterize specific compounds to further assess antioxidant activity.

  19. Evaluation of standard reagents for radial-immunodiffusion assays. In vitro control of rabies vaccines

    Directory of Open Access Journals (Sweden)

    MICELI Graciela S.

    2000-01-01

    Full Text Available The RID assay is one of the in vitro methods used for in-process control in the production of rabies vaccines for veterinary use. It has been shown to be very useful for determining antigen concentration in the final bulk product. The work presented in this paper, including the production and standardization of candidate standard reagents for use in the Radial Immunodiffusion Assay (RID was carried out at the Pan American Institute for Food Protection and Zoonoses (INPPAZ/PAHO/WHO. The study was completed with the cooperation of the Faculty of Veterinary Sciences, National University of La Plata (NULP, Argentina, where the validation of the proposed standards and the quality control of samples from 28 different batches of rabies vaccines produced with Pasteur strain rabies virus (PV in BHK cells were performed. The activity of the vaccines was determined by in vivo (NIH and in vitro (RIDassays. The results of the candidate reagents for the reagent standardization tests showed stability, sensitivity and reproducibility. The Relative Potency the 1.2 between the problem vaccines and the reference vaccine was estimated by variance and regression analysis. The results of our validation study show that the INPPAZ (PAHO/WHO is capable of producing and distributing the above-mentioned standard reagents, as well as of providing support for the incorporation of the RID technique (sensitive, rapid and inexpensive to the laboratories that manufacture rabies vaccines in Latin America and the Caribbean.

  20. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  1. Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Sjoerd Rijpkema

    2012-01-01

    Full Text Available Lethal toxin (LT of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8 is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb with toxin-neutralising (TN activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection.

  2. Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

    Directory of Open Access Journals (Sweden)

    Elena Serena

    Full Text Available INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin and functional properties. The spontaneous contraction frequency was (0.83±0.2 Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2O(2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2O(2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

  3. Development of an in vitro assay for teratogens: Final report, 1 July 1983 to 31 August 1986

    Energy Technology Data Exchange (ETDEWEB)

    Braun, A.G.

    1987-01-01

    The objectives of the work were to apply an in vitro teratogen assay system to samples of fossil fuel combustion products and to extend the assay system by incorporating metabolic activation. Potential teratogenicity was measured by the ability of a test compound to inhibit the attachment of tumor cells to lectin coated plastic surfaces.

  4. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    Science.gov (United States)

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

  5. In vitro genotoxicity of fipronil sister chromatid exchange, cytokinesis block micronucleus test, and comet assay.

    Science.gov (United States)

    Çelik, Ayla; Ekinci, Seda Yaprak; Güler, Gizem; Yildirim, Seda

    2014-03-01

    Fipronil (FP) is a phenylpyrazole pesticide developed by the transnational company Rhône-Poulenc Agro in 1987. Data on the genotoxicity and toxicity of FP are rather inadequate. In this study, we aimed to evaluate the potential genotoxic activity of FP using the single-cell microgel electrophoresis or comet assay, sister chromatid exchanges (SCEs), and micronuclei (MN) in human peripheral blood lymphocytes. In addition, the cytokinesis block proliferation index (CBPI) and proliferation index (PRI) were measured for cytotoxicity. In this study, three different doses of FP were used (0.7, 0.3, 0.1 μg/mL). Mitomycin C (2 μg/mL) and hydrogen peroxide were used as positive controls for SCE MN test systems, and comet assay, respectively. FP induced a statistically significant increase in the MN and SCE frequency and DNA damage in a dose-dependent manner in human peripheral blood lymphocytes (pcomet assay, we showed that all the doses of the FP induced DNA damage in human peripheral blood lymphocytes in vitro (p<0.05).

  6. A facile and green strategy for preparing newly-designed 3D graphene/gold film and its application in highly efficient electrochemical mercury assay.

    Science.gov (United States)

    Shi, Lei; Wang, Yan; Ding, Shiming; Chu, Zhenyu; Yin, Yu; Jiang, Danfeng; Luo, Jingyi; Jin, Wanqin

    2017-03-15

    In this work, we report a facile and green strategy for in situ and one step preparation of a novel 3D graphene/gold (G/Au) film. Triggering with unique driving force from hydrothermal growth, a 3D interlaced graphene framework with hierarchically porous structures was directly attached on a gold substrate pretreated with a self-assembled monolayer. Simultaneously, highly dispersive Au nanoparticles with tunable morphologies were anchored on the framework utilizing generated graphene as an endogenous reductant. Newly-designed 3D G/Au film possessed excellent properties of significantly large specific area, good electrical conductivity, high structure stability and substrate binding strength, etc. As a paradigm, an electrochemical Hg(2+) biosensor was constructed on 3D G/Au film, in which an exonuclease III-assisted target recycling was introduced. Impressively, an ultralow detection limit of 50 aM (S/N=3), a wide linear range from 0.1 fM to 0.1μM, a high selectivity and a good reliability for Hg(2+) assay in real water and serum samples were realized using prepared biosensor. It is highly envisioned that this work opens the door towards simply fabricating varying types of 3D graphene based hybrid films, and such G/Au film will have widespread applications in electroanalysis and electrocatalysis.

  7. Predictive endocrine testing in the 21st century using in vitro assays of estrogen receptor signaling responses.

    Science.gov (United States)

    Rotroff, Daniel M; Martin, Matt T; Dix, David J; Filer, Dayne L; Houck, Keith A; Knudsen, Thomas B; Sipes, Nisha S; Reif, David M; Xia, Menghang; Huang, Ruili; Judson, Richard S

    2014-01-01

    Thousands of environmental chemicals are subject to regulatory review for their potential to be endocrine disruptors (ED). In vitro high-throughput screening (HTS) assays have emerged as a potential tool for prioritizing chemicals for ED-related whole-animal tests. In this study, 1814 chemicals including pesticide active and inert ingredients, industrial chemicals, food additives, and pharmaceuticals were evaluated in a panel of 13 in vitro HTS assays. The panel of in vitro assays interrogated multiple end points related to estrogen receptor (ER) signaling, namely binding, agonist, antagonist, and cell growth responses. The results from the in vitro assays were used to create an ER Interaction Score. For 36 reference chemicals, an ER Interaction Score >0 showed 100% sensitivity and 87.5% specificity for classifying potential ER activity. The magnitude of the ER Interaction Score was significantly related to the potency classification of the reference chemicals (p vivo uterotrophic data, the ER Interaction Scores showed 91% sensitivity and 65% specificity. Overall, this study provides a novel method for combining in vitro concentration response data from multiple assays and, when applied to a large set of ER data, accurately predicted estrogenic responses and demonstrated its utility for chemical prioritization.

  8. In vitro cytotoxicity assays of solid lipid nanoparticles in epithelial and dermal cells

    Science.gov (United States)

    Ridolfi, D. M.; Marcato, P. D.; Machado, D.; Silva, R. A.; Justo, G. Z.; Durán, N.

    2011-07-01

    In recent years, the interest in nanostructured systems to drug delivery has increased because they offer several advantages over conventional dosage forms. Solid Lipid Nanoparticles (SLN) have been highlighted among these systems because they have advantages such as high physical stability, protection against drug degradation and ease of scale-up and manufacturing, without using organic solvent. The aim of this work was to evaluate the potential of SLN, by in vitro cytotoxicity assays, for dermal drug delivery. SLN of three different lipids were prepared by hot high pressure homogenization and the cytotoxicity was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test in mouse 3T3 fibroblasts and human HaCaT keratinocytes. SLN showed no cytotoxic potential suggesting a great potential for dermal application.

  9. In vitro cytotoxicity assays of solid lipid nanoparticles in epithelial and dermal cells

    Energy Technology Data Exchange (ETDEWEB)

    Ridolfi, D M; Marcato, P D; Duran, N [Instituto de Quimica, Universidade Estadual de Campinas (Brazil); Machado, D; Silva, R A [Instituto de Biologia, Universidade Estadual de Campinas (Brazil); Justo, G Z, E-mail: daniela_ridolfi@hotmail.com [Departamento de BioquImica, Universidade Federal de Sao Paulo (Brazil)

    2011-07-06

    In recent years, the interest in nanostructured systems to drug delivery has increased because they offer several advantages over conventional dosage forms. Solid Lipid Nanoparticles (SLN) have been highlighted among these systems because they have advantages such as high physical stability, protection against drug degradation and ease of scale-up and manufacturing, without using organic solvent. The aim of this work was to evaluate the potential of SLN, by in vitro cytotoxicity assays, for dermal drug delivery. SLN of three different lipids were prepared by hot high pressure homogenization and the cytotoxicity was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test in mouse 3T3 fibroblasts and human HaCaT keratinocytes. SLN showed no cytotoxic potential suggesting a great potential for dermal application.

  10. In vitro measurement of cell death with the annexin A5 affinity assay.

    Science.gov (United States)

    van Genderen, Hugo; Kenis, Heidi; Lux, Petra; Ungeth, Lisette; Maassen, Cecile; Deckers, Niko; Narula, Jagat; Hofstra, Leo; Reutelingsperger, Chris

    2006-01-01

    One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.

  11. Technical feasibility of 2D-3D coregistration for visualization of self-expandable microstents to facilitate coil embolization of broad-based intracranial aneurysms: an in vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Richter, Gregor [University of Erlangen-Nuernberg, Department of Neuroradiology, Erlangen (Germany); Kreisklinikum Siegen, Department of Radiology and Neuroradiology, Siegen (Germany); Pfister, Marcus [Siemens AG, Healthcare Sector, Forchheim (Germany); Struffert, Tobias; Engelhorn, Tobias; Doelken, Marc; Doerfler, Arnd [University of Erlangen-Nuernberg, Department of Neuroradiology, Erlangen (Germany); Spiegel, Martin; Hornegger, Joachim [University of Erlangen, Department of Informatics 5, Erlangen (Germany)

    2009-12-15

    The use of self-expandable microstents for treatment of broad-based intracranial aneurysms is widely spread. However, poor fluoroscopic visibility of the stents remains disadvantageous during the coiling procedure. Flat detector angiographic computed tomography (ACT) provides high resolution imaging of microstents even though integration of this imaging modality in the neurointerventional workflow has not been widely reported. An acrylic glass model was used to simulate the situation of a broad-based sidewall aneurysm. After insertion of a self-expandable microstent, ACT was performed. The resulting 3D dataset of the Microstent was subsequently projected into a conventional 2D fluoroscopic roadmap. This 3D visualization of the stent supported the coil embolization procedure of the in vitro aneurysm. In vitro 2D-3D coregistration with integration of 3D ACT data of a self-expandable microstent in a conventional 2D roadmap is feasible. Unsatisfying stent visibility constrains clinical cases with complex parent vessel anatomy and challenging aneurysm geometry; hence, this technique potentially may be useful in such cases. In our opinion, the clinical feasibility and utility of this new technique should be verified in a clinical aneurysm embolization study series using 2D-3D coregistration. (orig.)

  12. Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay

    Directory of Open Access Journals (Sweden)

    Zamurovic Natasa

    2004-03-01

    Full Text Available Abstract Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in α-MEM medium, supplemented with 10% of selected fetal calf serum, 1 μM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml, receptor activator of NFκB ligand (RANKL, 50 ng/ml, and transforming growth factor-β1 (TGF-β1, 5 ng/ml. Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase and activity (resorbed area and collagen fragments released from bone slices in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay.

  13. Sensitivity of two in vitro assays for evaluating plant activity against the infective stage of Haemonchus contortus strains.

    Science.gov (United States)

    Al-Rofaai, A; Rahman, W A; Abdulghani, Mahfoudh

    2013-02-01

    The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.

  14. Probing regenerative potential of Moringa oleifera aqueous extracts using In vitro cellular assays

    Directory of Open Access Journals (Sweden)

    Evangeline E Fernandes

    2016-01-01

    Full Text Available Background: Molecules stimulating regeneration and proliferation of cells are of significance in combating ailments caused due to tissue injury, inflammation, and degenerative disorders. Moringa oleifera is one of the most valued food plants having the profile of important nutrients and impressive range of medicinal uses. Objective: To evaluate the potential of M. oleifera aqueous leaf and flower extracts to promote the proliferation of cells and explore their effect on cancer cell lines for assessment of safety. Materials and Methods: Aqueous leaf and flower extracts of M. oleifera were investigated for effect on rat-derived primary fibroblast, mesenchymal stem cells (MSCs, and cancer cell lines using cell proliferation assay. They were also tested and compared for wound healing, angiogenesis, and hepatoprotective effect using in vitro assays. Results: Statistically significant increase in the proliferation of primary rat fibroblast, MSCs, and angiogenesis was observed after treatment with aqueous flower extract. The aqueous leaf extract determined a comparatively moderate increment in the proliferation of MSCs and angiogenesis. It however showed prominent cytotoxicity to cancer cell lines and a significant hepatoprotective effect. Conclusion: A very clear difference in response of the two extracts to different types of cells was detected in this study. The aqueous flower extract exhibited a higher potential to stimulate cell proliferation while not exerting the same effect on cancer cell lines. The leaf extract on the other hand, had a prominent antitumor and hepatoptotective effects.

  15. In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.

    Science.gov (United States)

    Shikanai, Toshiharu; Okuda, Kenji

    2011-01-01

    In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.

  16. Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, R.J. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Mestrado em Farmácia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mantovani, M.S.; Silva, A.F. da [Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR (Brazil); Pesarini, J.R. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mauro, M.O. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Doutorado em Biotecnologia e Biodiversidade - Rede Pró Centro-Oeste, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Ribeiro, L.R. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Programa de Pós-Graduação em Patologia, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP (Brazil)

    2014-03-28

    The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

  17. Experimental Study on Self-assembly of KLD-12 Peptide Hydrogel and 3-D Culture of MSC Encapsulated within Hydrogel In Vitro

    Institute of Scientific and Technical Information of China (English)

    Jianhua SUN; Qixin ZHENG

    2009-01-01

    o-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

  18. Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout, Oncorhynchus mykiss (Walbaum)

    DEFF Research Database (Denmark)

    Chettri, Jiwan Kumar; Holten-Andersen, Lars; Buchmann, Kurt

    Head kidney leukocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to a number of factors affecting the outcome of the assays. The present work...... describes the importance of temperature, cell concentration, immunostimulant, exposure time and immune-modulatory molecules on the respiratory burst activity of rainbow trout head kidney leukocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four...

  19. Antioxidant Activity of Seaweed Extracts: In Vitro Assays, Evaluation in 5 % Fish Oil-in-Water Emulsions and Characterization

    DEFF Research Database (Denmark)

    Farvin Habebullah, Sabeena; Jacobsen, Charlotte

    2015-01-01

    showed higher antioxidant activity both in in vitro assays and in 5 % oil-in-water emulsion in the presence or absence of iron. In spite of the higher phenolic content and very good antioxidant activity in some of the in vitro assays, the absolute ethanol extracts of both the species showed a pro......-oxidative tendency in 5 % fish oil-in-water emulsion in the presence or absence of iron. In order to investigate the reason for the higher antioxidant activity of 50 % ethanolic extracts of P. fucoides, these extracts were further fractionated into polyphenol-rich, protein-rich, polysaccharide-rich and low...

  20. THE EFFECTS OF OXIMES IN THE ASSAY OF ACETYLCHOLINESTERASE ACTIVITY IN LYSED ERYTHROCYTES IN VITRO

    Directory of Open Access Journals (Sweden)

    M. Abdollahi.

    1997-06-01

    Full Text Available Organophosphorus compounds are known to inhibit the esteratic site of acetylcholinesterase by phosphorylation. The phosphorylated esteratic site of acetylcholinesterase undergoes hydrolytic regeneration at a slow or negligible rate. Nucleophilic agents such as hydroxytamine, hydroxamic acids, and oximes reactivate the enzyme more erapidfy than does spontaneous hydrolysis. The red cell cholinesterose activity was assayed using dithio bis-2-nitrobenzoic acid (DTNB commonly known as Ellman's reagent. The principle of this assay method is the rate of hydrolysis of acetylthiocholine (substrate by a red celt suspension. Thiocholine that is produced, forms a yellow complex, when EUman's reagent (DTNB is used in the assay. This was tested in vitro in lysed erythrocyte samples of 35 healthy persons who had no known exposure to cholinesterose inhibitors, after the observation of immediate increase in absorption of light at 440 nm. All of data were statistically analyzed using one-way ANOVA and student t-test. A value of p<0.01 was considered. Results of this study show an increased absorbance in 440 nm, for pretreated samples with pratidoxime. This was observed by doses of (0.1, 0.5, 1,2 mmol, p<0.01. It was also a good dose dependent increase in absorbance at 440 nm for pralidoxime, (r=0.940, p<0.01. Also there is a significant increase in absorbance at 440 nm for samples pretreated by obidoxime at doses of (0.1, 0.5, 1,2 mmol. There is also a good correlation between absorbance at 440 nm and variou doses of obidoxime (r=0.946 , p<0.01. It is concluded that oximes can hydrofyzes the substrate, which then would be a source of error in determination of acetylcholinesterase activity and must be token into account.

  1. In vitro assay for HCV serine proteinase expressed in insect cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hou; Gui-Xin Du; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2003-01-01

    AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition.After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purifed by metalchelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS.Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5×107 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients′ sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity.CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

  2. Development of an in vitro binding assay for ecdysone receptor of mysid shrimp (Americamysis bahia)

    Energy Technology Data Exchange (ETDEWEB)

    Yokota, Hirofumi, E-mail: h-yokota@mail.kobe-c.ac.jp [Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505 (Japan); Eguchi, Sayaka [Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505 (Japan); Nakai, Makoto [Hita Laboratory, Chemicals Evaluation and Research Institute (CERI), 3-822, Ishii-machi, Hita-shi, Oita 877-0061 (Japan)

    2011-10-15

    Highlights: We successfully performed cDNA cloning of EcR and USP of mysid shrimp. We then expressed the ligand-binding domains of the corresponding receptor peptides. The translated peptides could bind to ecdysone agonists as heterodimers. These results indicate that they are functional hormone receptors of mysid shrimp. - Abstract: A global effort has been made to establish screening and testing methods that can identify the effects of endocrine-disrupting chemicals (EDCs) on invertebrates. The purpose of our study was to develop an in vitro receptor binding assay for ecdysone receptor (EcR) in mysid shrimp (Americamysis bahia). We cloned mysid shrimp EcR cDNA (2888 nucleotides) and ultraspiracle (USP) cDNA (2116 nucleotides), and determined that they encode predicted proteins of length 570 and 410 amino acids, respectively. The deduced amino acid sequences of these proteins shared 36-71% homology for EcR and 44-65% for USP with those of other arthropods. Phylogenetic analysis revealed that mysid shrimp EcR was classified into an independent cluster together with the EcRs of another mysid species, Neomysis integer and the cluster diverged early from those of the other taxonomic orders of crustaceans. We then expressed the ligand-binding domains (DEF regions) of mysid shrimp EcR (abEcRdef) and USP (abUSPdef) as glutathione S-transferase (GST)-fusion peptides in Escherichia coli. After purifying the fusion peptides by affinity chromatography and removing the GST labels, we subjected the peptides to a ligand-receptor binding assay. [{sup 3}H]-ponasterone A did not bind to abEcRdef or abUSPdef peptides alone but bound strongly to the abEcRdef/abUSPdef mixture with dissociation constant (K{sub d}) = 2.14 nM. Competitive binding assays showed that the IC{sub 50} values for ponasterone A, muristerone A, 20-hydroxyecdysone, and {alpha}-ecdysone were 1.2, 1.9, 35, and 1200 nM, respectively. In contrast, the IC{sub 50} values for two dibenzoylhydrazine ligands

  3. Soil quality in the Lomellina area using in vitro models and ecotoxicological assays

    Energy Technology Data Exchange (ETDEWEB)

    Baderna, Diego, E-mail: diego.baderna@marionegri.it [Laboratory of Environmental Chemistry and Toxicology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Via Giuseppe La Masa 19, 20156 Milan (Italy); Colombo, Andrea [Laboratory of Environmental Chemistry and Toxicology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Via Giuseppe La Masa 19, 20156 Milan (Italy); Romeo, Margherita [Department of Molecular Biochemistry and Pharmacology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Via Giuseppe La Masa 19, 20156 Milan (Italy); Cambria, Felice; Teoldi, Federico; Lodi, Marco [Laboratory of Environmental Chemistry and Toxicology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Via Giuseppe La Masa 19, 20156 Milan (Italy); Diomede, Luisa [Department of Molecular Biochemistry and Pharmacology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Via Giuseppe La Masa 19, 20156 Milan (Italy); Benfenati, Emilio [Laboratory of Environmental Chemistry and Toxicology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Via Giuseppe La Masa 19, 20156 Milan (Italy)

    2014-08-15

    Soil quality is traditionally evaluated by chemical characterization to determine levels of pollutants. Biological tools are now employed for soil monitoring since they can take account of the global biological effects induced by all xenobiotics. A combined monitoring of soils based on chemical analyses, human-related in vitro models and ecotoxicological assay was applied in the Lomellina, a semirural area of northern Italy. Chemical characterization indicated overall good quality of the soils, with low levels of toxic and carcinogenic pollutants such as heavy metals, PAHs, PCDD/Fs and PCBs. HepG2 cells were used as a model for the human liver and BALB/c 3T3 cells to evaluate carcinogenic potential. Cells were treated with soil extractable organic matter (EOM) and the MTS assay, DNA release and morphological transformation were selected as endpoints for toxicity and carcinogenicity. Soil EOMs induced dose-dependent inhibition of cell growth at low doses and cytotoxicity only at doses of 500 and 1000 mg soil equivalents/ml. Potential issues for human health can be hypothesized after ingestion of soil samples from some sites. No statistically significant inductions of foci were recorded after exposure to EOMs, indicating that the levels of the soil-extracted organic pollutants were too low to induce carcinogenesis in our experimental conditions. An acute phytotoxicity test and studies on Caenorhabditis elegans were used as ecotoxicological assays for plants and small invertebrates. No significant alerts for ecotoxicity were found. In this proposed case study, HepG2 cells detected differences in the toxicity of soil EOMs, indicating that this cell line could be appropriate to assess the potential harm caused by the ingestion of contaminated soil. Additional information on the carcinogenic potential of mixtures was provided by the cell transformation assay, strengthening the combined approach. - Highlights: • A combined approach for evaluation of soil quality is

  4. Comparison of the sensitivities of common in vitro and in vivo assays of estrogenic activity: application of chemical toxicity distributions.

    Science.gov (United States)

    Dobbins, Laura L; Brain, Richard A; Brooks, Bryan W

    2008-12-01

    A number of contaminants in municipal effluent discharges are estrogen agonists to fish. Whereas several in vitro and in vivo techniques have been developed to assess the estrogenic activity of these compounds or ambient environmental samples, previous comparisons of the relative sensitivities of these approaches remain inconclusive. We employed a probabilistic hazard assessment approach using chemical toxicity distributions (CTDs) to perform a novel evaluation of relative sensitivities of six common in vitro and in vivo assays. We predicted that there was an 8.3% (human breast ademocarcinoma cell line, MCF-7, assay), 6.3% (yeast estrogen screen assay), or 1.9% (fish hepatocyte vitellogenin, VTG, assay) probability of detecting a compound in aquatic systems that will elicit an estrogenic response at concentrations at or below 0.1 microg/L, suggesting that the MCF-7 assay was the most sensitive in vitro assay evaluated in this study. The probabilities of eliciting the estrogenic response of VTG induction at a concentration less than 0.1 microg/L in rainbow trout, fathead minnow, and Japanese medaka were determined at 29.9, 26.2, and 18.8%, respectively. Thus, rainbow trout VTG induction was the most sensitive in vivo assay assessed. Subsequently, CTDs may provide a useful technique for hazard assessment of chemical classes for which exposure data are limited and for chemicals with common toxicological mechanisms and modes of action.

  5. What level of estrogenic activity determined by in vitro assays in municipal waste waters can be considered as safe?

    Science.gov (United States)

    Jarošová, Barbora; Bláha, Luděk; Giesy, John P; Hilscherová, Klára

    2014-03-01

    In vitro assays are broadly used tools to evaluate the estrogenic activity in Waste Water Treatment Plant (WWTP) effluents and their receiving rivers. Since potencies of individual estrogens to induce in vitro and in vivo responses can differ it is not possible to directly evaluate risks based on in vitro measures of estrogenic activity. Estrone, 17beta-estradiol, 17alfa-ethinylestradiol and to some extent, estriol have been shown to be responsible for the majority of in vitro estrogenic activity of municipal WWTP effluents. Therefore, in the present study safe concentrations of Estrogenic Equivalents (EEQs-SSE) in municipal WWTP effluents were derived based on simplified assumption that the steroid estrogens are responsible for all estrogenicity determined with particular in vitro assays. EEQs-SSEs were derived using the bioassay and testing protocol-specific in vitro potencies of steroid estrogens, in vivo predicted no effect concentration (PNECs) of these compounds, and their relative contributions to the overall estrogenicity detected in municipal WWTP effluents. EEQs-SSEs for 15 individual bioassays varied from 0.1 to 0.4ng EEQ/L. The EEQs-SSEs are supposed to be increased by use of location-specific dilution factors of WWTP effluents entering receiving rivers. They are applicable to municipal wastewater and rivers close to their discharges, but not to industrial waste waters.

  6. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

    Science.gov (United States)

    Rochelle, Paul A; Marshall, Marilyn M; Mead, Jan R; Johnson, Anne M; Korich, Dick G; Rosen, Jeffrey S; De Leon, Ricardo

    2002-08-01

    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all

  7. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity.

    Science.gov (United States)

    Legler, Juliette; Zeinstra, Laura M; Schuitemaker, Femke; Lanser, Peter H; Bogerd, Jan; Brouwer, Abraham; Vethaak, A Dick; De Voogt, Pim; Murk, Albertinka J; Van der Burg, Bart

    2002-10-15

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.

  8. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    Science.gov (United States)

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  9. Pulmonary toxicity of nanomaterials: a critical comparison of published in vitro assays and in vivo inhalation or instillation studies.

    Science.gov (United States)

    Landsiedel, Robert; Sauer, Ursula G; Ma-Hock, Lan; Schnekenburger, Jürgen; Wiemann, Martin

    2014-11-01

    To date, guidance on how to incorporate in vitro assays into integrated approaches for testing and assessment of nanomaterials is unavailable. In addressing this shortage, this review compares data from in vitro studies to results from in vivo inhalation or intratracheal instillation studies. Globular nanomaterials (ion-shedding silver and zinc oxide, poorly soluble titanium dioxide and cerium dioxide, and partly soluble amorphous silicon dioxide) and nanomaterials with higher aspect ratios (multiwalled carbon nanotubes) were assessed focusing on the Organisation for Economic Co-Operation and Development (OECD) reference nanomaterials for these substances. If in vitro assays are performed with dosages that reflect effective in vivo dosages, the mechanisms of nanomaterial toxicity can be assessed. In early tiers of integrated approaches for testing and assessment, knowledge on mechanisms of toxicity serves to group nanomaterials thereby reducing the need for animal testing.

  10. Development of an in vitro assay for teratogens. Progress report, 1 July 1983-31 August 1985

    Energy Technology Data Exchange (ETDEWEB)

    Braun, A.G.

    1985-01-01

    An in vitro teratogen assay system was applied to samples of fossil fuel combustion products. Potential teratogenicity was measured by the ability of a test compound to inhibit the attachment of tumor cells to lectin coated plastic surfaces. 13 refs., 3 tabs. (DT)

  11. 78 FR 24425 - Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug...

    Science.gov (United States)

    2013-04-25

    ... HUMAN SERVICES Food and Drug Administration Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug Administration Staff; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food and Drug Administration (FDA) is announcing...

  12. Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays.

    Directory of Open Access Journals (Sweden)

    Samantha Forster

    Full Text Available Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.

  13. Efficacy Study of Broken Rice Maltodextrin in In Vitro Wound Healing Assay

    Directory of Open Access Journals (Sweden)

    Zahiah Mohamed Amin

    2015-01-01

    Full Text Available Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE range (DE 10–14 of maltodextrins at a concentration of 10% obtained from RB were found to be able to heal the wounds significantly faster (p<0.01 than maltodextrin with higher DE ranges (DE 15–19 and DE 20–24 and concentrations of 5% and 20%. The findings from both BrdU and MTT assay further confirmed its wound healing properties as the NIH 3T3 fibroblast wounded cells were able to proliferate without causing cytotoxic effect when wounded cell was treated with maltodextrin. All these findings indicated that the RB maltodextrin could perform better than the commercial maltodextrin at the same DE range. This study showed that RB maltodextrins had better functionality properties than other maltodextrin sources and played a beneficial role in wound healing application.

  14. Efficacy Study of Broken Rice Maltodextrin in In Vitro Wound Healing Assay.

    Science.gov (United States)

    Mohamed Amin, Zahiah; Koh, Soo Peng; Yeap, Swee Keong; Abdul Hamid, Nur Syazwani; Tan, Chin Ping; Long, Kamariah

    2015-01-01

    Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB) sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE) range (DE 10-14) of maltodextrins at a concentration of 10% obtained from RB were found to be able to heal the wounds significantly faster (p maltodextrin with higher DE ranges (DE 15-19 and DE 20-24) and concentrations of 5% and 20%. The findings from both BrdU and MTT assay further confirmed its wound healing properties as the NIH 3T3 fibroblast wounded cells were able to proliferate without causing cytotoxic effect when wounded cell was treated with maltodextrin. All these findings indicated that the RB maltodextrin could perform better than the commercial maltodextrin at the same DE range. This study showed that RB maltodextrins had better functionality properties than other maltodextrin sources and played a beneficial role in wound healing application.

  15. Antioxidant and Antiinflammatory Compounds in Nutmeg (Myristicafragrans) Pericarp as Determined by in vitro Assays.

    Science.gov (United States)

    Zhang, Chuan-rui; Jayashre, Ettannil; Kumar, Paramasivam Suresh; Nair, Muraleedharan G

    2015-08-01

    Nutmeg, Myristicafragrans, is known for its culinary and medicinal values. The nutmeg pericarp, abundant during the production of the seed, is also used in food and beverage preparations. In this study, the pericarp of M. fragrans was evaluated for its bioactive components using in vitro antioxidant and antiinflammatory assays. The hexane, ethyl acetate and methanolic extracts inhibited lipid peroxidation (LPO) by 82.5, 70.1 and 73.2%, and cyclooxygenase enzymes COX-1 by 44, 44 and 42% and COX-2 by 47, 41 and 36%, respectively, at 100 microg/mL. The bioassay-guided purifications of extracts yielded 20 compounds belonged to neolignans (0.13%), phenylpropanoids (0.28%), phenolic aldehyde (0.35%), triterpenoids (0.06%), triglycerides (0.20%), sugars (10.2%) and steroids (0.49%). Pure isolates 1-5 inhibited LPO by 70-99% and 3-12 inhibited COX-1 and -2 enzymes by 37-49%. This is the first report on the bioassay-guided characterization of constituents in nutmeg pericarp. Our results support the medicinal claims of nutmeg pericarp.

  16. Comparison of genotoxicity of textile dyestuffs in Salmonella mutagenicity assay, in vitro micronucleus assay, and single cell gel/comet assay.

    Science.gov (United States)

    Wollin, Klaus-M; Gorlitz, Bernd-D

    2004-01-01

    The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.

  17. The Use of MTT Assay, In Vitro and Ex Vivo, to Predict the Radiosensitivity of Colorectal Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Kim, Mi Sook; Kang, Chang Mo; Shin, Hye Kyung; Choi, Chul Won; Seo, Young Seok; Ji, Young Hoon [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kim, Jong Il [Seoul Women' s University College of Medicine, Seoul (Korea, Republic of)

    2008-09-15

    The measurement of radiosensitivity of individuals is useful in radiation therapy. Unfortunately, the measurement of radiation survival using a clonogenic assay, which is the established standard, can be difficult and time consuming. The aim of this study is to compare radiosensitivity results obtained from the MTT and clonogenic assays, and to evaluate whether the MTT assay can be used on clinical specimens. Materials and Methods: HCT-8, LoVo, CT-26, and WiDr were the colon cancer cell lines used for this study. The clonogenic assay was performed to obtain the cell survival curves and surviving fractions at a dose of 2 Gy (SF2) as the standard technique for radiosensitivity. Also, the MTT assay was performed for each of the cell lines (in vitro). To simulate clinical specimens, the cell lines were inoculated into nude mice, removed when the tumors reached 1 cm in diameter, and chopped. Next, the tumors were subjected to the same process involved with the MTT assay in vitro. The inhibition rates (IR) of 10 Gy or 20 Gy of irradiation for in vitro and ex vivo were calculated based on the optical density of the MTT assay, respectively. Results: According to SF2 and the cell survival curve, the HCT-8 and WiDr cell lines were more resistant to radiation than LoVo and CT-26 (p<0.05). The IR was measured by in vitro. The MTT assay IR was 17.3%, 21%, 30% and 56.5% for the WiDr, HCT-8, LoVo and CT-26 cell lines, respectively. In addition, the IR measured ex vivo by the MTT assay was 23.5%, 26%, 38% and 53% in the HCT-8, WiDr, LoVo and CT-26 tumors, respectively. Conclusion: The radiosensitivity measured by the MTT assay was correlated with the measures obtained from the clonogenic assay. This result highlights the possibility that the MTT assay could be used in clinical specimens for individual radiosensitivity assays.

  18. Genotoxicity Assessment of Chlorotrifluoroethylene Tetramer Acid using a Battery of In Vitro and In Vivo/In Vitro Assays

    Science.gov (United States)

    1990-12-01

    thy:’ f’yl)- phthalate: An industrial plastieizer induces hypolipidemia and 3nhncl:ci hepatic catalase and carnitine acetyltransferase activities in...assay, the top agar was melted and supplemented with a sterile solution that contained 0.5 mM L-histidine and 0.5 mM D-biotin (10% v/v). Minimal...Bottom Agar: The bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 0.2% (w/v) glucose. Nutrient Broth: The

  19. Synthesis and in-vitro evaluation of 2-amino-4-arylthiazole as inhibitor of 3D polymerase against foot-and-mouth disease (FMD).

    Science.gov (United States)

    Jeong, Kwi-Wan; Lee, Jung-Hun; Park, Sun-Mi; Choi, Joo-Hyung; Jeong, Dae-Youn; Choi, Dong-Hwa; Nam, Yeonju; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Su-Mi; Ku, Jin-Mo

    2015-09-18

    Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of livestock caused by a highly variable RNA virus, foot-and-mouth disease virus (FMDV). One of the targets to suppress expansion of and to control FMD is 3D polymerase (FMDV 3Dpol). In this study, 2-amino-4-arylthiazole derivatives were synthesized and evaluated for their inhibitory activity against FMDV 3Dpol. Among them, compound 20i exhibited the most potent functional inhibition (IC50 = 0.39 μM) of FMDV 3D polymerase and compound 24a (EC50 = 13.09 μM) showed more potent antiviral activity than ribavirin (EC50 = 1367 μM) and T1105 (EC50 = 347 μM) with IBRS-2 cells infected by the FMDV O/SKR/2010 strain.

  20. Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays.

    Science.gov (United States)

    Tsujita-Inoue, Kyoko; Hirota, Morihiko; Ashikaga, Takao; Atobe, Tomomi; Kouzuki, Hirokazu; Aiba, Setsuya

    2014-06-01

    The sensitizing potential of chemicals is usually identified and characterized using in vivo methods such as the murine local lymph node assay (LLNA). Due to regulatory constraints and ethical concerns, alternatives to animal testing are needed to predict skin sensitization potential of chemicals. For this purpose, combined evaluation using multiple in vitro and in silico parameters that reflect different aspects of the sensitization process seems promising. We previously reported that LLNA thresholds could be well predicted by using an artificial neural network (ANN) model, designated iSENS ver.1 (integrating in vitro sensitization tests version 1), to analyze data obtained from two in vitro tests: the human Cell Line Activation Test (h-CLAT) and the SH test. Here, we present a more advanced ANN model, iSENS ver.2, which additionally utilizes the results of antioxidant response element (ARE) assay and the octanol-water partition coefficient (LogP, reflecting lipid solubility and skin absorption). We found a good correlation between predicted LLNA thresholds calculated by iSENS ver.2 and reported values. The predictive performance of iSENS ver.2 was superior to that of iSENS ver.1. We conclude that ANN analysis of data from multiple in vitro assays is a useful approach for risk assessment of chemicals for skin sensitization.

  1. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    DEFF Research Database (Denmark)

    Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon;

    2015-01-01

    . Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food...... with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry...

  2. Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles: synthesis, characterization, and in vitro toxicity assay

    Directory of Open Access Journals (Sweden)

    Wang S

    2011-12-01

    Full Text Available Shige Wang1, Shihui Wen2, Mingwu Shen2, Rui Guo2, Xueyan Cao2, Jianhua Wang3, Xiangyang Shi1,2,41State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, 2College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 3Department of Biochemistry and Molecular Cell Biology, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China; 4Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, Funchal, PortugalBackground: We report on aminopropyltriethoxysilane (APTS-mediated surface modification of nanohydroxyapatite with different surface functional groups for potential biomedical applications. In this study, nanohydroxyapatite covalently linked with APTS (n-HA-APTS was reacted with acetic anhydride or succinic anhydride to produce neutralized (n-HA-APTS.Ac or negatively charged (n-HA-APTS.SAH nanohydroxyapatite, respectively. Nanohydroxyapatite formed with amine, acetyl, and carboxyl groups was extensively characterized using Fourier transform infrared spectroscopy, transmission electron microscopy, 1H nuclear magnetic resonance spectroscopy, X-ray diffraction, inductively coupled plasma-atomic emission spectroscopy, and zeta potential measurements.Results: In vitro 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric assay revealed that the slight toxicity of the amine-functionalized n-HA-APTS could be eliminated by post-functionalization of APTS amines to form acetyl and carboxyl groups. Blood compatibility assessment demonstrated that the negligible hemolytic activity of the pristine nanohydroxyapatite particles did not appreciably change after APTS-mediated surface functionalization.Conclusion: APTS-mediated functionalization of nanohydroxyapatite with different surface groups may be useful for further functionalization of nanohydroxyapatite with biologically active materials, thereby providing possibilities for a broad range of

  3. In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase.

    Science.gov (United States)

    Clark, Daniel N; Jones, Scott A; Hu, Jianming

    2017-01-01

    The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.

  4. Probing Regenerative Potential of Moringa oleifera Aqueous Extracts Using In vitro Cellular Assays

    Science.gov (United States)

    Fernandes, Evangeline E.; Pulwale, Anubha V.; Patil, Gauri A.; Moghe, Alpana S.

    2016-01-01

    Background: Molecules stimulating regeneration and proliferation of cells are of significance in combating ailments caused due to tissue injury, inflammation, and degenerative disorders. Moringa oleifera is one of the most valued food plants having the profile of important nutrients and impressive range of medicinal uses. Objective: To evaluate the potential of M. oleifera aqueous leaf and flower extracts to promote the proliferation of cells and explore their effect on cancer cell lines for assessment of safety. Materials and Methods: Aqueous leaf and flower extracts of M. oleifera were investigated for effect on rat-derived primary fibroblast, mesenchymal stem cells (MSCs), and cancer cell lines using cell proliferation assay. They were also tested and compared for wound healing, angiogenesis, and hepatoprotective effect using in vitro assays. Results: Statistically significant increase in the proliferation of primary rat fibroblast, MSCs, and angiogenesis was observed after treatment with aqueous flower extract. The aqueous leaf extract determined a comparatively moderate increment in the proliferation of MSCs and angiogenesis. It however showed prominent cytotoxicity to cancer cell lines and a significant hepatoprotective effect. Conclusion: A very clear difference in response of the two extracts to different types of cells was detected in this study. The aqueous flower extract exhibited a higher potential to stimulate cell proliferation while not exerting the same effect on cancer cell lines. The leaf extract on the other hand, had a prominent antitumor and hepatoptotective effects. SUMMARY Moringa oleifera flower extract showed significant ability to promote proliferation of rat fibroblast and mesenchymal stem cells. The extract also had prominent angiogenic and hepatoprotective effects.The extract did not influence proliferation of cancer cell lines indicating its safety for human consumption and use in pharmaceuticals.The Moringa oleifera leaf extract

  5. In Vitro Bioactivity in ToxCast Assays for Fruit and Vegetable Juices (TDS)

    Science.gov (United States)

    The ToxCast and Tox21 programs have generated in vitro screening data for over 1000 chemicals to aid in hazard identification and setting chemical testing priorities. These data, together with in vitro pharmacokinetic data, are used to infer possible toxic responses and external ...

  6. Comparison between 3D volumetric rendering and multiplanar slices on the reliability of linear measurements on CBCT images: an in vitro study

    Directory of Open Access Journals (Sweden)

    Thais Maria Freire FERNANDES

    2015-02-01

    Full Text Available OBJECTIVE: The purpose of this study was to determine the accuracy and reliability of two methods of measurements of linear distances (multiplanar 2D and tridimensional reconstruction 3D obtained from cone-beam computed tomography (CBCT with different voxel sizes. MATERIAL AND METHODS: Ten dry human mandibles were scanned at voxel sizes of 0.2 and 0.4 mm. Craniometric anatomical landmarks were identified twice by two independent operators on the multiplanar reconstructed and on volume rendering images that were generated by the software Dolphin®. Subsequently, physical measurements were performed using a digital caliper. Analysis of variance (ANOVA, intraclass correlation coefficient (ICC and Bland-Altman were used for evaluating accuracy and reliability (p<0.05. RESULTS: Excellent intraobserver reliability and good to high precision interobserver reliability values were found for linear measurements from CBCT 3D and multiplanar images. Measurements performed on multiplanar reconstructed images were more accurate than measurements in volume rendering compared with the gold standard. No statistically significant difference was found between voxel protocols, independently of the measurement method. CONCLUSIONS: Linear measurements on multiplanar images of 0.2 and 0.4 voxel are reliable and accurate when compared with direct caliper measurements. Caution should be taken in the volume rendering measurements, because the measurements were reliable, but not accurate for all variables. An increased voxel resolution did not result in greater accuracy of mandible measurements and would potentially provide increased patient radiation exposure.

  7. Relationship between the results of in vitro receptor binding assay to human estrogen receptor alpha and in vivo uterotrophic assay: comparative study with 65 selected chemicals.

    Science.gov (United States)

    Akahori, Yumi; Nakai, Makoto; Yamasaki, Kanji; Takatsuki, Mineo; Shimohigashi, Yasuyuki; Ohtaki, Masahiro

    2008-02-01

    For screening chemicals possessing endocrine disrupting potencies, the uterotrophic assay has been placed in a higher level in the OECD testing framework than the ER binding assay to detect ER-mediated activities. However, there are no studies that can demonstrate a clear relationship between these assays. In order to clarify the relationship between the in vitro ER binding and in vivo uterotrophic assays and to determine meaningful binding potency from the ER binding assay, we compared the results from these assays for 65 chemicals spanning a variety of chemicals classes. Under the quantitative comparison between logRBAs (relative binding affinities) and logLEDs (lowest effective doses), the log RBA was well correlated with both logLEDs of estrogenic and anti-estrogenic compounds at r(2)=0.67 (n=28) and 0.79 (n=23), respectively. The RBA of 0.00233% was found to be the lowest ER binding potency to elicit estrogenic or anti-estrogenic activities in the uterotrophic assay, accordingly this value is considered as the detection limit of estrogenic or anti-estrogenic activities in the uterotrophic assay. The usage of this value as cutoff provided the best concordance rate (82%). These findings are useful in a tiered approach for identifying chemicals that have potential to induce ER-mediated effects in vivo.

  8. Liposomal-benzocaine gel formulation: correlation between in vitro assays and in vivo topical anesthesia in volunteers.

    Science.gov (United States)

    Franz-Montan, Michelle; Cereda, Cintia Maria Saia; Gaspari, Adele; da Silva, Camila Morais Gonçalves; de Araújo, Daniele Ribeiro; Padula, Cristina; Santi, Patrizia; Narvaes, Eliene; Novaes, Pedro Duarte; Groppo, Francisco Carlos; de Paula, Eneida

    2013-03-01

    The aim of the present study was to characterize a liposome-based benzocaine (BZC) formulation designed for topical use on the oral mucosa and to evaluate its in vitro retention and permeation using the Franz-type diffusion cells through pig esophagus mucosa. To predict the effectiveness of new designed formulations during preclinical studies, a correlation between in vitro assays and in vivo efficacy was performed. Liposomal BZC was characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and morphology. Liposomal BZC (BL10) was incorporated into gel formulation and its performances were compared to plain BZC gel (B10) and the commercially available BZC gel (B20). BL10 and B10 presented higher flux and retention on pig esophagus mucosa with a shorter lag time, when compared to B20. BZC flux was strongly correlated with in vivo anesthetic efficacy, but not with topical anesthesia duration. The retention studies did not correlate with any of the in vivo efficacy parameters. Thus, in vitro permeation study can be useful to predict anesthetic efficacy during preclinical tests, because a correlation between flux and anesthetic efficacy was observed. Therefore, in vitro assays, followed by in vivo efficacy, are necessary to confirm anesthetic performance.

  9. Characterization of the in vitro propagation of epileptiform electrophysiological activity in organotypic hippocampal slice cultures coupled to 3D microelectrode arrays

    DEFF Research Database (Denmark)

    Pisciotta, Marzia; Morgavi, Giovanna; Jahnsen, Henrik

    2010-01-01

    Dynamic aspects of the propagation of epileptiform activity have so far received little attention. With the aim of providing new insights about the spatial features of the propagation of epileptic seizures in the nervous system, we studied in vitro the initiation and propagation of traveling epil......AnimalsAnimals, NewbornConvulsants/pharmacologyElectric Stimulation/methodsElectrophysiological Phenomena/drug effectsElectrophysiological Phenomena/physiology*Evoked Potentials/drug effectsEvoked Potentials/physiology*Hippocampus/anatomy & histologyHippocampus/drug effects......Hippocampus/physiology*Microelectrodes*Organ Culture TechniquesPicrotoxin/pharmacologyRatsRats, WistarReaction Time/drug effectsReaction Time/physiologyTime FactorsSubstancesConvulsantsPicrotoxin LinkOut - more resourcesFull Text SourcesElsevier ScienceEBSCOOhioLINK Electronic Journal CenterSwets Information ServicesMolecular Biology Databases...

  10. An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria.

    Science.gov (United States)

    Piersma, Sytse J; Leenaars, Marlies P P A M; Guzylack-Piriou, Laurence; Summerfield, Artur; Hendriksen, Coenraad F M; McCullough, Ken C

    2006-04-12

    Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.

  11. Assessment of Chemical Skin-Sensitizing Potency by an In Vitro Assay Based on Human Dendritic Cells

    OpenAIRE

    Lambrechts, Nathalie; Vanheel, Hanne; Nelissen, Inge; Witters, Hilda; VAN DEN HEUVEL Rosette; Van Tendeloo, Viggo; Schoeters, Greet; HOOYBERGHS, Jef

    2010-01-01

    The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemica...

  12. The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar Blue (resazurin) assay.

    Science.gov (United States)

    Clothier, Richard; Starzec, Gemma; Pradel, Lionel; Baxter, Victoria; Jones, Melanie; Cox, Helen; Noble, Linda

    2002-01-01

    A range of cosmetics formulations with human patch-test data were supplied in a coded form, for the examination of the use of a combined in vitro permeability barrier assay and cell viability assay to generate, and then test, a prediction model for assessing potential human skin patch-test results. The target cells employed were of the Madin Darby canine kidney cell line, which establish tight junctions and adherens junctions able to restrict the permeability of sodium fluorescein across the barrier of the confluent cell layer. The prediction model for interpretation of the in vitro assay results included initial effects and the recovery profile over 72 hours. A set of the hand-wash, surfactant-based formulations were tested to generate the prediction model, and then six others were evaluated. The model system was then also evaluated with powder laundry detergents and hand moisturisers: their effects were predicted by the in vitro test system. The model was under-predictive for two of the ten hand-wash products. It was over-predictive for the moisturisers, (two out of six) and eight out of ten laundry powders. However, the in vivo human patch test data were variable, and 19 of the 26 predictions were correct or within 0.5 on the 0-4.0 scale used for the in vivo scores, i.e. within the same variable range reported for the repeat-test hand-wash in vivo data.

  13. Relationship of two in vitro assays in protein efficiency ratio determination on selected agricultural by-products

    Directory of Open Access Journals (Sweden)

    Foster B. Wardlaw

    2006-03-01

    Full Text Available Utilization of agricultural by-products as food and feed has been of increasing interest. Protein is a crucial nutrient obtained from those sources. However, in vivo method (a rat study for protein efficiency ratio (PER determination is time consuming and expensive. C-PER and DC-PER are in vitro assays, which involve mathematical calculations using amino acid information from the sample. These two methods have been proven suitable for predicting protein quality of various samples with high correlation to the in vivo assay. Rapid prediction with less cost is the advantage of these methods. Theoretically, C-PER and DC-PER of each sample should have high correlation as they are computed from the same amino acid information. However, the efficiency of the methods is probably based on a range of certain information, especially protein digestibility. This study was conducted to demonstrate one of the limitations of the in vitro assays as shown in selected agricultural by-products. Three categories of selected agricultural by-products were feed-grade egg product (FGEP; 8 samples, distillers' dried grain (DDG; 4 samples, and defatted wheat germ (DWG; 8 samples. Protein contents, amino acid profiles, in vitro protein digestibility, C-PER and DC-PER were determined. Proteins in FGEP categories were significantly higher (P<0.05 than DWG and DDG, respectively. Both C-PER and DC-PER of all samples showed high correlation except in DWG-424. The low correlation in DWG-424 may be due to its low protein digestibility. It may also indicate the limitation of C-PER assay. The assay therefore may not be suitable for samples with low protein digestibility.

  14. Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

    Science.gov (United States)

    Luchansky, Matthew Sam

    In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample. In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of

  15. Comet assay measures of DNA damage as biomarkers of irinotecan response in colorectal cancer in vitro and in vivo.

    Science.gov (United States)

    Wood, Joanna P; Smith, Andrew J O; Bowman, Karen J; Thomas, Anne L; Jones, George D D

    2015-09-01

    The use of irinotecan to treat metastatic colorectal cancer (CRC) is limited by unpredictable response and variable toxicity; however, no reliable clinical biomarkers are available. Here, we report a study to ascertain whether irinotecan-induced DNA damage measures are suitable/superior biomarkers of irinotecan effect. CRC-cell lines (HCT-116 and HT-29) were treated in vitro with irinotecan and peripheral blood lymphocytes (PBL) were isolated from patients before and after receiving irinotecan-based chemotherapy. Levels of in vitro-, in vivo-, and ex vivo-induced DNA damage were measured using the Comet assay; correlations between damage levels with in vitro cell survival and follow-up clinical data were investigated. Irinotecan-induced DNA damage was detectable in both CRC cell-lines in vitro, with higher levels of immediate and residual damage noted for the more sensitive HT-29 cells. DNA damage was not detected in vivo, but was measurable in PBLs upon mitogenic stimulation prior to ex vivo SN-38 treatment. Results showed that, following corrections for experimental error, those patients whose PBLs demonstrated higher levels of DNA damage following 10 h of SN-38 exposure ex vivo had significantly longer times to progression than those with lower damage levels (median 291 vs. 173 days, P = 0.014). To conclude, higher levels of irinotecan-induced initial and residual damage correlated with greater cell kill in vitro and a better clinical response. Consequently, DNA damage measures may represent superior biomarkers of irinotecan effect compared to the more often-studied genetic assays for differential drug metabolism.

  16. Repair capacity of adult rat glial progenitor cells determined by an in vitro clonogenic assay after in vitro or in vivo fractionated irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Maazen, R.W.M. van der; Kleiboer, B.J.; Verhagen, I.; Kogel, A.J. van der (Nijmegen Univ. (Netherlands). Inst. of Radiotherapy)

    1993-05-01

    Demyelination is one condition identified as a late response of the central nervous system (CNS) to irradiation. In the present study the repair capacity of glial stem cells was investigated and compared with the repair capacity of the CNS in vivo using functional endpoints. For this purpose, glial stem cells, derived from the adult rat optic nerve, were subjected to fractionated irradiation in vivo and in vitro and their survival was quantified with an in vitro clonogenic assay. The data were analysed by three different methods all based on the LQ-model (single dose survival curve; '[beta][sub RR]', 'F[sub e]-plot'). The resulting value of the [beta]-parameter of adult glial stem cells is consistent with values obtained for functional endpoints after irradiation of the CNS in vivo. The [alpha]/[beta]-ratio (4.9-7.3 Gy) of adult glial stem cells, however, is higher than the [alpha]/[beta]-ratio ([approx] 2 Gy) obtained for CNS in vivo and is closer to that of an acute responding tissue. (Author).

  17. Gaining acceptance for the use of in vitro toxicity assays and QIVIVE in regulatory risk assessment.

    Science.gov (United States)

    Meek, M E Bette; Lipscomb, John C

    2015-06-05

    Testing strategies are anticipated to increasingly rely on in vitro data as a basis to characterize early steps or key events in toxicity at relevant dose levels in human tissues. Such strategies require quantitative in vitro to in vivo extrapolation to characterize dose-response as a basis for comparison with exposure to estimate risk. Current experience in the incorporation of mechanistic and in vitro data in risk assessment is considered here in the context of identified principles to increase the potential for timely acceptance of more progressive and tailored testing strategies by the regulatory community. These principles are outlined as transitioning in a familiar context, tiering to acquire experience and increase confidence, contextual knowledge transfer to facilitate interpretation and communication, coordination and development of expertise and continuing challenge. A proposed pragmatic tiered data driven framework which includes increasing reliance on in vitro data and quantitative in vitro to in vivo extrapolation is considered in the context of these principles. Based on this analysis, possible additional steps that might facilitate timely evolution and potentially, uptake are identified.

  18. 3D models of epithelial-mesenchymal transition in breast cancer metastasis: high-throughput screening assay development, validation, and pilot screen.

    Science.gov (United States)

    Li, Qun; Chen, Chaoyu; Kapadia, Amit; Zhou, Qiong; Harper, Mary Kay; Schaack, Jerome; LaBarbera, Daniel V

    2011-02-01

    Despite advancements in therapies developed for the treatment of cancer, patient prognosis and mortality rates have improved minimally, and metastasis remains the primary cause of cancer mortality worldwide. An underlying mechanism promoting metastasis in many types of cancer is epithelial-mesenchymal transition (EMT). Here the authors report a novel 3D model of EMT and metastatic breast cancer suitable for high-throughput screening (HTS) drug discovery. The primary assay incorporates the expression of the prognostic biomarker vimentin, as a luciferase reporter of EMT, in basil-like/triple-negative MDA-MB-231 breast carcinoma spheroids. Using this model, the authors developed a number of known antitumor agents as control modulators of EMT. U0126, PKC412, PF2341066, dasatinib, and axitinib downregulated vimentin expression by 70% to 90% as compared to untreated spheroids. Counterassays were developed to measure spheroid viability and the invasive potential of MDA-MB-231 spheroids after small-molecule treatment and used to confirm hits from primary screening. Finally, the authors conducted a pilot screen to validate this model for HTS using a purified library of marine secondary metabolites. From 230 compounds screened, they obtained a Z' score of 0.64, indicative of an excellent assay, and confirmed 4 hits, including isonaamidine B, papuamine, mycalolide E, and jaspamide. This HTS model demonstrates the potential to identify small-molecule modulators of EMT that could be used to discover novel antimetastatic agents for the treatment of cancer.

  19. Assay of tolnaftate in human skin samples after in vitro penetration studies using high performance liquid chromatography.

    Science.gov (United States)

    Kezutyte, Toma; Kornysova, Olga; Maruska, Audrius; Briedis, Vitalis

    2010-01-01

    Abstract: Tolnaftate, an antifungal of thiocarbamate class, is used topically in 1% formulations. Its penetration into skin layers is a prerequisite for tolnaftate action against dermatophytes. The aim of this work was to optimize and validate a simple, rapid, accurate and reproducible procedure for tolnaftate assay in human skin samples and to apply this procedure for in vitro tolnaftate penetration studies. High performance liquid chromatography (HPLC) method with UV detection was used to validate tolnaftate assay for linearity, specificity, accuracy, precision, limit of quantitation, limit of detection, drug extraction recovery and stability in skin extracts. In vitro tolnaftate penetration studies were carried out using flow-through diffusion cells, mounted with human skin. Epidermis and dermis, separated by heat-separation method, were extracted using ultrasonication in methanol. Linear range of the analytical procedure was within 0.6-100 pg/mL. The assay was specific, accurate (within-day and between-day recovery values were 98.2-104.2% and 98.7-101.4%, respectively) and precise (within-day and between-day imprecision was = 3.8%). Mean extraction recoveries of tolnaftate from epidermis and dermis were satisfactory and reaching 90%. In vitro skin penetration studies revealed that after application of 1% (w/w) tolnaftate solution in polyethylene glycol 400 for 24 hours, the mean amount of tolnaftate penetrating into the epidermis and dermis was 2.60 +/- 0.28 microg/cm2 and 0.92 +/- 0.12 microg/cm2, respectively. A validated reliable HPLC method could be recommended for biopharmaceutical evaluation of tolnaftate preparations and studies of pharmacokinetics in human skin after in vitro penetration studies.

  20. Assessment of chemical skin-sensitizing potency by an in vitro assay based on human dendritic cells.

    Science.gov (United States)

    Lambrechts, Nathalie; Vanheel, Hanne; Nelissen, Inge; Witters, Hilda; Van Den Heuvel, Rosette; Van Tendeloo, Viggo; Schoeters, Greet; Hooyberghs, Jef

    2010-07-01

    The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or-none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers.

  1. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  2. Quantitative predictivity of the transformation in vitro assay compared with the Ames test. [Hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Parodi, S.; Taningher, M.; Russo, P.; Pala, M.; Vecchio, D.; Fassina, G.; Santi, L.

    For 59 chemical compounds, homogeneous data on transformation in vitro, mutagenicity in the Ames test, and carcinogenicity was reviewed. The potency in inducing transformation in vitro in hamster fibroblast cells was compared with the carcinogenic potency and a modest correlation coefficient was found between the two parameters. For these same 59 compounds it was also possible to compare mutagenic potency in the Ames test with carcinogenic potency. The correlation level was very similar. The predictivity of transformation in vitro increased significantly when only compounds for which some kind of dose-response relationship was available were utilized. This result stresses the importance of the quantitative aspect of the response in predictivity studies. The present study is compared with previous studies on the quantitative predictivity of different short-term tests. The work is not definitive, but gives an idea of the possible type of approach to the problem of comparing quantitative predictivities.

  3. Antioxidant activites of Xanthosoma sagittifolium Schott using various in vitro assay models

    Institute of Scientific and Technical Information of China (English)

    Antony Nishanthini; Veerabahu Ramasamy Mohan

    2012-01-01

    Objective: To evaluate the total phenolic, flavonoid contents and in vitro antioxidant activity of methanol extract of Xanthosoma sagittifolium corm. Methods: Total phenolic content was estimated using the Folin Ciocalteu method. The flavonoid content was determined using aluminium chloride. In vitro antioxidant activities were evaluated by studying DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, ABTS radical cation scavenging activity and reducing power capacity were determined using standard procedure. Results:Xanthosoma sagittifolium corm exhibited 0.32g100g-1 total phenolic; 0.26g100g-1 flavonoid and better scavenging activity of DPPH (78.22±0.56%), hydroxyl radical (69.11±0.21%), superoxide radical (83.27±0.08%) and ABTS radical cations(76.11±0.07%).Conclusions: The present studies confirm the methanol extracts have potential in vitro antioxidant activity.

  4. Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Abuzar Elnager

    2015-01-01

    Full Text Available Background. Caffeic acid phenethyl ester (CAPE has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM. After 3 hours, D-dimer (DD levels and WB clot weights were measured for each concentration. Thromboelastography (TEG parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM. The 50% effective dose (ED50 of CAPE (based on DD was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

  5. Physicomechanical, In Vitro and In Vivo Performance of 3D Printed Doped Tricalcium Phosphate Scaffolds for Bone Tissue Engineering and Drug Delivery

    Science.gov (United States)

    Tarafder, Solaiman

    Although tricalcium phosphate (TCP) is widely used in bone tissue engineering, the strength degradation kinetics is not well controlled. This study focuses on the underlying mechanism of strength degradation kinetics by incorporating trace elements in TCP. The objective of this research is to modify the mechanical properties of TCP to achieve the desired degradation rate for the specific need, and improve the in vivo bioactivity for early wound healing by incorporating trace elements such as strontium (Sr2+), magnesium (Mg2+) and silicon (Si4+) as dopants. The hypothesis of this research is that the presence of different trace elements in TCP will influence its phase stability, microstructure, mechanical strength, and both in vitro and in vivo bioactivity. Direct three dimensional printing (3DP) was used to fabricate designed interconnected macroporous pure and doped TCP scaffolds. Microwave sintering as opposed to conventional sintering was also used for better densification and higher mechanical strength. A maximum compressive strength of 10.95 +/- 1.28 MPa and 12.01 +/- 1.56 MPa were achieved for pure and Sr2+-Mg2+ doped TCP scaffolds with 500 microm designed pores (˜400 microm after sintering) sintered in microwave furnace, respectively. Substitution of Mg2+ and Sr2+ into calcium (Ca2+) sites of TCP crystal lattice contributed to phase stability and controlled gradual degradation. On the other hand, Si4+ substitution into phosphorous (P5+) sites destabilized the crystal structure and accelerated degradation of TCP. Interconnected macroporous beta-TCP scaffolds facilitated in vivo guided bone tissue regeneration through infiltration of cells and extracellular matrix into the designed pores. Presence of Sr2+, Mg2+ and Si4+ into beta-TCP induced increased in vivo early bone formation and better bone remodeling through increased extracellular matrix production such as, collagen and osteocalcin, when tested in rat and rabbit distal femur model. The presence of Si4

  6. PENGHAMBATAN CAJUPUTS CANDY TERHADAP VIABILITAS KHAMIR Candida albicans SECARA IN VITRO [Inhibition of Cajuputs Candy Toward the Viability of Candida albicans by using In Vitro Assay

    Directory of Open Access Journals (Sweden)

    C. Hanny Wijaya1*

    2014-12-01

    Full Text Available The utilization of cajuput essential oil as a flavor in candy may produce a physiological active added value. Some compounds of cajuput plant (Melaleuca cajuputi L have been reported for their anti-microbial activities. Candida albicans is a normal commensal organism in human mouth. However, it may become virulent and responsible for oral diseases known as oral candidiasis. This study aimed to determine the effect of cajuput and peppermint oil in cajuputs candy in inhibiting the C. albicans biofilms formation by using in vitro biofilm assay and viability assay. Furthermore, the influence of concentration of cajuput oil on the anti-microbial activities had been analyzed. All the tested concentration of cajuput oil in cajuputs candy was effective to inhibit the viability of C. albicans. The provision of flavor components of cajuput and peppermint oil could produce synergistic effects compared to a single flavor component. The addition of cajuput oil at 0.6% was able to inhibit the viability of C. albicans. The activities of the cajuput oil showed positive correlation to the concentration. The variable of plus and minus 0.1% addition of the cajuput oil concentration, however, produced no significant difference to inhibit the growth of C. albicans in biofilm. Sensory test, hedonic test, was conducted to evaluate the flavor, aroma, and overall attributes, resulting in no significant difference between 0.6 to 0.8% additions of cajuput oil upon the sensory acceptance.

  7. A rapid colorimetric assay for the quantitation of the viability of free-living larvae of nematodes in vitro.

    Science.gov (United States)

    James, Catherine E; Davey, Mary W

    2007-09-01

    With increasing drug resistance in gastrointestinal parasites, identification of new anthelmintics is essential. The non-parasitic nematode Caenorhabditis elegans is used extensively as a model to identify drug targets and potential novel anthelmintics because it can be readily cultured in vitro. Traditionally, the assessment of worm viability has relied on labour-intensive developmental and behavioral assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) colorimetric assay uses metabolic activity as a marker of viability in mammalian cell culture systems and has been applied for use with filarial nematodes. In the present study, this assay has been optimized and validated to rapidly assess the viability of C. elegans after drug treatment. Living, but not dead, C. elegans take up MTT and reduce it to the blue formazan, providing visual, qualitative, and quantitative assessment of viability. MTT at a concentration of 5 mg/ml with 3 h incubation was optimal for detecting changes in viability with drug treatment. We have applied this assay to quantitate the effects of ivermectin and short-chain alcohols on the viability of C. elegans. This assay is also applicable to first-stage larvae of the parasitic nematode Haemonchus contortus. The advantage of this assay is the rapid quantitation in screening drugs to identify potential anthelmintics.

  8. Identification of Chemical Vascular Disruptors During Development Using An Integrative Predictive Toxicity Model and Zebrafish and in Vitro Functional Angiogenesis Assays.

    Science.gov (United States)

    Identification of chemical vascular disruptors during development using an integrative predictive toxicity model and zebrafish and in vitro functional angiogenesis assays Chemically-induced vascular toxicity during embryonic development can result in a wide range of adverse pre...

  9. Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single—Chain protease

    Institute of Scientific and Technical Information of China (English)

    Rong-BinGuan; Yi-GangTong; Hai-TaoWang; Gui-XinDu; Li-HuaHou

    2002-01-01

    AIM:To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine prtease based on the recombinant protease and substrate,and to evaluate its feasibility in screening the enzyme inhibitors.

  10. Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

    Science.gov (United States)

    We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

  11. Unsaturated compounds induce up-regulation of CD86 on dendritic cells in the in vitro sensitization assay LCSA.

    Science.gov (United States)

    Frohwein, Thomas Armin; Sonnenburg, Anna; Zuberbier, Torsten; Stahlmann, Ralf; Schreiner, Maximilian

    2016-04-01

    Unsaturated compounds are known to cause false-positive reactions in the local lymph node assay (LLNA) but not in the guinea pig maximization test. We have tested a panel of substances (succinic acid, undecylenic acid, 1-octyn-3-ol, fumaric acid, maleic acid, linoleic acid, oleic acid, alpha-linolenic acid, squalene, and arachidonic acid) in the loose-fit coculture-based sensitization assay (LCSA) to evaluate whether unspecific activation of dendritic cells is a confounder for sensitization testing in vitro. Eight out of 10 tested substances caused significant up-regulation of CD86 on dendritic cells cocultured with keratinocytes and would have been classified as sensitizers; only succinic acid was tested negative, and squalene had to be excluded from data analysis due to poor solubility in cell culture medium. Based on human data, only undecylenic acid can be considered a true sensitizer. The true sensitizing potential of 1-octyn-3-ol is uncertain. Fumaric acid and its isomer maleic acid are not known as sensitizers, but their esters are contact allergens. A group of 18- to 20-carbon chain unsaturated fatty acids (linoleic acid, oleic acid, alpha-linolenic acid, and arachidonic acid) elicited the strongest reaction in vitro. This is possibly due to the formation of pro-inflammatory lipid mediators in the cell culture causing nonspecific activation of dendritic cells. In conclusion, both the LLNA and the LCSA seem to provide false-positive results for unsaturated fatty acids. The inclusion of T cells in dendritic cell-based in vitro sensitization assays may help to eliminate false-positive results due to nonspecific dendritic cell activation. This would lead to more accurate prediction of sensitizers, which is paramount for consumer health protection and occupational safety.

  12. In vitro androgenetic cultures of Hyoscyamus niger L., H. albus L. and alkaloid content assay

    Directory of Open Access Journals (Sweden)

    Maria Wesołwska

    2014-01-01

    Full Text Available In vitro cultures of Hyoscyamus niger L. and H. albus L. anthers were initiated which resulted in obtaining androgenectic plants and callus cultures. The leaves of these pants and the callus cultures were subjected to analysis (TLC, GC for the presence of alkaloids, derivatives of tropane. In the studied material, alkaloids of different qualitative and quantitative composition from that of ground-grown plants were found.

  13. Efficacy Study of Broken Rice Maltodextrin in In Vitro Wound Healing Assay

    OpenAIRE

    Zahiah Mohamed Amin; Soo Peng Koh; Swee Keong Yeap; Nur Syazwani Abdul Hamid; Chin Ping Tan; Kamariah Long

    2015-01-01

    Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB) sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE) range (DE 10–14) of maltodextrins at ...

  14. Activity of sertraline against Cryptococcus neoformans: in vitro and in vivo assays.

    Science.gov (United States)

    Treviño-Rangel, Rogelio de J; Villanueva-Lozano, Hiram; Hernández-Rodríguez, Pedro; Martínez-Reséndez, Michel F; García-Juárez, Jaime; Rodríguez-Rocha, Humberto; González, Gloria M

    2016-03-01

    Cryptococcus neoformans infection is an important cause of meningitis in HIV/AIDS endemic regions. Antifungals for its management include amphotericin B, flucytosine, and fluconazole. Recently, treatment of this mycosis with sertraline has been studied with variable clinical outcomes. The aim of the study was to assess the in vitro antifungal effect of sertraline against clinical isolates of Cryptococcus spp. as well as its in vivo activity in a murine model of cryptococcal meningoencephalitis. The in vitro susceptibility to fluconazole, amphotericin B, voriconazole and sertraline of 153 Cryptococcus spp. strains were evaluated according to CLSI procedures. Fungal tissue burden, serum antigenaemia and histopathology, together with the therapeutic efficacy of amphotericin B (3 mg/kg), fluconazole (15 mg/kg), and sertraline (3, 10, and 15 mg/kg) were evaluated in mice intracranially inoculated with one isolate of Cryptococcus neoformans. All strains were susceptible to the antifungals studied and exhibited growth inhibition with sertraline at clinically relevant concentrations. Sertraline at a dose of 15 mg/kg reduced the fungal burden in the brain and spleen with an efficacy comparable to that of fluconazole. In conclusion, sertraline exhibited an excellent in vitro-in vivo anti-cryptococcal activity, representing a possible new alternative for the clinical management of meningeal cryptococcosis.

  15. Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.

    Science.gov (United States)

    Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro

    2016-12-20

    Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP ). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

  16. Endocrine activity of persistent organic pollutants accumulated in human silicone implants — Dosing in vitro assays by partitioning from silicone

    DEFF Research Database (Denmark)

    Gilbert, Dorothea; Mayer, Philipp; Pedersen, Mikael;

    2015-01-01

    Persistent organic pollutants (POPs) accumulated in human tissues may pose a risk for human health by interfering with the endocrine system. This study establishes a new link between actual human internal POP levels and the endocrine active dose in vitro, applying partitioning-controlled dosing...... from silicone to the H295R steroidogenesis assay: (1) Measured concentrations of POPs in silicone breast implants were taken from a recent study and silicone disks were loaded according to these measurements. (2) Silicone disks were transferred into H295R cell culture plates in order to control...

  17. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Science.gov (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  18. An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

    Directory of Open Access Journals (Sweden)

    Vermeulen Michiel

    2004-01-01

    Full Text Available Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

  19. Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals.

    Science.gov (United States)

    Körner, W; Hanf, V; Schuller, W; Bartsch, H; Zwirner, M; Hagenmaier, H

    1998-01-01

    The E-Screen assay serves as an in vitro tool for the detection of estrogenic activity of chemicals and extracts of environmental samples. Based on the induction of proliferation in human estrogen receptor-positive MCF-7 breast cancer cells we could substantially simplify the assay. As one important step of validation we applied the modified assay for testing nine known xenoestrogens. We could confirm the results of other groups assuring the reproducibility of the E-Screen assay. The results provide evidence that the E-Screen assay is suitable for determination of estradiol equivalency factors (EEFs) for environmental estrogens to rank their estrogenic potency relative to the natural estrogen 17 beta-estradiol. Further, we used the optimized proliferation test to screen nine halogenated phenolic compounds for their possible estrogenic potency. Three widely applied chemicals expressed a weak receptor-mediated estrogenic activity: the flame retardant Tetrabromo-Bisphenol-A, the disinfectant 4-chloro-3-methylphenol, and the herbicide educt 4-chloro-2-methylphenol. Their estrogenic potencies were five to six orders of magnitude lower than that of 17 beta-estradiol.

  20. Assays for the in vitro establishment of Swietenia macrophylla and Cedrela odorata

    Directory of Open Access Journals (Sweden)

    Julián Pérez Flores

    2012-08-01

    Full Text Available Normal 0 21 false false false ES-CO X-NONE X-NONE MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} Título en español: Ensayos para el establecimiento in vitro de Swietenia macrophylla y Cedrela odorata Abstract: Recalcitrance and contamination in Mahogany (Swietenia macrophylla King and Spanish cedar (Cedrela odorata L. stem tissues are the main causes of its ineffective in vitro propagation. The objectives of this research were: a to evaluate sodium hypochlorite (NaOCl and plant preservative mixture (PPM® as surface disinfectants and/or added to the culture medium for the in vitro establishment of nodal explants taken from 10-year-old Mahogany and Spanish cedar plants, and b to evaluate the in vitro response of such explants treated with N6-benzylaminopurine (BAP (0, 2.2, 4.4, 8.8, 17.7 μM, silver nitrate (AgNO3 (0, 3 mg l-1, activated charcoal (0, 1 g l-1 and vented caps. All the experiments were arranged in a completely randomized design. The NaOCl at 15%, for 20 min, as a surface sterilization or PPM® at 2 ml l-1  into the culture medium, were the best treatments to reduce contamination for both species. For Mahogany explants, BAP at 17.7 μM resulted in higher percentages of bud breaks than Spanish cedar (64% and 25%, respectively. Leaves on elongated shoots dropped off by 20 days after

  1. p53 Promoter-based Reporter Gene in vitro Assays for Quick Assessment of Agents with Genotoxic Potential

    Institute of Scientific and Technical Information of China (English)

    Huaixing LI; Ke SHI; Ruiwen CHEN; Yan HE; Dan WU; Shuhan SUN

    2007-01-01

    The p53 promoter-based green fluorescent protein (GFP) and luciferase reporter gene assays have been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or 5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cells exposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of the cells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reporter gene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assessment or screening of agents with genotoxic potential.

  2. Suitability of the in vitro Caco-2 assay to predict the oral absorption of aromatic amine hair dyes.

    Science.gov (United States)

    Obringer, Cindy; Manwaring, John; Goebel, Carsten; Hewitt, Nicola J; Rothe, Helga

    2016-04-01

    Oral absorption is a key element for safety assessments of cosmetic ingredients, including hair dye molecules. Reliable in vitro methods are needed since the European Union has banned the use of animals for the testing of cosmetic ingredients. Caco-2 cells were used to measure the intestinal permeability characteristics (Papp) of 14 aromatic amine hair dye molecules with varying chemical structures, and the data were compared with historical in vivo oral absorption rat data. The majority of the hair dyes exhibited Papp values that indicated good in vivo absorption. The moderate to high oral absorption findings, i.e. ≥60%, were confirmed in in vivo rat studies. Moreover, the compound with a very low Papp value (APB: 3-((9,10-dihydro-9,10-dioxo-4-(methylamino)-1-anthracenyl)amino)-N,N-dimethyl-N-propyl-1-propanaminium) was poorly absorbed in vivo as well (5% of the dose). This data set suggests that the Caco-2 cell model is a reliable in vitro tool for the determination of the intestinal absorption of aromatic amines with diverse chemical structures. When used in combination with other in vitro assays for metabolism and skin penetration, the Caco-2 model can contribute to the prediction and mechanistic interpretation of the absorption, metabolism and elimination properties of cosmetic ingredients without the use of animals.

  3. Development and Use of Anucleate Bacterial Cells to Assay the in vitro Activity of Pollutants

    Science.gov (United States)

    1982-12-07

    1052-1064. 2. Reeve, J. N., R. A. Weigand, and J. W. Greenawalt. 1971. Presence of bacteriophace-like inhz.bitory particles in Escherichi a coli . J...H3; 1am193;LG3 infected E. coli cells DNase digestion. Bring to 0.3M NIH4Cl S Slow speed centrifugation High speed centrifugation + Pass supernatant...7 ’I -.&. - - ~ ,-..4 R IP-PAC;E: 3 S-1lethxonine Labullcd P. coli B3 Extract ,44r~~.a~~c2d 3a3b 3P. 3d 0 40- FIGURE 3 la = Uninfected E. coli

  4. Investigations of plant-derived products with the in vitro comet assay

    Directory of Open Access Journals (Sweden)

    Luc Verschaeve

    2015-08-01

    It is impossible to perform a wide range of tests for screening purposes and often only the Ames assay is performed, which is insufficient. Furthermore, this test is most probably not the best choice when plant extracts need to be tested, because they often contain high amounts of histidine and have antibacterial properties. We have participated in many screening programs of medicinal plants and used different genotoxicity tests (mainly Ames assay, Vitotox test, micronucleus test and comet assay. Or results revealed that a combination of the Vitotox test and comet assay provides sufficiently reliable data with respect to genotoxicity as well as antigenotoxicity. This holds true for the testing of (medicinal plant extracts but also other plant derived products, for example those aimed at identifying novel TB chemotherapeutic drugs. The investigation of smoke and smoke compounds as enhancers of seed germination and smoke treated plants provides another example in which the comet assay proved to be valuable in the assessment of potential adverse health effects resulting from such treatment.

  5. Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro.

    Science.gov (United States)

    Li, Jie; Zhang, Donglan; Ward, Kathleen M; Prendergast, George C; Ayene, Iraimoudi S

    2012-06-01

    Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

  6. Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis.

    Science.gov (United States)

    Ihara, Masaru; Kitamura, Tomokazu; Kumar, Vimal; Park, Chang-Beom; Ihara, Mariko O; Lee, Sang-Jung; Yamashita, Naoyuki; Miyagawa, Shinichi; Iguchi, Taisen; Okamoto, Seiichiro; Suzuki, Yutaka; Tanaka, Hiroaki

    2015-05-19

    The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ERα reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the following: estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ERα reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17β-estradiol, estriol, and 17α-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ERα reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ERα reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ERα reporter gene assay.

  7. Genotoxic investigation of a thiazolidinedione PPARγ agonist using the in vitro micronucleus test and the in vivo homozygotization assay.

    Science.gov (United States)

    Morais, Janicélle Fernandes; Sant'Anna, Juliane Rocha de; Pereira, Tais Susane; Franco, Claudinéia Conationi da Silva; Mathias, Paulo Cezar de Freitas; de Castro-Prado, Marialba Avezum Alves

    2016-07-01

    Pioglitazone (PTZ) is an oral antidiabetic agent whose anti-cancer properties have been described recently. Since PTZ increases the production of reactive oxygen species in mammalian cells, the aim of current study was to evaluate the cytotoxic, mutagenic and recombinogenic effects of PTZ using respectively the in vitro mitotic index assay and the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans, which detects the loss of heterozygosity due to somatic recombination. Although the lowest PTZ concentrations (4-36 μM) did not show any significant rise in the micronucleus production, the higher PTZ concentration (108 μM) produced a statistically higher number of micronuclei than the negative control and significantly altered the cell-proliferation kinetics, demonstrating the mutagenic and antiproliferative effects of PTZ, respectively. The recombinogenic activity of PTZ, demonstrated here for the first time, was observed at the highest tested concentration (400 μM) through the homozygotization index rates significantly different from the negative control. Taken together, our results show that PTZ is genotoxic at a concentration higher than the therapeutic plasma concentration. This PTZ genotoxicity may be a potential benefit to its previously described antitumour activity.

  8. Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

    Science.gov (United States)

    Chen, Qiyu; Jia, Ai; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

    2016-02-01

    Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants.

  9. In vitro activity assays for MYST histone acetyltransferases and adaptation for high-throughput inhibitor screening

    Science.gov (United States)

    McCullough, Cheryl E.; Marmorstein, Ronen

    2016-01-01

    Lysine acetylation is a post-translational modification that is carried out by acetyltransferases. The MYST proteins form the largest and most diverse family of acetyltransferases, which regulate gene expression, DNA repair, and cell cycle homeostasis, among other activities, by acetylating both histone and non-histone proteins. This chapter will describe methods for the preparation and biochemical characterization of MYST family acetyltransferases, including protocols for the preparation of recombinant protein, enzyme assays for measuring steady state parameters and binding assays to measure cofactor and inhibitor binding. We also provide details on adapting these assays for high throughput screening for small molecule MYST inhibitors. This chapter seeks to prepare researchers for some hurdles that they may encounter when studying the MYST proteins so that there may be better opportunity to plan appropriate controls and obtain high quality data. PMID:27372752

  10. Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals

    DEFF Research Database (Denmark)

    Körner, Wolfgang; Vinggaard, Anne; Terouanne, B.;

    2004-01-01

    , the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50......) values ranging from 1.1 x 10(-7) M to 4.7 x 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values...

  11. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro.

    Science.gov (United States)

    Paczesny, Sophie; Shi, Honhgzhen; Saito, Hiroaki; Mannoni, Patrice; Fay, Joseph; Banchereau, Jacques; Palucka, A Karolina

    2005-01-01

    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  12. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

    NARCIS (Netherlands)

    Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

    2002-01-01

    Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-

  13. In vitro Assays of Staphylococcus epidermidis Characteristics and Outcome in an Endocarditis Model

    Directory of Open Access Journals (Sweden)

    Betty Herndon

    1993-01-01

    Full Text Available Objective: Staphylococcus epidermidis adherence to indwelling polymers is important in prosthetic valve endocarditis. Earlier studies have related streptococcal endocarditis to isolates with high levels of cell-associated hexoses. The objective of the present study was to determine if a relationship exists between an S epidermidis isolate assay score and production/severity of experimental endocarditis.

  14. Colony formation in agar: in vitro assay for haemopoietic stem cells

    NARCIS (Netherlands)

    Dicke, K.A.; Platenburg, M.G.C.; Bekkum, D.W. van

    1971-01-01

    Using a method in which embryo fibroblasts were used as feeder layers, the colony forming capacity in agar of a variety of mouse haemopoietic suspensions was compared with their CFUs content. A striking parallelism between the results of the two assays was found. In addition, under certain condition

  15. Quantifying the effect of experimental design choices for in vitro scratch assays.

    Science.gov (United States)

    Johnston, Stuart T; Ross, Joshua V; Binder, Benjamin J; Sean McElwain, D L; Haridas, Parvathi; Simpson, Matthew J

    2016-07-07

    Scratch assays are often used to investigate potential drug treatments for chronic wounds and cancer. Interpreting these experiments with a mathematical model allows us to estimate the cell diffusivity, D, and the cell proliferation rate, λ. However, the influence of the experimental design on the estimates of D and λ is unclear. Here we apply an approximate Bayesian computation (ABC) parameter inference method, which produces a posterior distribution of D and λ, to new sets of synthetic data, generated from an idealised mathematical model, and experimental data for a non-adhesive mesenchymal population of fibroblast cells. The posterior distribution allows us to quantify the amount of information obtained about D and λ. We investigate two types of scratch assay, as well as varying the number and timing of the experimental observations captured. Our results show that a scrape assay, involving one cell front, provides more precise estimates of D and λ, and is more computationally efficient to interpret than a wound assay, with two opposingly directed cell fronts. We find that recording two observations, after making the initial observation, is sufficient to estimate D and λ, and that the final observation time should correspond to the time taken for the cell front to move across the field of view. These results provide guidance for estimating D and λ, while simultaneously minimising the time and cost associated with performing and interpreting the experiment.

  16. Statistical procedures for the design and analysis of in vitro mutagenesis assays

    Energy Technology Data Exchange (ETDEWEB)

    Kaldor, J.

    1983-03-01

    In previous statistical treatments of a certain class of mutagenesis assays, stochastic models of mutation and cell growth have not been utilized. In this paper, we review the assumptions under which these models are derived, introduce some further assumptions, and propose ways to estimate and test hypotheses regarding the parameters of the models from assay data. It is shown via simulation and exact calculation that if the models are valid, the proposed statistical procedures provide very accurate Type I error rates for hypothesis tests, and coverage probabilities for confidence intervals. The cases of a linear dose response relationship for mutagenesis, and a comparison of a set of treated cell cultures with a set of control cultures are treated in detail. Approximate power functions for hypothesis tests of interest are then derived, and these are also shown to be satisfactorily close to the true power functions. The approximations are used to develop guidelines for planning aspects of a mutagenesis assay, including the number, spacing and range of dose levels employed. Examples of applications of the procedures are provided, and the paper concludes with a discussion of future statistical work which may be carried out in the area of mutagenesis assays. 38 references, 8 figures, 7 tables.

  17. Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay?

    Science.gov (United States)

    Enciso, José Manuel; Sánchez, Oscar; López de Cerain, Adela; Azqueta, Amaya

    2015-01-01

    The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.

  18. Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

    NARCIS (Netherlands)

    Westerink, W.M.; Schirris, T.J.J.; Horbach, G.J.; Schoonen, W.G.

    2011-01-01

    In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these

  19. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Tomitaka, Asahi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)], E-mail: d07gd158@ynu.ac.jp; Hirukawa, Atsuo; Yamada, Tsutomu [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Morishita, Shin [Department of Mechanical Engineering and Materials Science, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Takemura, Yasushi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)

    2009-05-15

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe{sub 3}O{sub 4} (20-30 nm), ZnFe{sub 2}O{sub 4} (15-30 nm) and NiFe{sub 2}O{sub 4} (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe{sub 3}O{sub 4} sample was found to be biocompatible on HeLa cells. While ZnFe{sub 2}O{sub 4} and NiFe{sub 2}O{sub 4} were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 {mu}g/ml nanoparticles.

  20. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    Science.gov (United States)

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-03

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

  1. High content screening as high quality assay for biological evaluation of photosensitizers in vitro.

    Directory of Open Access Journals (Sweden)

    Gisela M F Vaz

    Full Text Available A novel single step assay approach to screen a library of photdynamic therapy (PDT compounds was developed. Utilizing high content analysis (HCA technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4. The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT. The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells. A high correlation was found between the high content screening (HCS and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR study for photosensitizer libraries.

  2. Correlation of In Vivo and In Vitro Assay Results for Assessment of Free Radical Scavenging Activity of Green Tea Nutraceuticals.

    Science.gov (United States)

    Abd-ElSalam, Heba-Alla H; Al-Ghobashy, Medhat A; Al-Shorbagy, Muhammad; Nassar, Noha; Zaazaa, Hala E; Ibrahim, Mohamed A

    2016-07-01

    Green tea (GT)-derived catechins; epigallocatechin gallate (EGCG) in particular are commonly used nutraceuticals for their free-radical scavenging activity (FRSA). The influence of photodegradation on the protective power of GT nutracenticals against oxidative stress was thoroughly explored. Photodegradation of GT extracts was carried out and monitored using orthogonal stability-indicating testing protocol; in vitro and in vivo assays. Total polyphenol content (TPC) and FRSA were determined spectrophotometrically while EGCG was selectively monitored using SPE-HPLC. In vivo assessment of photodegraded samples was investigated via measuring a number of biomarkers for hepatic oxidative stress and apoptosis (caspase-3, inducible nitric oxide synthase, nitric oxide, mitogen-activated protein kinase, glutathione, thiobarbituric acid reactive substances, nuclear factor kappa beta, and nuclear factor erythroid 2-related factor) as well as liver damage (alanine transaminase and aspartate transaminase) in serum of rats previously subjected to oxidative stress. Results showed complete degradation of EGCG in photodegraded green tea samples with no correlation with either TPC or FRSA. On the other hand, in vivo assay results revealed not only loss of activity but formation of harmful pro-oxidants. Photostability was found crucial for the protective effect of GT extract against lead acetate insult. Results confirmed that careful design of quality control protocols requires correlation of chemical assays to bioassays to verify efficacy, stability, and most importantly safety of nutraceuticals.

  3. Development and characterization of a Pseudomonas aeruginosa in vitro coupled transcription-translation assay system for evaluation of translation inhibitors

    Science.gov (United States)

    Fyfe, Corey; Sutcliffe, Joyce A.; Grossman, Trudy H.

    2013-01-01

    Bacterial transcription and translation have proven to be effective targets for broad-spectrum antimicrobial therapies owing to the critical role they play in bacterial propagation and the overall conservation of the associated machinery involved. Escherichia coli is the most common source of S30 extract used in bacterial in vitro coupled transcription-translation assays, however, transcription-translation assays in other important pathogens including Staphylococcus aureus and Streptococcus pneumoniae have been described (Murray et al., 2001; Dandliker et al., 2003). Pseudomonas aeruginosa is an important and difficult-to-treat Gram-negative pathogen. In a drug discovery program, to de-risk any potential species specificity of novel inhibitors, we developed and optimized a robust method for the preparation of S30 extract from P. aeruginosa strain PAO1. Further, a P. aeruginosa transcription-translation assay using a firefly luciferase reporter plasmid was validated and compared to an E. coli S30-based system using a wide range of antibiotics encompassing multiple classes of translation inhibitors. Results showed a similar ranking of the activities of known inhibitors, illustrative of the high degree of conservation between the transcription-translation pathways in both organisms. PMID:22677604

  4. New hosts of Myrothecium spp. in Brazil and a preliminary in vitro assay of fungicides

    Directory of Open Access Journals (Sweden)

    A.M. Quezado Duval

    2010-03-01

    Full Text Available Myrothecium roridum and M. verrucaria are two plant pathogenic species causing foliar spots in a large number of cultivated plants. This paper aims to study the causal agents of foliar spots in vegetable crops (sweet pepper, tomato and cucumber, ornamental plants (Spathiphyllum wallisii, Solidago canadensis, Anthurium andreanum, Dieffenbachia amoena and a solanaceous weed plant (Nicandra physaloides. Most of the isolates were identified as M. roridum; only the isolate 'Myr-02' from S. canadensis was identified as M. verrucaria. All the isolates were pathogenic to their original plant hosts and also to some other plants. Some fungicides were tested in vitro against an isolate of M. roridum and the mycelial growth recorded after seven days. Fungicides with quartenary ammonium, tebuconazole and copper were highly effective in inhibiting the mycelial growth of M. roridum. This paper confirms the first record of M. roridum causing leaf spots in sweet pepper, tomato, Spathiphyllum, Anthurium, Dieffenbachia and N. physaloides in Brazil. We also report M. roridum as causal agent of cucumber fruit rot and M. verrucaria as a pathogen of tango plants.

  5. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay.

    Science.gov (United States)

    Miura, Kazutoyo; Zhou, Hong; Diouf, Ababacar; Moretz, Samuel E; Fay, Michael P; Miller, Louis H; Martin, Laura B; Pierce, Mark A; Ellis, Ruth D; Mullen, Gregory E D; Long, Carole A

    2009-07-01

    Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP1(42)) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP1(42)-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP1(42) IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab(50)) were compared for rabbit and human antibodies. The Ab(50)s of rabbit and human anti-MSP1(42) IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab(50) data against FVO parasites also demonstrated significant differences. We further investigated the Ab(50)s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab(50) varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP1(42)-based vaccine development efforts in preclinical and clinical trials.

  6. Genotoxic and Cytotoxic Safety Evaluation of Papain (Carica papaya L. Using In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Claudia R. da Silva

    2010-01-01

    This work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced-H2O2 oxidative stress in Escherichia coli strains. Cytotoxicity assay, Growth inhibition test, WP2-Mutoxitest and Plasmid-DNA treatment, and agarose gel electrophoresis were used to investigate if papain would present any toxic or mutagenic potential as well as if papain would display antioxidant properties. Papain exhibited negative results for all tests. This agent presented an activity protecting cells against H2O2-induced mutagenesis.

  7. A Demonstration of the Uncertainty in Predicting the Estrogenic Activity of Individual Chemicals and Mixtures From an In Vitro Estrogen Receptor Transcriptional Activation Assay (T47D-KBluc) to the In Vivo Uterotrophic Assay Using Oral Exposure.

    Science.gov (United States)

    Conley, Justin M; Hannas, Bethany R; Furr, Johnathan R; Wilson, Vickie S; Gray, L Earl

    2016-10-01

    In vitro estrogen receptor assays are valuable tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently unable to fully account for absorption, distribution, metabolism, and excretion. To explore this issue, we calculated relative potency factors (RPF), using 17α-ethinyl estradiol (EE2) as the reference compound, for several chemicals and mixtures in the T47D-KBluc estrogen receptor transactivation assay. In vitro RPFs were used to predict rat oral uterotrophic assay responses for these chemicals and mixtures. EE2, 17β-estradiol (E2), benzyl-butyl phthalate (BBP), bisphenol-A (BPA), bisphenol-AF (BPAF), bisphenol-C (BPC), bisphenol-S (BPS), and methoxychlor (MET) were tested individually, while BPS + MET, BPAF + MET, and BPAF + BPC + BPS + EE2 + MET were tested as equipotent mixtures. In vivo ED50 values for BPA, BPAF, and BPC were accurately predicted using in vitro data; however, E2 was less potent than predicted, BBP was a false positive, and BPS and MET were 76.6 and 368.3-fold more active in vivo than predicted from the in vitro potency, respectively. Further, mixture ED50 values were more accurately predicted by the dose addition model using individual chemical in vivo uterotrophic data (0.7-1.5-fold difference from observed) than in vitro data (1.4-86.8-fold). Overall, these data illustrate the potential for both underestimating and overestimating in vivo potency from predictions made with in vitro data for compounds that undergo substantial disposition following oral administration. Accounting for aspects of toxicokinetics, notably metabolism, in in vitro models will be necessary for accurate in vitro-to-in vivo extrapolations.

  8. IDENTIFICATION OF GLUCOSE TRANSPORTER-1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

    Institute of Scientific and Technical Information of China (English)

    章精; 刘志红; 刘栋; 黎磊石

    2001-01-01

    Objective. To evaluate the role of glucose transporter-l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

  9. Endocrine activity of persistent organic pollutants accumulated in human silicone implants — Dosing in vitro assays by partitioning from silicone

    DEFF Research Database (Denmark)

    Gilbert, Dorothea; Mayer, Philipp; Pedersen, Mikael

    2015-01-01

    Persistent organic pollutants (POPs) accumulated in human tissues may pose a risk for human health by interfering with the endocrine system. This study establishes a new link between actual human internal POP levels and the endocrine active dose in vitro, applying partitioning-controlled dosing...... from silicone to the H295R steroidogenesis assay: (1) Measured concentrations of POPs in silicone breast implants were taken from a recent study and silicone disks were loaded according to these measurements. (2) Silicone disks were transferred into H295R cell culture plates in order to control...... increases in progestagen and corticosteroid levels at Cfree of individual POPs in or below the femtomolar range. Silicone acted not only as source of the POPs but also as a sorption sink for lipophilic hormones, stimulating the cellular hormone production. Methodologically, the study showed that silicone...

  10. Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but has no detectable effects on telomerase activity in vivo.

    Science.gov (United States)

    Wadhwa, Renu; Colgin, Lorel; Yaguchi, Tomoko; Taira, Kazunari; Reddel, Roger R; Kaul, Sunil C

    2002-08-01

    MKT-077, a cationic rhodacyanine dye analogue, causes selective toxicity to cancer cells. Its cellular targets elucidated thus far include oncogenic Ras, F-actin, mortalin (hmot-2)/mthsp70, and telomerase. Here we report that MKT-077 causes growth arrest of cancer cells in culture independent of their Ras, p53, or telomerase status. Telomerase activity is inhibited in vitro by MKT-077 in the telomerase assay used. However, the in vivo toxicity seen in telomerase-positive cancer cells was not accompanied by inhibition of telomerase activity or telomere shortening. Furthermore, cells with an alternative mechanism for lengthening of telomeres were also sensitive to MKT-077 and showed enhanced formation of alternative mechanism for lengthening of telomeres-associated PML bodies in their nuclei. The data suggested that inhibition of telomerase activity is unlikely to be a prime cause of MKT-077-induced toxicity in cancer cells.

  11. Analysis of In Vitro DNA Interactions of Brassinosteroid-Controlled Transcription Factors Using Electrophoretic Mobility Shift Assay.

    Science.gov (United States)

    Unterholzner, Simon J; Rozhon, Wilfried; Poppenberger, Brigitte

    2017-01-01

    Most signaling cascades ultimately lead to changes in gene expression by modulating the activity of transcription factors (TFs). The electrophoretic mobility shift assay (EMSA) is a simple but powerful in vitro method for investigation of specific protein-DNA interactions. It makes use of the fact that protein-DNA complexes have a lower electrophoretic mobility in gels than free DNA has. The application of labeled probes in combination with unlabeled competitors allows investigation of DNA-binding specificity and identification of binding motifs with single base-pair resolution. Here we describe the application of EMSAs for the study of interactions of the brassinosteroid-regulated TFs, BRASSINAZOLE-RESISTANT1, (BZR1), BRI1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1)/BZR2, and CESTA with putative binding sites. The classical approach using radiolabeled probes, as well as the more recent application of fluorescent probes, is described and the advantages and disadvantages of both methods are discussed.

  12. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette

    2002-01-01

    culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause...... stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads...... for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay....

  13. Varying assay geometry to emulate connective tissue planes in an in vitro model of acupuncture needling.

    Science.gov (United States)

    Julias, Margaret; Buettner, Helen M; Shreiber, David I

    2011-02-01

    During traditional acupuncture, fine needles are inserted subcutaneously and rotated, which causes loose fascial tissue to wind around the needle. This coupling is stronger at acupuncture points, which tend to fall above intermuscular fascial planes, than control points, which lay above skeletal muscle. These different anatomical constraints may affect the mechanical coupling. Fascia at acupuncture points is bounded on two sides by skeletal muscle, but at control points is essentially unbounded. These differences were approximated in simple in vitro models. To emulate the narrower boundary within the intermuscular plane, type I collagen was cast in circular gels of different radii. To model the channel-like nature of these planes, collagen was cast in elliptical gels with major and minor axes matching the large and small circular gels, respectively, and in planar gels constrained on two sides. Acupuncture needles were inserted into the gels and rotated via a computer-controlled motor while capturing the evolution of fiber alignment under cross-polarization. Small circular gels aligned faster, but failed earlier than large circular gels. Rotation in elliptical and planar gels generated more alignment-per-revolution than circular gels. Planar gels were particularly resistant to failure. Fiber alignment in circular gels was isotropic, but was stronger in the direction of the minor axis in elliptical and planar gels. In fibroblast-populated gels, cells followed the alignment of the collagen fibers, and also became denser in regions of stronger alignment. These results suggest that the anatomy at acupuncture points provides unique boundaries that accentuate the mechanical response to needle manipulation.

  14. Skin sensitizers in cosmetics and beyond: potential multiple mechanisms of action and importance of T-cell assays for in vitro screening.

    Science.gov (United States)

    Vukmanović, Stanislav; Sadrieh, Nakissa

    2017-03-22

    Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity (DTH) reaction induced by repeated contact with sensitizers. The ability of a chemical to act as a sensitizer has most frequently been tested in animals. As the use of animals for these purposes is gradually and globally being phased out, there is a need for reliable in vitro surrogate assays. Currently proposed in vitro assays are designed to test four key events of the adverse outcome pathway (AOP) involving covalent modification of self-proteins by sensitizers (haptenation) and presentation of new antigens (hapten/carrier complexes) to the immune system. There appears to be imperfect alignment of in vitro assays with clinical and/or animal data, suggesting possibly additional mechanisms of ACD development. Indeed, studies on allergies to small drugs, small chemical-induced HLA-peptide exchange for vaccination purposes and cosmetic ingredient-induced exposure of autoantigens suggest a possibility of DTH response promotion by hapten/carrier-independent mechanisms. Therefore, there is a need for additional appropriate in vitro assays, in order to achieve maximal concordance between clinical and/or animal data and in vitro assays. In this paper, we will review evidence supporting the idea of diverse mechanisms of ACD development. We will also discuss the impact of these multiple mechanisms, on the AOP and on the in vitro assays that should be used for allergen detection. We will propose alloreactivity-like reactions, aided by computer modeling and biochemical tests of compound-HLA binding, as additional tools for better prediction of DTH reactions, resulting from exposure to ingredients in cosmetic products. The combination of the proposed tests, along with the existing assays, should further enhance animal-free assessment of sensitizing potential of individual chemicals.

  15. Comparison of in vitro hormone activities of selected phthalates using reporter gene assays.

    Science.gov (United States)

    Shen, Ouxi; Du, Guizhen; Sun, Hong; Wu, Wei; Jiang, Yi; Song, Ling; Wang, Xinru

    2009-12-01

    Phthalates are widely used in the plastic industry and food packaging, imparting softness and flexibility to normally rigid plastic medical devices and children's toys. Even though phthalates display low general toxicity, there is increasing concern on the effects of endocrine system induced by some of phthalate compounds. The hormone activity of dibutyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP) were assessed using the luciferase reporter gene assays. The results showed that DBP, MBP and DEHP, not only exhibited potent antiandrogenic activity, with IC(50) value of 1.05x10(-6), 1.22x10(-7)M and exceeding 1x10(-4)M respectively, but also showed the androgenic activity with EC(50) value of 6.17x10(-6), 1.13x10(-5)M and exceeding 1x10(-4)M. We also found that all the three related chemicals possessed thyroid receptor (TR) antagonist activity with IC(50) of 1.31x10(-5), 2.77x10(-6)M and exceeding 1x10(-4)M respectively, and none showed TR agonist activity. These results indicate that TR might be the targets of industrial chemicals. In the ER mediate reporter gene assay, three chemicals showed no agonistic activity except for DBP, which appeared weakly estrogenic at the concentration of 1.0x10(-4)M. Together, the findings demonstrate that the three phthalates could simultaneously disrupt the function of two or more hormonal receptors. Therefore, these phthalates should be considered in risk assessments for human health.

  16. Experimental Assessment of Moringa oleifera Leaf and Fruit for Its Antistress, Antioxidant, and Scavenging Potential Using In Vitro and In Vivo Assays

    OpenAIRE

    Suaib Luqman; Suchita Srivastava; Ritesh Kumar; Anil Kumar Maurya; Debabrata Chanda

    2012-01-01

    We have investigated effect of Moringa oleifera leaf and fruit extracts on markers of oxidative stress, its toxicity evaluation, and correlation with antioxidant properties using in vitro and in vitro assays. The aqueous extract of leaf was able to increase the GSH and reduce MDA level in a concentration-dependent manner. The ethanolic extract of fruit showed highest phenolic content, strong reducing power and free radical scavenging capacity. The antioxidant capacity of ethanolic extract of ...

  17. Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.

    Science.gov (United States)

    Wang, Juan; Zhao, Shasha; Zhou, Ying; Wei, Yun; Deng, Wensheng

    2015-01-01

    Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.

  18. Fluorescent ester dye-based assays for the in vitro measurement of Neospora caninum proliferation.

    Science.gov (United States)

    Mota, Caroline M; Ferreira, Marcela D; Costa, Lourenço F; Barros, Patrício S C; Silva, Murilo V; Santiago, Fernanda M; Mineo, José R; Mineo, Tiago W P

    2014-09-15

    Techniques for the measurement of parasite loads in different experimental models have evolved throughout the years. The quantification of stained slides using regular cytological stains is currently the most common technique. However, this modality of evaluation is labor-intensive, and the interpretation of the results is subjective because the successes of the assays mainly rely on the abilities of the professionals involved. Moreover, the novel genetic manipulation techniques that are commonly applied for closely related Toxoplasma gondii have not yet been developed for Neospora caninum. Thus, we aimed to develop a simple protocol for parasite quantification using pre-stained N. caninum tachyzoites and fluorescent probes based on ester compounds (i.e., CFSE and DDAO). For this purpose, we employed a quantification procedure based on flow cytometry analysis. Pre-stained parasites were also examined with a fluorescent microscope, which revealed that both dyes were detectable. Direct comparison of the numbers of CFSE+ and DDAO+ cells to the values obtained with classical cytology techniques yielded statistically comparable results that also accorded with genomic DNA amplification results. Although the fluorescence emitted by DDAO was more intense and provided better discrimination between the populations of parasitized cells, CFSE+ tachyzoites were detected for several days. In conclusion, this study describes a simple, fast, low-cost and reproducible protocol for N. caninum quantification that is based on parasite pre-staining with fluorescent ester-based probes.

  19. Comparative Study of Cigarette Smoke Cytotoxicity Using Two In Vitro Assay Systems

    Directory of Open Access Journals (Sweden)

    Fukushima Toshiro

    2014-09-01

    Full Text Available L'objet de la présente étude fut de comparer les résultats obtenus à partir de deux essais de cytotoxicité in vitro s'appuyant sur des mécanismes/modes d'action différents. Le test de fixation du rouge neutre (Neutral Red Uptake - NRU se fonde sur l'endocytose tandis que le test des sels de tetrazolium hydrosolubles (WST-1 s'appuie sur l'activité de la déshydrogénase mitochondriale. Ces deux tests furent analysés à la lumière de leur fréquence d'utilisation et de leur validation documentée. La matière particulaire totale (MPT et la phase gaz/vapeur (PGV de la fumée principale produite par les cigarettes de référence Kentucky 3R4F et les dix cigarettes testées composées à 100% de tabac Burley ou à 100% de tabac jaune furent appliquées individuellement dans les deux essais utilisant des cellules CHO-K1. En outre, les constituants de fumée de cigarette et les agents cytotoxiques connus, dont la capacité à affecter certains indicateurs de résultat est documentée, furent évalués lors des deux tests. Bien que le test de fixation du rouge neutre se révéla, dans un premier temps, plus sensible que le test aux WST-1, les deux essais livrèrent des résultats comparables en termes de classement par ordre de rang de la cytotoxicité des échantillons de fumée de cigarette. Eu égard à la cytotoxicité des constituants de fumée de cigarette, l'acroléine, l'hydroquinone et la catéchine présentèrent de claires diminutions de viabilité cellulaire proportionnelles à la dose (un indicateur de résultat commun aux deux essais. Par ailleurs, les inhibiteurs enzymatiques de la chaîne respiratoire mitochondriale et les produits chimiques portant atteinte à la membrane cellulaire présentèrent également des réactions similaires, indépendamment de l'indicateur de résultat spécifique visé lors du test de cytotoxicité. En conclusion, les résultats glanés lors du test de fixation du rouge neutre et du test aux sels de

  20. In vitro assessment of genotoxic effects of electric arc furnace dust on human lymphocytes using the alkaline comet assay.

    Science.gov (United States)

    Garaj-Vrhovac, Vera; Orescanin, Visnja; Ruk, Damir; Gajski, Goran

    2009-02-15

    In vitro genotoxic effects of leachates of electric arc furnace dust (EAFD) on human peripheral lymphocytes, assessed prior and following the treatment with a strong alkaline solution were investigated using the alkaline comet assay. Prior and following the treatment, lymphocytes were incubated with leachate of EAFD for 6 and 24 hours at 37 degrees C. Negative controls were also included. Mean values of the tail lengths established in the samples treated with the leachate stemming from the original dust for 6 and 24 hours, were 15.70 microm and 16.78 microm, respectively, as compared to 12.33 microm found in the control sample. Slight, but significant increase in the tail length was also found with the dust treated with a strong alkaline solution (13.37 microm and 13.60 microm). In case of high heavy metal concentrations (the extract of the original furnace dust), the incubation period was revealed to be of significance as well. The obtained results lead to the conclusion that alkaline comet assay could be used as a rapid, sensitive and low-cost tool when assessing genotoxicity of various waste materials, such as leachates of the electric arc furnace dust.

  1. Specific in vitro toxicity of crude and refined petroleum products: 3. Estrogenic responses in mammalian assays.

    Science.gov (United States)

    Vrabie, Cozmina M; Candido, Angelica; van den Berg, Hans; Murk, Albertinka J; van Duursen, Majorie B M; Jonker, Michiel T O

    2011-04-01

    Current petroleum risk assessment considers only narcosis as the mode of action, but several studies have demonstrated that oils contain compounds with dioxin-like, estrogenic or antiestrogenic, and androgenic or antiandrogenic activities. The present study is the third in a series investigating the specific toxic effects of 11 crude oils and refined products. By employing recombinant mammalian cells stably transfected with the human estrogen receptor alpha (ERα) or beta (ERβ), and expressing the luciferase protein (ERα-U2OS-Luc and ERβ-U2OS-Luc assay), the estrogenicity or antiestrogenicity of oils was studied. All oils, except for two refined oils and one crude oil, induced estrogenic responses. The calculated estrogenic potencies of the oils were six to nine orders of magnitude lower than the potency of 17β-estradiol (E2). Upon coexposure to a fixed concentration of E2 and increasing concentrations of oils, additive, antagonistic, and synergistic effects were revealed. One nautical fuel oil was tested in the human breast carcinoma cell line MCF-7, in which it induced cell proliferation up to 70% relative to the maximal induction by E2. At its minimum effect concentration of 25 mg/L, the oil was also capable of inducing mRNA expression of the estrogen-dependent protein pS2 by a factor of two. The present results indicate that oils naturally contain potentially endocrine-disrupting compounds that are able to influence the estrogenicity of other compounds and may cause biological responses beyond receptor binding.

  2. A human in vitro whole blood assay to predict the systemic cytokine response to therapeutic oligonucleotides including siRNA.

    Directory of Open Access Journals (Sweden)

    Christoph Coch

    Full Text Available Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR or RIG-I like helicases (RLH are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4 was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.

  3. A 155-plex high-throughput in vitro coregulator binding assay for (anti-)estrogenicity testing evaluated with 23 reference compounds.

    Science.gov (United States)

    Wang, Si; Houtman, René; Melchers, Diana; Aarts, Jac; Peijnenburg, Ad; van Beuningen, Rinie; Rietjens, Ivonne; Bovee, Toine F

    2013-01-01

    To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor α was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERα binding to coregulator peptides in the presence of ligands on the PamChip® plate, were compared to the relative estrogenic potencies as determined in the in vivo uterotrophic assay. The results show that the estrogenic potencies predicted by the 57 coactivators on the peptide microarray for 18 compounds that display a clear E2 dose-dependent response (goodness of fit of a logistic dose-response model of 0.90 or higher) correlated very well with their in vivo potencies in the uterotrophic assay, i.e., coefficient of determination values for 30 coactivators higher than or equal to 0.85. Moreover, this coregulator binding assay is able to distinguish ER agonists from ER antagonists: profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct from those of pure ER agonists, such as dienestrol. Combination of this coregulator binding assay with other types of in vitro assays, e.g., reporter gene assays and the H295R steroidogenesis assay, will frame an in vitro test panel for screening and prioritization of chemicals, thereby contributing to the reduction and ultimately the replacement of animal testing for (anti-)estrogenic effects.

  4. Total antioxidant potential of juices, beverages and hot drinks consumed in Egypt screened by DPPH in vitro assay

    Directory of Open Access Journals (Sweden)

    Ramadan-Hassanien, Mohamed Fawzy

    2008-09-01

    Full Text Available Plant foods contain different classes and types of antioxidants and knowledge of their total antioxidant potential (TAP, which is the cumulative capacity of food components to scavenge free radicals, would be useful for epidemiological purposes.To accomplish this, a variety of fruit juices, hot drinks and beverages commonly consumed in Egypt were analyzed using in vitro DPPH assay. The order of effectiveness of fruit juices in inhibiting free radicals was as follows: red grapes juice > mango juice > guava juice > cocktail juice > pineapple juice > orange juice > cherry juice > apple juice. Among beverages and hot drinks, teas followed by coffees had the greatest TAP. These data confirm grape juice, teas and coffees as good dietary sources of antioxidants.Las plantas comestibles contienen diferentes clases y tipos de antioxidantes y el conocimiento de su potencial antioxidante total (TAP, que es la capacidad acumulativa de los componentes de los alimentos para captar radicales libres, debería ser útil en estudios epidemiológicos. De acuerdo a esto, una variedad de zumos de fruta, bebidas calientes y bebidas consumidas habitualmente en Egipto fueron analizadas usando un ensayo in vitro con DPPH. El orden de efectividad de los zumos de frutas en inhibir los radicales libres fue el siguiente: zumo de uva tinta > zumo de mango > zumo de guayaba > zumo de macedonia de frutas > zumo de piña >zumo de naranja > zumo de cereza > zumo de manzana. Entre las bebidas y bebidas calientes, el té seguido por el café son los que tuvieron mayores TAPs. Estos datos confirman que el zumo de uva, el té y el café son buenas fuentes de antioxidantes.

  5. Experimental Assessment of Moringa oleifera Leaf and Fruit for Its Antistress, Antioxidant, and Scavenging Potential Using In Vitro and In Vivo Assays

    Directory of Open Access Journals (Sweden)

    Suaib Luqman

    2012-01-01

    Full Text Available We have investigated effect of Moringa oleifera leaf and fruit extracts on markers of oxidative stress, its toxicity evaluation, and correlation with antioxidant properties using in vitro and in vitro assays. The aqueous extract of leaf was able to increase the GSH and reduce MDA level in a concentration-dependent manner. The ethanolic extract of fruit showed highest phenolic content, strong reducing power and free radical scavenging capacity. The antioxidant capacity of ethanolic extract of both fruit and leaf was higher in the in vitro assay compared to aqueous extract which showed higher potential in vivo. Safety evaluation studies showed no toxicity of the extracts up to a dose of 100 mg/kg body weight. Our results support the potent antioxidant activity of aqueous and ethanolic extract of Moringa oleifera which adds one more positive attribute to its known pharmacological importance.

  6. Experimental Assessment of Moringa oleifera Leaf and Fruit for Its Antistress, Antioxidant, and Scavenging Potential Using In Vitro and In Vivo Assays.

    Science.gov (United States)

    Luqman, Suaib; Srivastava, Suchita; Kumar, Ritesh; Maurya, Anil Kumar; Chanda, Debabrata

    2012-01-01

    We have investigated effect of Moringa oleifera leaf and fruit extracts on markers of oxidative stress, its toxicity evaluation, and correlation with antioxidant properties using in vitro and in vitro assays. The aqueous extract of leaf was able to increase the GSH and reduce MDA level in a concentration-dependent manner. The ethanolic extract of fruit showed highest phenolic content, strong reducing power and free radical scavenging capacity. The antioxidant capacity of ethanolic extract of both fruit and leaf was higher in the in vitro assay compared to aqueous extract which showed higher potential in vivo. Safety evaluation studies showed no toxicity of the extracts up to a dose of 100 mg/kg body weight. Our results support the potent antioxidant activity of aqueous and ethanolic extract of Moringa oleifera which adds one more positive attribute to its known pharmacological importance.

  7. Bioprinting of 3D hydrogels.

    Science.gov (United States)

    Stanton, M M; Samitier, J; Sánchez, S

    2015-08-07

    Three-dimensional (3D) bioprinting has recently emerged as an extension of 3D material printing, by using biocompatible or cellular components to build structures in an additive, layer-by-layer methodology for encapsulation and culture of cells. These 3D systems allow for cell culture in a suspension for formation of highly organized tissue or controlled spatial orientation of cell environments. The in vitro 3D cellular environments simulate the complexity of an in vivo environment and natural extracellular matrices (ECM). This paper will focus on bioprinting utilizing hydrogels as 3D scaffolds. Hydrogels are advantageous for cell culture as they are highly permeable to cell culture media, nutrients, and waste products generated during metabolic cell processes. They have the ability to be fabricated in customized shapes with various material properties with dimensions at the micron scale. 3D hydrogels are a reliable method for biocompatible 3D printing and have applications in tissue engineering, drug screening, and organ on a chip models.

  8. In vitro mutagenicity testing. I. Kermide 601 resin, Sylgard 184 encapsulating resin, and Sylgard 184 curing agent. [Ames Salmonella assay system used

    Energy Technology Data Exchange (ETDEWEB)

    Wang, S.Y.; Smith, D.M.

    1978-08-01

    Five compounds, Kerimide 601 resin, Sylgard 184 encapsulating resin, Sylgard 184 curing agent, benzo(a)pyrene, and acridine orange were tested for in vitro mutagenicity using the Ames Salmonella assay system. Kerimide 601 resin, Sylgard 184 encapsulating resin, and Sylgard 184 curing agent were not mutagenic under the described experimental conditions, while benzo(a)pyrene and acridine orange were both mutagenic.

  9. Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons

    NARCIS (Netherlands)

    Kappers, W.A.; Och, F.M.M. van; Groene, E.M. de; Horbach, G.J.

    2000-01-01

    Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro- polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2- nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs

  10. Biological measurement of estrogenic activity in urine and bile conjugates with the in vitro ER-CALUX reporter gene assay.

    Science.gov (United States)

    Legler, Juliette; Jonas, Arjen; Lahr, Joost; Vethaak, A Dick; Brouwer, Abraham; Murk, Albertinka J

    2002-03-01

    Although estrogens are excreted as biologically inactive conjugates, they can be reconverted to an active form, possibly by bacteria. A simple method was developed to deconjugate estrogen metabolites present in human urine and fish bile back to active estrogens by enzymatic hydrolysis with beta-glucuronidase or live Escherichia coli cells. Deconjugated extracts were tested for estrogenic activity in the in vitro stable estrogen receptor-mediated chemical-activated luciferase gene expression (ER-CALUX) assay. Estrogen glucuronides in urine obtained from human males and females were effectively converted to active forms after incubation with beta-glucuronidase or E. coli. The highest estrogenic activity was found in deconjugated metabolites from urine of a pregnant woman, in which levels up to 3,000 nmol estradiol equivalents per liter of urine were found after overnight incubation of urine with E. coli. Bile sampled from male bream and flounder from various freshwater and marine locations was also deconjugated and a good correlation was found between high biliary estrogenic activity and elevated levels of xenoestrogenic activity in surface water as well as in plasma vitellogenin. Therefore, the measurement of deconjugated bile could form a useful (indirect) biomarker for internal dose of xenoestrogens in male fish.

  11. In vitro Antimicrobial Assay of Actinomycetes in Rice AgainstXanthomonas oryzae pv. oryzicola and as Potential Plant Growth Promoter

    Directory of Open Access Journals (Sweden)

    Erneeza Mohd Hata

    2015-12-01

    Full Text Available ABSTRACT The aim of this work was to invitro assay the antimicrobial activity of actinomycetes in rice against Xanthomonas oryzae pv. oryzicola and as potential plant growth promoter. A total of 92 actinomycete strains were isolated from different rice plant components and field locations. Of these, only 21.74% showed antagonistic activity against the Xoc pathogen. Molecular identification via 16s rRNA amplification revealed that 60% of the active antagonistic strains belonged to the genus Streptomyces. Isolates that demonstrated the highest antagonistic activity were also able to produce hydrolytic enzymes and plant growth-promoting hormones. Combination of preliminary screening based on in vitro antagonistic, hydrolytic enzyme and plant growth hormone activity facilitated the best selection of actinomycete candidates as evidenced by strains classification using cluster analysis (Ward's Method. Results from the preliminary screening showed that actinomycetes, especially Streptomycetes, could offer a promising source for both biocontrol and plant growth-promotion agents against BLS disease in rice.

  12. Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics.

    Science.gov (United States)

    Vanparys, Philippe; Corvi, Raffaella; Aardema, Marilyn J; Gribaldo, Laura; Hayashi, Makoto; Hoffmann, Sebastian; Schechtman, Leonard

    2012-04-11

    Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals.

  13. In vitro cytotoxicity studies on Carissa congesta, Polyalthia longifolia, and Benincasa hispida extracts by Sulforhodamine B assay method

    Directory of Open Access Journals (Sweden)

    Gaurav Mahesh Doshi

    2015-01-01

    Full Text Available Background: Indian medicinal plants have contributed to the growth of world′s ethnopharmacological heritage. Roots of Carissa congesta (CC powder are mixed with horse urine, lime juice, and camphor and used as remedies for relieving itching conditions, Polyalthia longifolia (PL leaves are aromatic and used for decoration in festivals as sonamukhi and Benincasa hispida (BH seeds provide treatment for cough and vitiated conditions of pitta. Aims of the Study: In the current studies, crude petroleum ether extracts (BH and CC and ethanolic extract of (PL were screened for in vitro cytotoxicity activity using different cell lines. Settings and Design: In the experiment, human colon cancer HCT15, human breast cancer MCF7 and human leukemia MOLT4 cell lines were studied on the extracts. Materials and Methods: The method used was Sulforhodamine B (SRB assay method in which growth inhibition of 50% (GI 50 was analyzed by comparing it with standard drug Adriamycin (ADR (doxorubicin. Results: The CC and PL extracts showed equivalent activity to ADR (doxorubicin for human breast cancer cell line MCF7 and human leukemia cell line MOLT4 respectively. BH extract did not show satisfactory activity on selected cell lines. Conclusion: In the future, new cell lines may be screened in order to check the potency of CC, PL, and BH extracts.

  14. Evaluation of the in vitro activities of ceftobiprole and comparators in staphylococcal colony or microtitre plate biofilm assays.

    Science.gov (United States)

    Abbanat, Darren; Shang, Wenchi; Amsler, Karen; Santoro, Colleen; Baum, Ellen; Crespo-Carbone, Steven; Lynch, A Simon

    2014-01-01

    The aim of this study was to evaluate the in vitro efficacy of ceftobiprole and comparator antibiotics, either alone or in combination, in staphylococcal MBEC™ (minimum biofilm eradication concentration) and colony biofilm assays at dilutions of the maximum free-drug plasma concentration attained during clinical use (fCmax). Staphylococci tested included meticillin-susceptible and meticillin-resistant Staphylococcus aureus (n=6) and Staphylococcus epidermidis (n=2). Relative to no-drug controls, after 7 days of exposure ceftobiprole concentrations from 1/4 fCmax to fCmax generally decreased CFUs in MBEC or colony biofilms of S. aureus isolates by ca. 1.5log10 to ≥2.5log10. Gentamicin reduced colony biofilm CFUs by ≥1.4log10 at these concentrations with gentamicin-susceptible isolates. Following 7 days of exposure, vancomycin and rifampicin were ineffective as single agents or in combination in the colony model, but yielded CFU decreases from 0 to 5log10 in the MBEC model. Treatment of biofilms with rifampicin for 7 days yielded rifampicin-resistant mutants, and the selection of rifampicin resistance was inhibited by co-treatment with ceftobiprole. Thus, ceftobiprole alone or in combination demonstrated promising activity against biofilms of meticillin-susceptible and -resistant staphylococci at clinically relevant concentrations. In contrast, vancomycin and rifampicin, two agents used clinically for the treatment of biofilm infections, tested separately or together gave inconsistent results and generally had little impact on cell viability.

  15. Quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions.

    Science.gov (United States)

    Wilhelmsen, Kevin; Farrar, Katherine; Hellman, Judith

    2013-08-23

    The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

  16. Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. General considerations, the nitrosoureas and alkylating agents.

    Science.gov (United States)

    Bosanquet, A G

    1985-01-01

    In vitro drug sensitivity of tumour biopsies is currently being determined using a variety of methods. For these chemosensitivity assays many drugs are required at short notice, and this in turn means that the drugs must generally be stored in solution. There are, however, a number of potential problems associated with dissolving and storing drugs for in vitro use, which include (a) drug adsorption; (b) effects of freezing; (c) drug stability under the normal conditions of dilution and setting up of an in vitro assay; and (d) insolubility of drugs in normal saline (NS) or phosphate-buffered saline (PBS). These problems are considered in general, and some recommendations for use of solutions of drugs in in vitro assays are suggested. The nitrosoureas and alkylating agents are also investigated in greater detail in this respect. The nitrosoureas are found to be very labile in PBS at pH 7, with 5% degradation (t0.95) occurring in 10-50 min at room temperature. These values are increased about 10-fold on refrigeration and about 5- to 10-fold on reduction of the pH of the medium to pH 4-5. At pH 7 and room temperature, t0.95 is observed in under 1 h with the alkylating agents nitrogen mustard, chlorambucil, melphalan, 2,5-diaziridinyl-3,6-bis(2-hydroxyethylamino)-1,4-benzoquinone (BZQ), dibromodulcitol, dibromomannitol, treosulphan, and procarbazine. Of the other alkylating agents, 4-hydroperoxycylophosphamide (sometimes used in vitro in place of cyclophosphamide), busulphan, dianhydrogalactitol, aziridinylbenzoquinone (AZQ), and dacarbazine have a t0.95 of between 2 and 24 h, while ifosfamide and pentamethylmelamine are both stable in aqueous solution for greater than 7 days. About half the drugs studied in detail have been stored frozen in solution for in vitro use, although very little is known about their stability under these conditions.

  17. Combination of retinyl palmitate and UV-filters: phototoxic risk assessment based on photostability and in vitro and in vivo phototoxicity assays.

    Science.gov (United States)

    Benevenuto, Carolina Gomes; Guerra, Lucas Offenbecker; Gaspar, Lorena Rigo

    2015-02-20

    This study aimed to assess the phototoxic potential of combined UV-filters and retinyl palmitate (RP) in the presence or not of bemotrizinol (BMTZ), employing photostability and in vitro and in vivo phototoxicity assays. The formulations tested contained octocrylene (OCT), octyl methoxycinnamate (OMC), benzophenone-3 (BZP-3) and RP (photostable) or octocrylene (OCT), octyl methoxycinnamate (OMC), avobenzone (AVO) and RP (less photostable). Both formulations were supplemented with bemotrizinol. Photostability was evaluated by exposing, or not, formulations spread on a glass plate to UVA/UVB irradiation. The resulting products were quantified by HPLC analysis. In vitro phototoxicity of UV-filters and combinations were evaluated using 3T3 viable monolayer fibroblast cultures submitted, or not, to irradiation according to OECD TG 432. In vivo photoallergy and photoxicity were assessed by clinical studies (photopatch test). Photostability assays showed that UV-filter bemotrizinol was a better photostabilizer for RP/benzophenone-3 than for RP/avobenzone. The in vitro phototoxicity of the combination RP/avobenzone was reduced by bemotrizinol. Clinical studies did not indicate phototoxic or photoallergenic potentials in all formulations tested. It is concluded that the 3T3 NRU phototoxicity test may be considered a supplementary assay in formulation developments, since it can detect chemically unstable and potentially phototoxic combinations. However, extrapolation of in vitro positive results to human photopatch tests may be performed only to a limited extent.

  18. 3D Animation Essentials

    CERN Document Server

    Beane, Andy

    2012-01-01

    The essential fundamentals of 3D animation for aspiring 3D artists 3D is everywhere--video games, movie and television special effects, mobile devices, etc. Many aspiring artists and animators have grown up with 3D and computers, and naturally gravitate to this field as their area of interest. Bringing a blend of studio and classroom experience to offer you thorough coverage of the 3D animation industry, this must-have book shows you what it takes to create compelling and realistic 3D imagery. Serves as the first step to understanding the language of 3D and computer graphics (CG)Covers 3D anim

  19. 3D video

    CERN Document Server

    Lucas, Laurent; Loscos, Céline

    2013-01-01

    While 3D vision has existed for many years, the use of 3D cameras and video-based modeling by the film industry has induced an explosion of interest for 3D acquisition technology, 3D content and 3D displays. As such, 3D video has become one of the new technology trends of this century.The chapters in this book cover a large spectrum of areas connected to 3D video, which are presented both theoretically and technologically, while taking into account both physiological and perceptual aspects. Stepping away from traditional 3D vision, the authors, all currently involved in these areas, provide th

  20. In vitro adhesion assay of lactic acid bacteria, Escherichia coli and Salmonella sp. by microbiological and PCR methods

    Directory of Open Access Journals (Sweden)

    Didier Montet

    2006-03-01

    Full Text Available In vitro adhesion assay using Lactobacillus reuteri KUB-AC5 as a test strain has been studied by applying simple PCR reaction together with image analysis and plate count techniques. Critical factor affecting the PCR method was quality and quantity of DNA. The cell lysis technique was modified to optimize this method. Thus, lysozyme and proteinase K were added to lyse the cells, followed by SDS solution to obtain a complete cell lysis. Only PCR products from total cells (TC were obtained, with low consistency, but none from cells bound to mucus (BC at either 0.1 or 0.5 mg/mL concentration. It was hypothesized that the attached cells might not be extracted into the cell suspension. Therefore, 1% SDS solution and 0.1M NaOH were used directly in the extraction. As expected, PCR products were observed when both TC and BC were used as a DNA template. Adhesion appeared at a wide range of 0-45%, with low consistency. Therefore, a simple microbiological method (plate count was used. The extraction of bound cells into cell suspension was critical in this method. Extraction times of 20, 60, 120 and 150 min were tried. Results showed that maximum cell number was obtained with 120 min extraction. L. reuteri KUB-AC5, L. reuteri KUB-AC16, L. reuteri KUB-AC20, L. salivarius KUB-AC21, L. acidophilus KV-1, Escherichia coli E010, Salmonella sp. S003, E. coli ATCC8739, and S. typhimurium ATCC 13311 exhibited adhesion activity of 21.6%, 0.8%, 5.7%, 1.1%, 23.1%, 10.7%, 10.3%, 4.4% and 3.2%, respectively. Among the 9 types of microorganisms tested L. acidophilus KV-1 and L. reuteri KUB-AC5 showed higher adhesion activity than the others.

  1. Screening of Antioxidant Potential of Selected Barks of Indian Medicinal Plants by Multiple in vitro Assays LI

    Institute of Scientific and Technical Information of China (English)

    ARCHANA KUMARI; POONAM KAKKAR

    2008-01-01

    Objective To evaluate the antioxidant potential in herbal extract barks of five therapeutically important medicinal plants native to India,i.e.Crataeva nurvala Buch.-Ham.,Buchanania lanzan Spreng.,Aegle marmelos Corr.,Dalbergia sissoo Roxb.ex DC.,and Cedrela toona Roxb.Methods Standardized aqueous alcoholic extracts from the selected barks having different target radicals,such as superoxide radical,nitric oxide,ABTS radical,and peroxidative decomposition of phosphohpids.were prepared and screened bv multiple in vitro assays.These extracts were also tested for total phenolic and tannin content and correlated with antioxidant capacity. Results Tbtal phenolic and tannin contents were found to be the highest in C. nurvala (195 GAE mg/g and 218.3 mg/g CE).SOD mimetic activity was found to be the highest in Crataeva nurvula,although all barks showed activity more than 100 units/mg extract.Lipid peroxidation inhibitory potential was found to be the highest in Crataeva nurvala(83.4% inhibition of MDA formation/10 μg extract),and also showed a comparatively high NO quenching capacity (45.5% per 10 μg extract).The highest NO quenching potential was found in Aegle marmelos(47.3% per 10 μg extract).Cedrela toona showed the lowest LPO inhibitory potential and NO quenching capacity(50.5% and 30.5%,respectively).Buchanania lanzan,a medicinal plant extensively used for inflammatory disorders and Dalbergia sissoo also showed 72.5% and 69.1% LPO inhibitory potential/10 μg extract.Trolox equivalent antioxidant capacity ranged from 0.24 to 0.39 mmol/L TEAC/mg extract,indicating that all the barks tested had ABTS+ radical quenching capacity.Conclusion Bark of Crataeva nurvulahas the highest antioxidant capacity and a positive correlation between antioxidant activity and their plendic content was found.

  2. EVALUATION OF THE ANTITUMOR ACTIVITY OF HUMAN IL-4 BY IN VITRO AND IN V1VO ASSAYS

    Institute of Scientific and Technical Information of China (English)

    王彤钢; 陈慰峰

    1994-01-01

    The characteristics of rhuIL-4 induced cytotoxicity was detected in vitro by using 51 Cr release assay and the anti-tumor activity of rhuIL-4 induced killer cell was evaluated in vivo by using a human tumor model in nudemice.huIL-4 can induce LAK activity from peripheral blood lymphocytes(PBMC) stimulated with phytohemagglutinin(PHA).Compared with the LAK activity induced by rhuIL-2,the cytotoxicity of the killer cells induced by rhuIL-4 to K562 and Raji cells was lower ,but that to TBL-E,a human lymphoid leukemia cell line established in our laboratory,and PHA-activated blast cells(PHA-blasts) was of similar magnitude.In the cytotoxicity assay using PHA-blasts,the addition of PHA increased the IL-4 induced killer cell cytotoxicity by 131%,but had no effect on IL-2-induced ki8ller cell cytotoxicity.This implies that IL-4 mainly induces CTL-like activity,while IL-2 mainly induces NK-like activity,An experimental human tumor model in nude mice was established by injection of TBL-E human leukemia cells.The anti-tumor activity of rhuIL-4 was evaluated by injection of haman LAK cells induced from PHA-blasts by rhuIL-2+rhuIL-4 and human cytokines into tumor-bearing nude mice.The results showed that human LAK cells effectively inhibit the tumorigenicity of TBL-E cells in nude mice with an inhibition rate of 61%.The antitumor effect of rhuIL-2 was better than that of rIL-4 ,and the antitumor effect of rhuIL-2+rhuIL-4 was similar to that of rhuIL-2 ,though the former delayed the occurence of tumors.Our data imply the potential application of human IL-4 in clinic,and provide an animal model to evaluate the anti-tumor activity of human cytokine(s) with species specificity.

  3. Development of paper-based microfluidic analytical device for iron assay using photomask printed with 3D printer for fabrication of hydrophilic and hydrophobic zones on paper by photolithography.

    Science.gov (United States)

    Asano, Hitoshi; Shiraishi, Yukihide

    2015-07-09

    This paper describes a paper-based microfluidic analytical device for iron assay using a photomask printed with a 3D printer for fabrication of hydrophilic and hydrophobic zones on the paper by photolithography. Several designed photomasks for patterning paper-based microfluidic analytical devices can be printed with a 3D printer easily, rapidly and inexpensively. A chromatography paper was impregnated with the octadecyltrichlorosilane n-hexane solution and hydrophobized. After the hydrophobic zone of the paper was exposed to the UV light through the photomask, the hydrophilic zone was generated. The smallest functional hydrophilic channel and hydrophobic barrier were ca. 500 μm and ca. 100 μm in width, respectively. The fabrication method has high stability, resolution and precision for hydrophilic channel and hydrophobic barrier. This test paper was applied to the analysis of iron in water samples using a colorimetry with phenanthroline.

  4. Non-small cell lung cancer 95D cells co-cultured with 3D-bioprinted scaffold to construct a lung cancer model in vitro%95 D细胞与三维打印支架共培养构建体外肺癌模型

    Institute of Scientific and Technical Information of China (English)

    牟好; 王剑; 胡慧珍; 徐炜; 陈清勇

    2015-01-01

    Objective To fabricate an innovative scaffold for lung cancer cell culture and establish a three⁃dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two⁃dimensional and three⁃dimensional culture conditions. Methods We chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co⁃cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis. Results Cells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D⁃cultured cells for 2⁃6 days were significantly lower than, but those cultured for 8⁃9 days were significantly higher than that of the 2D⁃cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8⁃9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down⁃regulated in 3D scaffold⁃cultured group (0.529± 0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D⁃cultured group (3.063±0.139, 1.738 ±0.122 vs. 2.472±0.151)(P may provide a promising model for lung cancer research in vitro.%目的:探讨非小细胞肺癌细胞在二维和三维培养条件下的生物学行为差异。方法选用琼脂和海藻酸钠材料,利用三维(3D)打印技术制备细胞支架,并与肺癌95D细胞共培养,利用光学显微镜、电子显微镜、HE染色、四甲

  5. Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke.

    Science.gov (United States)

    Roemer, Ewald; Zenzen, Volker; Conroy, Lynda L; Luedemann, Kathrin; Dempsey, Ruth; Schunck, Christian; Sticken, Edgar Trelles

    2015-01-01

    Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

  6. Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay.

    Science.gov (United States)

    Mao, Weifeng; Niikura, Masahiro; Silva, Robert F; Cheng, Hans H

    2008-03-01

    Marek's disease, a T cell lymphoma, is an economically important disease of poultry caused by the Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus. A greater understanding of viral gene function and the contribution of sequence variation to virulence should facilitate efforts to control Marek's disease in chickens. To characterize a naturally occurring single nucleotide polymorphism (SNP; AY510475:g.108,206C>T) in the MDV UL41 gene that results in a missense mutation (AAS01683:p.Arg377Cys), bacterial artificial chromosome (BAC)-derived MDVs that differed only in the UL41 SNP were evaluated using a head-to-head competition assay in vitro. Monitoring the frequency of each SNP by pyrosequencing during virus passage determined the ratio of each viral genome in a single monolayer, which is a very sensitive method to monitor viral fitness. MDV with the UL41*Cys allele showed enhanced fitness in vitro. To evaluate the mechanism of altered viral fitness caused by this SNP, the virion-associated host shutoff (vhs) activity of both UL41 alleles was determined. The UL41*Cys allele had no vhs activity, which suggests that enhanced fitness in vitro for MDV with inactive vhs was due to reduced degradation of viral transcripts. The in vitro competition assay should be applicable to other MDV genes and mutations.

  7. In vitro immunomodulation of a whole blood IFN-γ release assay enhances T cell responses in subjects with latent tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Rajiv L Gaur

    Full Text Available BACKGROUND: Activation of innate immunity via pathogen recognition receptors (PRR modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ release assays (IGRAs are functional T cell assays used to diagnose latent tuberculosis infection (LTBI; however, novel approaches are needed to improve their sensitivity. METHODS: In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube with Toll-like receptor agonists poly(I:C, LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls. RESULTS: In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells. CONCLUSIONS: In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

  8. In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions); Effets in vitro du piracetam sur la radiosensibilite des cellules hypoxiques (adapatation du test au MTT aux conditions d`hypoxie)

    Energy Technology Data Exchange (ETDEWEB)

    Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T. [Universitaire Instelling Antwerpen, Antwerp (Belgium); Lagarde, P. [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Institut Bergonie, 33 - Bordeaux (France); Pooter, C.M.J. de [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Hopital de Middelheim, Anvers (Belgium); Chomy, F. [Institut Bergonie, 33 - Bordeaux (France)

    1995-12-31

    This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs.

  9. Evaluation of Dimethyl Methylphosphonate and Exo-Tetrahydrodi- (Cyclopentadiene) in a Battery of in Vitro Short-Term Assays

    Science.gov (United States)

    1983-02-01

    sites of both chromatids of a chromosome . These include breaks, fragments, markers (i.e., dicentrics , rings), and gaps. 2. Chromatid aberrations - changes...Ames Salmonella/mammalian microsomal mutagenicity assay, the CHO/HGPRT gene mutation assay, the CHO/sister chromatid exchange assay, the CHO/ chromosome ...exhibited a low clastogenic activity in the CHO/ chromosome aberrations assay and was negative in the remaining four assays. Compound JP-1O also had a

  10. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.

    Science.gov (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng

    2015-08-03

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  11. Evaluation of Abelmoschus moschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using in vitro assays

    Directory of Open Access Journals (Sweden)

    Qureshi Insaf A

    2011-08-01

    Full Text Available Abstract Background Abelmoschus moschatus Medik. leaves and seeds are considered as valuable traditional medicine. The aromatic seeds of this plant are aphrodisiac, ophthalmic, cardio tonic, antispasmodic and used in the treatment of intestinal complaints and check queasiness. To give a scientific basis for traditional usage of this medicinal plant, the seed and leaf extracts were evaluated for their antioxidant, free radical scavenging, antimicrobial and antiproliferative activities. Methods In this study, antioxidant, antimicrobial and antiproliferative activities of A. moschatus extracts were evaluated in a series of in vitro assay involving free radicals, reactive oxygen species and their IC50 values were also determined. The antioxidant activities of the seed and leaf extracts of A. moschatus were determined by total antioxidant, DPPH, and ferrous reducing antioxidant property (FRAP methods. In addition, the antiproliferative activity was also evaluated using colorectal adenocarcinoma and retinoblastoma human cancer cell lines. Moreover, six bacterial reference strains, two gram-positive (Bacillus subtilis and Staphylococcus aureus, four gram-negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Salmonella enterica paratyphi and one fungal strain (Candida albicans were used to evaluate its antimicrobial activity. Results The results from this study showed that the antioxidant activities of A. moschatus as determined by the total phenol, flavonoids, total antioxidant and FRAP methods were higher in leaf than that of the seed extracts. On the other hand, the aqueous overnight seed extract (AMS-I has shown significant radical scavenging activity as in 1, 1- Diphenyl-2-picrylhydrazyl (DPPH, hydrogen peroxide, hydroxyl radical, superoxide and lipid peroxidation as compared to other seed and leaf extracts. The AMS-I and AML-IV have shown activity against six and seven microorganisms respectively. Simulteneously, AMS-IV and AML

  12. A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans.

    Science.gov (United States)

    Wolf, Babette; Morgan, Hannah; Krieg, Jennifer; Gani, Zaahira; Milicov, Adriana; Warncke, Max; Brennan, Frank; Jones, Stewart; Sims, Jennifer; Kiessling, Andrea

    2012-12-01

    The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine

  13. Development of an in vitro assay and demonstration of Plasmodium berghei liver-stage inhibition by TRAP-specific CD8+ T cells.

    Directory of Open Access Journals (Sweden)

    Rhea J Longley

    Full Text Available The development of an efficacious vaccine against the Plasmodium parasite remains a top priority. Previous research has demonstrated the ability of a prime-boost virally vectored sub-unit vaccination regimen, delivering the liver-stage expressed malaria antigen TRAP, to produce high levels of antigen-specific T cells. The liver-stage of malaria is the main target of T cell-mediated immunity, yet a major challenge in assessing new T cell inducing vaccines has been the lack of a suitable pre-clinical assay. We have developed a flow-cytometry based in vitro T cell killing assay using a mouse hepatoma cell line, Hepa1-6, and Plasmodium berghei GFP expressing sporozoites. Using this assay, P. berghei TRAP-specific CD8+ T cell enriched splenocytes were shown to inhibit liver-stage parasites in an effector-to-target ratio dependent manner. Further development of this assay using human hepatocytes and P. falciparum would provide a new method to pre-clinically screen vaccine candidates and to elucidate mechanisms of protection in vitro.

  14. Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications.

    Science.gov (United States)

    Pessina, Augusto; Bonomi, Arianna; Baglio, Carolina; Cavicchini, Loredana; Gribaldo, Laura

    2009-09-01

    The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.

  15. IZDELAVA TISKALNIKA 3D

    OpenAIRE

    Brdnik, Lovro

    2015-01-01

    Diplomsko delo analizira trenutno stanje 3D tiskalnikov na trgu. Prikazan je razvoj in principi delovanja 3D tiskalnikov. Predstavljeni so tipi 3D tiskalnikov, njihove prednosti in slabosti. Podrobneje je predstavljena zgradba in delovanje koračnih motorjev. Opravljene so meritve koračnih motorjev. Opisana je programska oprema za rokovanje s 3D tiskalniki in komponente, ki jih potrebujemo za izdelavo. Diploma se oklepa vprašanja, ali je izdelava 3D tiskalnika bolj ekonomična kot pa naložba v ...

  16. In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy.

    Science.gov (United States)

    Yao, Jianhua; Gao, Qian; Mi, Qili; Li, Xuemei; Miao, Mingming; Cheng, Peng; Luo, Ying

    2013-08-15

    The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200μg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.

  17. MTS colorimetric assay in combination with a live-dead assay for testing encapsulated L929 fibroblasts in alginate poly-L-lysine microcapsules in vitro

    NARCIS (Netherlands)

    Bunger, CM; Jahnke, A; Stange, J; de Vos, P; Hopt, UT

    2002-01-01

    Biomaterials such as applied in microcapsules may have harmful effects on encapsulated cells. Up to now, there are no adequate assays available for testing the function and viability of cells in capsules. Therefore, we investigated whether the combination of MTS proliferation assay and live-dead via

  18. A 155-plex High-Throughput In Vitro Coregulator Binding Assay for (Anti-) Estrogenicity Testing Evaluated with 23 Reference Compounds

    NARCIS (Netherlands)

    Wang, S.; Houtman, R.; Melchers, D.; Aarts, J.; Peijnenburg, A.A.C.M.; Beuningen, van R.; Rietjens, I.M.C.M.; Bovee, T.F.H.

    2013-01-01

    To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor a was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERa bind

  19. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

    NARCIS (Netherlands)

    Godoy, Patricio; Hewitt, Nicola J.; Albrecht, Ute; Andersen, Melvin E.; Ansari, Nariman; Bhattacharya, Sudin; Bode, Johannes Georg; Bolleyn, Jennifer; Borner, Christoph; Boettger, Jan; Braeuning, Albert; Budinsky, Robert A.; Burkhardt, Britta; Cameron, Neil R.; Camussi, Giovanni; Cho, Chong-Su; Choi, Yun-Jaie; Rowlands, J. Craig; Dahmen, Uta; Damm, Georg; Dirsch, Olaf; Teresa Donato, Maria; Dong, Jian; Dooley, Steven; Drasdo, Dirk; Eakins, Rowena; Ferreira, Karine Sa; Fonsato, Valentina; Fraczek, Joanna; Gebhardt, Rolf; Gibson, Andrew; Glanemann, Matthias; Goldring, Chris E. P.; Jose Gomez-Lechon, Maria; Groothuis, Geny M. M.; Gustavsson, Lena; Guyot, Christelle; Hallifax, David; Hammad, Seddik; Hayward, Adam; Haeussinger, Dieter; Hellerbrand, Claus; Hewitt, Philip; Hoehme, Stefan; Holzhuetter, Hermann-Georg; Houston, J. Brian; Hrach, Jens; Ito, Kiyomi; Jaeschke, Hartmut; Keitel, Verena; Kelm, Jens M.; Park, B. Kevin; Kordes, Claus; Kullak-Ublick, Gerd A.; LeCluyse, Edward L.; Lu, Peng; Luebke-Wheeler, Jennifer; Lutz, Anna; Maltman, Daniel J.; Matz-Soja, Madlen; McMullen, Patrick; Merfort, Irmgard; Messner, Simon; Meyer, Christoph; Mwinyi, Jessica; Naisbitt, Dean J.; Nussler, Andreas K.; Olinga, Peter; Pampaloni, Francesco; Pi, Jingbo; Pluta, Linda; Przyborski, Stefan A.; Ramachandran, Anup; Rogiers, Vera; Rowe, Cliff; Schelcher, Celine; Schmich, Kathrin; Schwarz, Michael; Singh, Bijay; Stelzer, Ernst H. K.; Stieger, Bruno; Stoeber, Regina; Sugiyama, Yuichi; Tetta, Ciro; Thasler, Wolfgang E.; Vanhaecke, Tamara; Vinken, Mathieu; Weiss, Thomas S.; Widera, Agata; Woods, Courtney G.; Xu, Jinghai James; Yarborough, Kathy M.; Hengstler, Jan G.

    2013-01-01

    This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being

  20. Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models.

    Science.gov (United States)

    Costa, Elisabete C; Gaspar, Vítor M; Coutinho, Paula; Correia, Ilídio J

    2014-08-01

    Three-dimensional (3D) cell culture models of solid tumors are currently having a tremendous impact in the in vitro screening of candidate anti-tumoral therapies. These 3D models provide more reliable results than those provided by standard 2D in vitro cell cultures. However, 3D manufacturing techniques need to be further optimized in order to increase the robustness of these models and provide data that can be properly correlated with the in vivo situation. Therefore, in the present study the parameters used for producing multicellular tumor spheroids (MCTS) by liquid overlay technique (LOT) were optimized in order to produce heterogeneous cellular agglomerates comprised of cancer cells and stromal cells, during long periods. Spheroids were produced under highly controlled conditions, namely: (i) agarose coatings; (ii) horizontal stirring, and (iii) a known initial cell number. The simultaneous optimization of these parameters promoted the assembly of 3D characteristic cellular organization similar to that found in the in vivo solid tumors. Such improvements in the LOT technique promoted the assembly of highly reproducible, individual 3D spheroids, with a low cost of production and that can be used for future in vitro drug screening assays.

  1. 3D and Education

    Science.gov (United States)

    Meulien Ohlmann, Odile

    2013-02-01

    Today the industry offers a chain of 3D products. Learning to "read" and to "create in 3D" becomes an issue of education of primary importance. 25 years professional experience in France, the United States and Germany, Odile Meulien set up a personal method of initiation to 3D creation that entails the spatial/temporal experience of the holographic visual. She will present some different tools and techniques used for this learning, their advantages and disadvantages, programs and issues of educational policies, constraints and expectations related to the development of new techniques for 3D imaging. Although the creation of display holograms is very much reduced compared to the creation of the 90ies, the holographic concept is spreading in all scientific, social, and artistic activities of our present time. She will also raise many questions: What means 3D? Is it communication? Is it perception? How the seeing and none seeing is interferes? What else has to be taken in consideration to communicate in 3D? How to handle the non visible relations of moving objects with subjects? Does this transform our model of exchange with others? What kind of interaction this has with our everyday life? Then come more practical questions: How to learn creating 3D visualization, to learn 3D grammar, 3D language, 3D thinking? What for? At what level? In which matter? for whom?

  2. Comparing an in vivo egg reduction test and in vitro egg hatching assay for different anthelmintics against Fasciola species, in cattle.

    Science.gov (United States)

    Arafa, Waleed M; Shokeir, Khalid M; Khateib, Abdelrahman M

    2015-11-30

    This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A-E). On day zero; groups A-D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0 μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100 μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect.

  3. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    NARCIS (Netherlands)

    Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; Rotterdam, van B.; Derzelle, S.

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely o

  4. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes.

    Science.gov (United States)

    Trubetskoy, Olga V; Gibson, Jasmin R; Marks, Bryan D

    2005-02-01

    Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

  5. The relevance of chemical interactions with CYP17 enzyme activity: Assessment using a novel in vitro assay

    Energy Technology Data Exchange (ETDEWEB)

    Roelofs, Maarke J.E., E-mail: m.j.e.roelofs@uu.nl [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands); Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Piersma, Aldert H. [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands); Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Berg, Martin van den; Duursen, Majorie B.M. van [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands)

    2013-05-01

    The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. - Highlights: ► DHEA, produced by CYP17, plays a key role in sexual functioning and development. ► No rapid screening assay for effects on CYP17 is available yet. ► A novel assay using porcine adrenal cortex microsomes (PACMs) was described. ► Endocrine disrupting compounds (EDCs) targeting CYP17 interact differently with CYP19. ► A

  6. TEHNOLOGIJE 3D TISKALNIKOV

    OpenAIRE

    Kolar, Nataša

    2016-01-01

    Diplomsko delo predstavi razvoj tiskanja skozi čas. Podrobneje so opisani 3D tiskalniki, ki uporabljajo različne tehnologije 3D tiskanja. Predstavljene so različne tehnologije 3D tiskanja, njihova uporaba in narejeni prototipi oz. končni izdelki. Diplomsko delo opiše celoten postopek, od zamisli, priprave podatkov in tiskalnika do izdelave prototipa oz. končnega izdelka.

  7. A novel mycobacterial In Vitro infection assay identifies differences of induced macrophage apoptosis between CD4+ and CD8+ T cells

    Science.gov (United States)

    Nkwouano, Vanesa; Witkowski, Sven; Rehberg, Nidja; Kalscheuer, Rainer; Nausch, Norman; Mayatepek, Ertan

    2017-01-01

    Macrophages are natural host cells for pathogenic mycobacteria, like Mycobacterium tuberculosis (M.tb). Immune surveillance by T cells and interaction with M.tb infected macrophages is crucial for protection against M.tb reactivation and development of active tuberculosis. Several factors play a role in the control of M.tb infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional in vitro assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional in vitro assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a Mycobacterium bovis BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated in vitro and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. M.tb protein specific CD4+ and CD8+ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4+ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional in vitro assay has the potential to contribute to the

  8. Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays

    DEFF Research Database (Denmark)

    Weisser, Johan Juhl; Hansen, Cecilie Hurup; Poulsen, Rikke

    2016-01-01

    Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination...... of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis...

  9. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    OpenAIRE

    Ågren, Joakim; Raditijo A Hamidjaja; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; van Rotterdam, Bart; Derzelle, Sylviane

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. ...

  10. Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration.

    Science.gov (United States)

    Goncharova, Elena A; Goncharov, Dmitry A; Krymskaya, Vera P

    2006-01-01

    Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.

  11. Improving the in vitro ethoxyresorufin-O-deethylase (EROD) assay with RTL-W1 by metabolic normalization and use of β-naphthoflavone as the reference substance.

    Science.gov (United States)

    Heinrich, Patrick; Diehl, Ulrike; Förster, Franziska; Braunbeck, Thomas

    2014-08-01

    The ethoxyresorufin-O-deethylase (EROD) assay is a widely applied method for the evaluation of the dioxin-like activity of single substances and environmental samples. As for most enzyme assays, the specific activity is normally related to total protein contents, the determination of which has clear limitations in high-throughput assays. EROD induction potentials are usually expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents, a substance highly toxic to humans. In order to compensate for these shortcomings, two modifications of the EROD protocol are proposed: (1) EROD activity is normalized to the metabolic activity of the cells as determined by a modified thiazolyl blue tetrazolium (MTT) assay and expressed as metabolic cell equivalents (MCE) based on MTT data rather than to protein contents. Via MCE data, cytotoxicity information can always be reported in parallel to EROD data; with the protocol presented here, MTT and EROD data are collected simultaneously. (2) Among several reference substances tested (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), β-naphthoflavone and benzo[a]pyrene), β-naphthoflavone proved to be the most suitable reference for the routine in vitro EROD assay, although TCDD has generally been preferred for purely scientific reasons.

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

    Science.gov (United States)

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-11-15

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

  13. 3D virtuel udstilling

    DEFF Research Database (Denmark)

    Tournay, Bruno; Rüdiger, Bjarne

    2006-01-01

    3d digital model af Arkitektskolens gård med virtuel udstilling af afgangsprojekter fra afgangen sommer 2006. 10 s.......3d digital model af Arkitektskolens gård med virtuel udstilling af afgangsprojekter fra afgangen sommer 2006. 10 s....

  14. Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

    Science.gov (United States)

    Alépée, N; Hibatallah, J; Klaric, M; Mewes, K R; Pfannenbecker, U; McNamee, P

    2016-06-01

    Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods.

  15. Radiosensitivity of glial progenitor cells of the perinatal and adult rat optic nerve studied by an in vitro clonogenic assay

    Energy Technology Data Exchange (ETDEWEB)

    Maazen, R.W.M. van der; Verhagen, I.; Kleiboer, B.J.; Kogel, A.J. van der (Nijmegen University (Netherlands). Institute of Radiotherapy)

    1991-04-01

    The cellular basis of radiation-induced demyelination and white matter necrosis of the central nervous system (CNS), is poorly understood. Glial cells responsible for myelination in the CNS might be the target cells of this type of damage. Glial cells with stem cell properties derived from the perinatal and adult rat CNS can be cultured in vitro. These cells are able to differentiate into oligodendrocytes or type-2 astrocytes (O-2A) depending on the culture conditions. Growth factors produced by monolayers of type-1 astrocytes inhibit premature differentiation of O-2A progenitor cells and allow colony formation. A method which employs these monolayers of type-1 astrocytes to culture O-2A progenitor cells has been adapted to allow the analysis of colonies of surviving cells after X-irradiation. In vitro survival curves were obtained for glial progenitor cells derived from perinatal and adult optic nerves. The intrinsic radiosensitivity of perinatal and adult O-2A progenitor cells showed a large difference. Perinatal O-2A progenitor cells are quite radiosensitive, in contrast to adult O-2A progenitor cells. For both cell types an inverse relationship was found between the dose and the size of colonies derived from surviving cells. Surviving O-2A progenitor cells maintain their ability to differentiate into oligo-dendrocytes or type-2 astrocytes. This system to assess radiation-induced damage to glial progenitor cells in vitro systems to have a great potential in unraveling the cellular basis of radiation-induced demyelinating syndromes of the CNS. (author). 28 refs.; 4 figs.; 1 tab.

  16. Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens.

    Science.gov (United States)

    Gutendorf, B; Westendorf, J

    2001-09-14

    Many chemicals in surface waters and sediments have recently been discovered to have estrogenic/antiestrogenic activity. Among these compounds, known as 'endocrine disrupters', are natural and synthetic hormones, phytoestrogenes and a variety of industrial chemicals, such as certain detergents and pesticides. These substances are supposed to affect the development and reproduction in wildlife and humans and may also be involved in the induction of cancer. In order to assess the estrogenic/antiestrogenic potential of pure compounds and complex environmental samples we compared an array of in vitro test systems, (i) two luciferase reporter gene assays using transgenic human MVLN cells (derived from MCF-7 cells) and HGELN cells (derived from HeLa cells); (ii) a competitive binding assay with recombinant human estrogen receptors (ER) alpha and beta; and (iii) a proliferation assay with MCF7-cells (E-Screen). The sensitivity of the assays for 17-beta-estradiol decreased in the order: MVLN-cells=E-Screen>HGELN-cells>binding to ER-alpha>binding to ER-beta. A good correlation was obtained between the estrogenic potencies of 11 compounds (17-beta-estradiol (E(2)), estrone (E(1)), estriol (E(3)), ethinylestradiol (EE(2)), diethylstilbestrol (DES), coumestrol, beta-sitosterol, genistein, 4-nonylphenol, 4-octylphenol, bisphenol A) in the three tissue culture assays. The relative potencies of the compounds obtained by the cell free binding assays were one to two orders of magnitude higher compared with the cell culture assays. The phytoestrogens showed a preference to bind to ER-beta, but only genistein showed a much lower activity in the E-Screen (growth induction in breast cancer cells) compared with the luciferase induction in MVLN and HGELN-cells.

  17. In vitro xanthine oxidase and albumin denaturation inhibition assay of Barringtonia racemosa L. and total phenolic content analysis for potential anti-inflammatory use in gouty arthritis

    Science.gov (United States)

    Osman, Nurul Izzati; Sidik, Norrizah Jaafar; Awal, Asmah; Adam, Nurul Athirah Mohamad; Rezali, Nur Inani

    2016-01-01

    Aim: This study was conducted to evaluate the in vitro anti-inflammatory activities and total phenolic content (TPC) of methanolic extracts of infloresence axes, endosperms, leaves, and pericarps of Barringtonia racemosa L. Methods: The anti-inflammatory study was conducted by assessing the potential through xanthine oxidase (XO) and albumin denaturation inhibition assays. Meanwhile, the TPC in the extracts were assessed by Folin-Ciocalteu assay. Results: In the XO inhibition assay, the infloresence axes extract was found to exert the highest inhibition capacity at 0.1% (w/v) with 59.54 ± 0.001% inhibition followed by leaves (58.82 ± 0.001%), pericarps (57.99 ± 0.003%), and endosperms (57.20 ± 0.003%) extracts. Similarly in the albumin denaturation inhibition assay, the infloresence axes extract had shown the greatest inhibition capacity with 70.58 ± 0.004% inhibition followed by endosperms (66.80 ± 0.024%), leaves (65.29 ± 0.006%), and pericarps extracts (43.33 ± 0.002%). Meanwhile, for TPC analysis, leaves extract was found to have the highest phenolic content (53.94 ± 0.000 mg gallic acid equivalent [GAE]/g DW) followed by infloresence axes (31.54 ± 0.001 mg GAE/g DW), endosperms (22.63 ± 0.001 mg GAE/g DW), and the least was found in pericarps (15.54 ± 0.001 mg GAE/g DW). Conclusion: The results indeed verified the in vitro anti-inflammatory activities of B. racemosa and supported its potential to be used in alleviating gouty arthritis and XO-related diseases. PMID:27757263

  18. Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment.

    Science.gov (United States)

    Bryce, Steven M; Avlasevich, Svetlana L; Bemis, Jeffrey C; Tate, Matthew; Walmsley, Richard M; Saad, Frédéric; Van Dijck, Kris; De Boeck, Marlies; Van Goethem, Freddy; Lukamowicz-Rajska, Magdalena; Elhajouji, Azeddine; Dertinger, Stephen D

    2013-04-01

    An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥ 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints-relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity.

  19. Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus.

    Science.gov (United States)

    Alonso-Díaz, M A; Torres-Acosta, J F J; Sandoval-Castro, C A; Hoste, H

    2011-09-27

    The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 μg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (Palicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L(3) larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.

  20. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

    Science.gov (United States)

    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

    2014-01-01

    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was

  1. In Vitro genotoxic and antigenotoxic studies of Thai Noni fruit juice by chromosomal aberration and sister chromatid exchange assays in human lymphocytes

    Directory of Open Access Journals (Sweden)

    Treetip Ratanavalachai

    2008-09-01

    Full Text Available The genotoxic and antigenotoxic effects of Noni fruit juice produced in Thailand have been studied in human lymphocytes for chromosome aberration assay and sister chromatid exchange (SCE assay in vitro. Treatment of Noni fruit juice(3.1-50 mg/ml alone for 3 h did not significantly induce chromosomal aberration or SCE (p<0.05. Noni fruit juice at 6.2 mg/ml is the optimum dose for cell survival and cell replication as demonstrated by the highest value of mitotic index and proliferation index (P.I.. Interestingly, pretreatment of Noni fruit juice at the same concentration of 6.2 mg/ml for 2 hfollowed by mitomycin C treatment at 3 μg/ml for 2 h significantly reduced SCE level induced by mitomycin C (p<0.05. However, these treatments did not show significant decrease in chromatid-type aberrations. Our data indicate that Thai Noni fruit juice is not genotoxic against human lymphocytes in vitro. In addition, pretreatment of Noni fruit juice at 6.2 mg/ml demonstrated no anticlastogenic effect while had some antigenotoxic effects as demonstrated by significant decrease in the SCE level induced by mitomycin C (p<0.05. Therefore, the optimum dose of Noni fruit juice used as a traditional medicine is required and needs to be studied further for the benefit of human health.

  2. Risk assessment of human myelotoxicity of anticancer drugs: a predictive model and the in vitro colony forming unit granulocyte/macrophage (CFU-GM) assay.

    Science.gov (United States)

    Masubuchi, N

    2006-02-01

    Myelotoxicity is one of the major limitations to the use of anticancer drugs. It is desirable to evaluate human myelotoxicity before a Phase I study, however, this is difficult because of the differences in susceptibility between humans and animals. The purpose of this study was to establish a reliable method to predict the human maximum tolerated dose (MTD) of five camptothecin derivatives: SN-38, DX-8951f, topotecan (TPT), 9-aminocamptothecin (9-AC), and camptothecin (CAM). The myelotoxicity of camptothecin derivatives was evaluated on bone marrow from mice, dogs, and humans using a 14-day colony-forming unit-granulocyte/macrophage (CFU-GM) assay to determine the 50%, 75%, and 90% inhibitory concentration values (IC50, IC75, and IC90, respectively). Then, using human and murine IC90 values for myelotoxicity of these compounds, in vivo toxicological data, and pharmacokinetic parameters (data referred to the literature), human MTDs were predicted retrospectively. The mechanism-based prediction model which is proposed uses the in vitro CFU-GM assay and in vivo parameters on the basis of free fraction of area under the concentration-curve (AUC) at the MTD (r2 = 0.887) and suggests that the human MTDs were well predicted for the five camptothecin derivatives by this model rather than by other models. The application of this model for in vitro hematotoxicology could be very useful in the development of new anticancer agents.

  3. Development of a combined in vitro cell culture--quantitative PCR assay for evaluating the disinfection performance of pulsed light for treating the waterborne enteroparasite Giardia lamblia.

    Science.gov (United States)

    Garvey, Mary; Stocca, Alessia; Rowan, Neil

    2014-09-01

    Giardia lamblia is a flagellated protozoan parasite that is recognised as a frequent cause of water-borne disease in humans and animals. We report for the first time on the use of a combined in vitro HCT-8 cell culture-quantitative PCR assay for evaluating the efficacy of using pulsed UV light for treating G. lamblia parasites. Findings showed that current methods that are limited to using vital stains before and after cyst excystation are not appropriate for monitoring or evaluating cyst destruction post PUV-treatments. Use of the human ileocecal HCT-8 cell line was superior to that of the human colon Caco-2 cell line for in vitro culture and determining PUV sensitivity of treated cysts. G. lamblia cysts were also shown to be more resistant to PUV irradiation compared to treating similar numbers of Cryptosporidium parvum oocysts. These observations also show that the use of this HCT-8 cell culture assay may replace use of animal models for determining disinfection performances of PUV for treating both C. parvum and G. lamblia.

  4. An in vitro skin sensitization assay termed EpiSensA for broad sets of chemicals including lipophilic chemicals and pre/pro-haptens.

    Science.gov (United States)

    Saito, Kazutoshi; Takenouchi, Osamu; Nukada, Yuko; Miyazawa, Masaaki; Sakaguchi, Hitoshi

    2016-12-10

    To evaluate chemicals (e.g. lipophilic chemicals, pre/pro-haptens) that are difficult to correctly evaluate using in vitro skin sensitization tests (e.g. DPRA, KeratinoSens or h-CLAT), we developed a novel in vitro test termed "Epidermal Sensitization Assay: EpiSensA" that uses reconstructed human epidermis. This assay is based on the induction of multiple marker genes (ATF3, IL-8, DNAJB4 and GCLM) related to two keratinocyte responses (inflammatory or cytoprotective) in the induction of skin sensitization. Here, we first confirmed the mechanistic relevance of these marker genes by focusing on key molecules that regulate keratinocyte responses in vivo (P2X7 for inflammatory and Nrf2 for cytoprotective responses). The up-regulation of ATF3 and IL-8, or DNAJB4 and GCLM induced by the representative sensitizer 2,4-dinitrochlorobenzene in human keratinocytes was significantly suppressed by a P2X7 specific antagonist KN-62, or by Nrf2 siRNA, respectively, which supported mechanistic relevance of marker genes. Moreover, the EpiSensA had sensitivity, specificity and accuracy of 93%, 100% and 93% for 29 lipophilic chemicals (logKow≥3.5), and of 96%, 75% and 88% for 43 hydrophilic chemicals including 11 pre/pro-haptens, compared with the LLNA. These results suggested that the EpiSensA could be a mechanism-based test applicable to broad sets of chemicals including lipophilic chemicals and pre/pro-haptens.

  5. The in vitro effects of artificial and natural sweeteners on the immune system using whole blood culture assays.

    Science.gov (United States)

    Rahiman, F; Pool, E J

    2014-01-01

    This article investigates the effects of commercially available artificial (aspartame, saccharin, sucralose) and natural sweeteners (brown sugar, white sugar, molasses) on the immune system. Human whole blood cultures were incubated with various sweeteners and stimulated in vitro with either phytohemagglutinin or endotoxin. Harvested supernatants were screened for cytotoxicity and cytokine release. Results showed that none of the artificial or natural sweeteners proved to be cytotoxic, indicating that no cell death was induced in vitro. The natural sweetener, sugar cane molasses (10 ug/mL), enhanced levels of the inflammatory biomarker IL-6 while all artificial sweeteners (10 ug/mL) revealed a suppressive effect on IL-6 secretion (P sweeteners under stimulatory conditions reduced levels of the biomarker of humoral immunity, Interleukin-10 (P < 0.001). The cumulative suppression of Interleukin-6 and Interleukin-10 levels induced by sucralose may contribute to the inability in mounting an effective humoral response when posed with an exogenous threat.

  6. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine

    2013-01-01

    on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S......Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely......-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal...

  7. Blender 3D cookbook

    CERN Document Server

    Valenza, Enrico

    2015-01-01

    This book is aimed at the professionals that already have good 3D CGI experience with commercial packages and have now decided to try the open source Blender and want to experiment with something more complex than the average tutorials on the web. However, it's also aimed at the intermediate Blender users who simply want to go some steps further.It's taken for granted that you already know how to move inside the Blender interface, that you already have 3D modeling knowledge, and also that of basic 3D modeling and rendering concepts, for example, edge-loops, n-gons, or samples. In any case, it'

  8. Unique Nanoparticle Optical Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Screening and Ranking

    Science.gov (United States)

    Nanoparticles (NPs) are novel materials having at least one dimension less than 100 nm and display unique physicochemical properties due to their nanoscale size. An emphasis has been placed on developing high throughput screening (HTS) assays to characterize and rank the toxiciti...

  9. Unique Nanoparticle Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Testing and Ranking

    Science.gov (United States)

    Nanomaterials are a diverse collection of novel materials that exhibit at least one dimension less than 100 nm and display unique chemical and physical properties due to their nanoscale size. An emphasis has been put on developing high throughput screening (HTS) assays to charac...

  10. Endocrine potency of wastewater: Contents of endocrine disrupting chemicals and effects measured by in vivo and in vitro assays

    DEFF Research Database (Denmark)

    Kusk, Kresten Ole; Krüger, Tanja; Long, Manhai

    2011-01-01

    properties: phthalate metabolites, parabens, industrial phenols, ultraviolet screens, and natural and synthetic steroid estrogens. The endocrine disrupting bioactivity and toxicity of the extracts were analyzed in cell culture assay for the potency to affect the function of the estrogen, androgen, aryl...

  11. Effect of oxygen plasma treatment on surface charge and wettability of PVC blood bag—In vitro assay

    Science.gov (United States)

    Khorasani, M. T.; Mirzadeh, H.

    2007-06-01

    Wettability and zeta potential studies were performed to characterize the hydrophobicity and surface charge of PVC blood bag samples and evaluate the effect of these properties on fibroblast cells growth. The surface properties of PVC and plasma treated PVC were compared by water drop contact angle and zeta potential measurement. Light microscopy was used to study the behavior of cell attachment and growth on these surfaces. Water drop contact angle measurement shows that the plasma treated PVC becomes more hydrophilic and wettability increased. Zeta potential and in vitro cell culture measurements noticed that the plasma treated PVC surface is more negatively charge and consequently attachment of the L929 fibroblast cells decreased on this surface.

  12. Effect of oxygen plasma treatment on surface charge and wettability of PVC blood bag-In vitro assay

    Energy Technology Data Exchange (ETDEWEB)

    Khorasani, M.T. [Biomaterial Department of Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran (Iran, Islamic Republic of)]. E-mail: M.Khorasani@ippi.ac.ir; Mirzadeh, H. [Biomaterial Department of Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran (Iran, Islamic Republic of)

    2007-06-15

    Wettability and zeta potential studies were performed to characterize the hydrophobicity and surface charge of PVC blood bag samples and evaluate the effect of these properties on fibroblast cells growth. The surface properties of PVC and plasma treated PVC were compared by water drop contact angle and zeta potential measurement. Light microscopy was used to study the behavior of cell attachment and growth on these surfaces. Water drop contact angle measurement shows that the plasma treated PVC becomes more hydrophilic and wettability increased. Zeta potential and in vitro cell culture measurements noticed that the plasma treated PVC surface is more negatively charge and consequently attachment of the L929 fibroblast cells decreased on this surface.

  13. 3D Digital Modelling

    DEFF Research Database (Denmark)

    Hundebøl, Jesper

    wave of new building information modelling tools demands further investigation, not least because of industry representatives' somewhat coarse parlance: Now the word is spreading -3D digital modelling is nothing less than a revolution, a shift of paradigm, a new alphabet... Research qeustions. Based...... on empirical probes (interviews, observations, written inscriptions) within the Danish construction industry this paper explores the organizational and managerial dynamics of 3D Digital Modelling. The paper intends to - Illustrate how the network of (non-)human actors engaged in the promotion (and arrest) of 3......D Modelling (in Denmark) stabilizes - Examine how 3D Modelling manifests itself in the early design phases of a construction project with a view to discuss the effects hereof for i.a. the management of the building process. Structure. The paper introduces a few, basic methodological concepts...

  14. An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds.

    Science.gov (United States)

    Festag, Matthias; Viertel, Bruno; Steinberg, Pablo; Sehner, Claudia

    2007-12-01

    The embryonic stem cell test (EST) developed by Spielmann et al. [Spielmann, H., Pohl, I., Doering, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro. Toxicol. 10, 119-127] is currently the most promising in vitro assay to predict the embryotoxic potential of compounds. In this assay the disturbance of the differentiation of embryonic stem (ES) cells into contracting cardiomyocytes by test compounds as well as the direct cytotoxicity of the test compounds on ES cells and 3T3 fibroblasts is analyzed. On the basis of these results and by applying a biostatistical prediction model (PM) [Genschow, E., Scholz, G., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., Spielmann, H., 2000. Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study. In Vitr. Mol. Toxicol. 13, 51-66; Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209-244; Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151-176] test compounds can be classified as non-embryotoxic, weakly or strongly embryotoxic. In order to introduce a further endpoint into the EST, the disturbance of vasculogenesis and/or angiogenesis, a protocol to differentiate ES cells into endothelial cells, was established in the accompanying paper. PECAM-1 and VE

  15. Usefulness of in vitro combination assays of mitochondrial dysfunction and apoptosis for the estimation of potential risk of idiosyncratic drug induced liver injury.

    Science.gov (United States)

    Goda, Keisuke; Takahashi, Tadakazu; Kobayashi, Akio; Shoda, Toshiyuki; Kuno, Hideyuki; Sugai, Shoichiro

    2016-01-01

    Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.

  16. Professional Papervision3D

    CERN Document Server

    Lively, Michael

    2010-01-01

    Professional Papervision3D describes how Papervision3D works and how real world applications are built, with a clear look at essential topics such as building websites and games, creating virtual tours, and Adobe's Flash 10. Readers learn important techniques through hands-on applications, and build on those skills as the book progresses. The companion website contains all code examples, video step-by-step explanations, and a collada repository.

  17. AE3D

    Energy Technology Data Exchange (ETDEWEB)

    2016-06-20

    AE3D solves for the shear Alfven eigenmodes and eigenfrequencies in a torodal magnetic fusion confinement device. The configuration can be either 2D (e.g. tokamak, reversed field pinch) or 3D (e.g. stellarator, helical reversed field pinch, tokamak with ripple). The equations solved are based on a reduced MHD model and sound wave coupling effects are not currently included.

  18. Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity

    NARCIS (Netherlands)

    Louisse, J.; Gönen, S.; Rietjens, I.M.C.M.; Verwei, M.

    2011-01-01

    The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental toxicit

  19. Effect of phytate reduction of sorghum, through genetic modification, on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay, Caco-2 cell uptake assay, and suckling rat pup absorption model.

    Science.gov (United States)

    Kruger, Johanita; Taylor, John R N; Du, Xiaogu; De Moura, Fabiana F; Lönnerdal, Bo; Oelofse, André

    2013-11-15

    Improved iron and zinc availability from sorghum, a commonly consumed staple, will benefit many malnourished communities in rural Africa burdened with high prevalence of iron and zinc deficiency. This research compared the effect of genetic phytate reduction in sorghum on iron and zinc bioaccessibility and uptake measured by in vitro dialysability and Caco-2 cell uptake assays to that of iron and zinc absorption measured by a suckling rat pup model. The phytate reduction (80-86%) in these sorghums significantly increased zinc availability. The Caco-2 cell method, but not the dialysability assay, proved useful in estimating zinc absorption. The measured increase in iron availability differed between the methods, possibly due to the effect of varying mineral (Ca, Fe, Zn, P) contents of the sorghums. This effect was most prominent in the iron uptake results. More research is needed to determine the effect of naturally occurring variations in mineral contents of sorghum on the iron uptake by Caco-2 cells.

  20. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    Science.gov (United States)

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K.V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-01-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors. PMID:27995961

  1. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    Science.gov (United States)

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-12-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  2. Development and Testing of an In Vitro Assay for Screening of Potential Therapeutic Agents Active against Na Channel Neurotoxins

    Science.gov (United States)

    1991-04-12

    to produce approximately half-maximal effects mediated through these different sodium channel sites in the assay. Thus, the binding of [3HJBTX-B should...experiments with I3H]STX, yielding the unexpected result that effects of HM-197 are not mediated through the TTX/STX sodium channel binding site. Additional...Scorpion toxin; Screening; nA Pyrethroids; Radioligand binding; Synaptoneurosomes; RA 1 ; nA I ~ I ITherapeutic agents; Sodium channel 19. ABSTRACT

  3. An enzymatic fluorimetric assay for glucose-6-phosphate: application in an in vitro Warburg-like effect

    OpenAIRE

    Zhu, Aiping; Romero, Roberto; Petty, Howard R.

    2009-01-01

    Recently, there has been a resurgence of interest in the regulatory role of cell metabolism in tumor biology and immunology. To assess changes in metabolite levels in cell populations and tissues, especially from small clinical samples, highly sensitive assays are required. Based upon glucose 6-phosphate’s reaction and the diaphorase-resazurin amplifying system, we have developed a fluorescence methodology to measure glucose 6-phosphate concentrations in cell extracts. In this approach, gluco...

  4. Genotoxicity evaluation of locally produced dental porcelain--an in vitro study using the Ames and Comet assays.

    Science.gov (United States)

    Noushad, Mohammed; Kannan, Thirumulu Ponnuraj; Husein, Adam; Abdullah, Haswati; Ismail, Abdul Rashid

    2009-09-01

    The aim of this study was to determine the genotoxicity of a locally produced dental porcelain (Universiti Sains Malaysia, Malaysia) using the Ames and Comet assays. In the Ames assay, four genotypic variants of the Salmonella strains (TA98, TA100, TA1537 and TA1535) carrying mutations in several genes were used. The dental porcelain was incubated with these four strains in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with locally produced dental porcelain was less than double as compared to that of negative control. In the Comet assay, L929 (CCL-1 ATCC, USA) mouse fibroblast cells were treated with the dental porcelain in three different concentrations along with concurrent negative and positive controls. The tail moment which was used as a measurement of DNA damage was almost equal to that of the negative control, suggesting that the locally produced dental porcelain did not induce any DNA damage. The results indicated that the locally produced dental porcelain is non-genotoxic under the present test conditions.

  5. Estrogen agonist/antagonist properties of dibenzyl phthalate (DBzP) based on in vitro and in vivo assays.

    Science.gov (United States)

    Zhang, Zhaobin; Hu, Ying; Zhao, Liang; Li, Jun; Bai, Huicheng; Zhu, Desheng; Hu, Jianying

    2011-11-10

    The most commonly used phthalates have been banned or restricted for use as plasticizers in toys in some countries because of their endocrine-disrupting properties. Dibenzyl phthalate (DBzP) has been proposed as a possible alternative for the banned/restricted phthalates. In this study, the estrogen agonist/antagonist properties of DBzP were predicted by molecular docking and confirmed by yeast estrogen screen (YES) and immature mouse uterotrophic assays. The YES assay results showed a dose-dependent increase in DBzP estrogen agonist activity from 10⁻⁶ to 10⁻⁴ M, and at concentrations from 1.95×10⁻⁶ M to higher, DBzP significantly inhibited the agonist activity of 10⁻⁹ M 17β-estradiol (E₂), inhibiting 10⁻⁹ M E₂ by 74.5% at its maximum effectiveness. The in vivo estrogen agonist/antagonist activities of DBzP were demonstrated in immature mouse uterotrophic assays. The antagonist activity of DBzP inhibited E₂-induced uterine growth promoted at 40 and 400 μg/kg bw (body weight) (Pestrogen agonist/antagonist potentials of benzyl butyl phthalate (BBP) by YES, and found both were weaker than those of DBzP, suggesting DBzP would be more toxic than BBP and should not be used as an alternative plasticizer.

  6. In vitro estrogen receptor binding of PCBs: measured activity and detection of hydroxylated metabolites in a recombinant yeast assay.

    Science.gov (United States)

    Layton, Alice C; Sanseverino, John; Gregory, Betsy W; Easter, James P; Sayler, Gary S; Schultz, T Wayne

    2002-05-01

    The estrogenic activities of 17beta-estradiol, biphenyl, chlorinated biphenyls, and Aroclor mixtures 1221, 1242, and 1248 were measured with a modified recombinant yeast estrogen assay (i.e., a Saccharomyces cerevisiae-based lac-Z (beta-galactosidase) reporter assay). Modifications of the assay included the use of glass vials instead of plastic microtiter plates and the addition of the medium and yeast before the test substrate. 14C-labeled compounds were used to follow improvements in the assay procedures. 14C-17beta-estradiol recovery from plastic microtiter plates and glass vials using the standard or the modified procedure was approximately 89%. However, 14C-4-CB (4-chlorobiphenyl) recovery was considerably less, ranging from 3% in plastic microtiter plates using the standard procedure to 26% in vials using the modified procedure. These results suggest that the toxicity of strongly hydrophobic chemicals may be underestimated. Using the modified yeast estrogen assay, full agonist activity was observed for 4-CB, 2,4,6-CB, and 2,5-CB while each of the Aroclor mixtures were only partial agonists. The equivalent EC50 values in ppm were in environmentally relevant concentrations for biphenyl (19 ppm), 4-CB (4.5 ppm), 2,5-CB (21 ppm), 2,4,6-CB (0.8 ppm), Aroclor 1221 (2.9 ppm), Aroclor 1242 (0.65 ppm), and Aroclor 1248 (2.3 ppm). Estrogen receptor binding for the individual PCB congeners was 25- to 650-fold less than the reported estrogen binding for the corresponding hydroxylated PCB metabolite. Gas chromatographic/mass spectrometric analysis of yeast extracts indicated that S. cerevisiae hydroxylated the individual PCB congeners in the ppb range. With the exception of biphenyl, the concentration of hydroxylated metabolites obtained from incubation of S. cerevisiae with PCB congeners was consistent with the concentration necessary to elicit a positive estrogen receptor-binding response. This work provides evidence that S. cerevisiae are capable of metabolic

  7. Conducting Polymer 3D Microelectrodes

    Directory of Open Access Journals (Sweden)

    Jenny Emnéus

    2010-12-01

    Full Text Available Conducting polymer 3D microelectrodes have been fabricated for possible future neurological applications. A combination of micro-fabrication techniques and chemical polymerization methods has been used to create pillar electrodes in polyaniline and polypyrrole. The thin polymer films obtained showed uniformity and good adhesion to both horizontal and vertical surfaces. Electrodes in combination with metal/conducting polymer materials have been characterized by cyclic voltammetry and the presence of the conducting polymer film has shown to increase the electrochemical activity when compared with electrodes coated with only metal. An electrochemical characterization of gold/polypyrrole electrodes showed exceptional electrochemical behavior and activity. PC12 cells were finally cultured on the investigated materials as a preliminary biocompatibility assessment. These results show that the described electrodes are possibly suitable for future in-vitro neurological measurements.

  8. Two-stage in vitro digestibility assay, a tool for formulating non-starch polysaccharide degrading enzyme combinations for commonly used feed ingredients of poultry rations

    Directory of Open Access Journals (Sweden)

    Y. Ramana Reddy

    2013-05-01

    Full Text Available Aim: An attempt was made to assess the effect of pure enzyme combinations with the objective of formulating customized enzyme mixtures based on sugar release when subjected to two-stage in vitro digestion assay. Materials and Methods: A two-stage in vitro digestibility assay was carried out for commonly used feed ingredients for poultry viz., maize, soy bean meal, sunflower cake, and de-oiled rice bran supplemented with three concentrations of xylanase (5000; 7500 and 10000 IU/kg, cellulase (50; 100 and 400 IU/kg and â-D-glucanase (100; 200 and 400 IU/kg were used to formulate various NSP enzymes combinations. In total 27 NSP enzyme combinations (3x3x3 were formulated and the sugar released due to NSP digestion was quantified by phenol sulphuric acid method. Results: The total sugar release was significantly (P<0.05 higher with supplementation of various enzymes combinations for maize, sunflower cake and de-oiled rice bran where as no significant (P<0.05 interaction of various NSP enzymes combinations was observed for soy bean meal. The NSP digestibility was highest in combination (xylanase-5000, cellulase-50 and â-D-glucanase-400 IU/kg, (xylanase-10000, cellulase-50 and â-D-glucanase-200 IU/kg and (xylanase-7500, cellulase- 100 and â-D-glucanase-100 IU/kg for maize, sunflower cake and de-oiled rice bran respectively. In case of sunflower cake, significant (P<0.01 three way interaction was observed among the xylanase, cellulose, and â-D-glucanase enzymes and the two-way interactions between the enzymes were also significant (P<0.01. Conclusion: It is concluded that 'n' number of non-starch Polysaccharide enzymes combinations can be screened for their efficiency to digest non-starch Polysaccharides present in various feed ingredients commonly used in poultry rations by employing two-stage in vitro digestibility assay as a tool. [Vet World 2013; 6(8.000: 525-529

  9. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    Science.gov (United States)

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-06-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

  10. Evaluation of leishmanicidal effect of Euphorbia erythadenia extract by in vitro leshmanicidal assay using promastigotes of Leishmania major

    Institute of Scientific and Technical Information of China (English)

    Reza Kazemi Oskuee; Mahmoud Reza Jaafari; Sara Amani; Mohammad Ramezani

    2014-01-01

    Objective:To evaluate leishmanicidal effects of Euphorbia erythadenia plant extract. Methods:Extraction was done using methanolic Soxhlet of dried and ground aerial parts of the plant. Then, five different extract concentrations, in addition of positive, negative and solvent controls were prepared and added to a 24-well plate containing 40 000 parasites/well. The extract concentrations were 1, 0.5, 0.25, 0.125 and 0.062 5 mg/mL. Amphotricin B (0.5 mg/mL) was used as positive control while negative control contained only culture medium. After 3 d incubation at 25 °C the amount of parasites in each well was determined on each day of experiment microscopially using Neubar chamber. Results:Soxhlet extract as well as amphotricin B killed all parasites at concentration of 1 mg/mL. The leshmanicidal activity of lower doses of extract was dose-dependent. The EC50 for Soxhlet extracts in dimethylsulfoxide was 0.30 mg/mL. The EC50 for Soxhlet extracts in methanol was 0.23 mg/mL. No obvious effects from the control solvent on the Leishmania major promastigotes were observed. Conclusions: The Soxhlet extract of Euphorbia erythadenia showed suitable leishmanicidal activity, especially in higher concentration fractions.

  11. Comparative Evaluation of Marginal and Internal Gap of Co-Cr Copings Fabricated from Conventional Wax Pattern, 3D Printed Resin Pattern and DMLS Tech: An In Vitro Study.

    Science.gov (United States)

    Bhaskaran, Eswaran; Azhagarasan, N S; Miglani, Saket; Ilango, T; Krishna, G Phani; Gajapathi, B

    2013-09-01

    Accuracy of the fit of the restoration has always remained as one of the primary factors in determining success of the restoration. A well fitting restoration needs to be accurate both along its margins and internal surface. This study was conducted to comparatively evaluate the marginal gap and internal gap of cobalt-chromium (Co-Cr) copings fabricated by conventional casting procedures and with direct metal laser sintering (DMLS) technique. Among the total of 30 test samples 10 cast copings were made from inlay casting wax and 10 from 3D printed resin pattern. 10 copings were obtained from DMLS technique. All the 30 test samples were then cemented sequentially on stainless steel model using pressure indicating paste and evaluated for vertical marginal gap in 8 predetermined reference areas. All copings were then removed and partially sectioned and cemented sequentially on same master model for evaluation of internal gap at 4 predetermined reference areas. Both marginal gap and internal gap were measured in microns using video measuring system (VMS2010F). The results obtained for both marginal and internal gap were statistically analyzed and the values fall within the clinically acceptable range. The DMLS technique had an edge over the other two techniques used, as it exhibited minimal gap in the marginal region which is an area of chief concern.

  12. Roughness and pH changes of enamel surface induced by soft drinks in vitro-applications of stylus profilometry, focus variation 3D scanning microscopy and micro pH sensor.

    Science.gov (United States)

    Fujii, Mie; Kitasako, Yuichi; Sadr, Alireza; Tagami, Junji

    2011-01-01

    This study aimed to evaluate enamel surface roughness (Ra) and pH before and after erosion by soft drinks. Enamel was exposed to a soft drink (cola, orange juice or green tea) for 1, 5 or 60 min; Ra was measured using contact-stylus surface profilometry (SSP) and non-contact focus variation 3D microscope (FVM). Surface pH was measured using a micro pH sensor. Data were analyzed at significance level of alpha=0.05. There was a significant correlation in Ra between SSP and FVM. FVM images showed no changes in the surface morphology after various periods of exposure to green tea. Unlike cola and orange juice, exposure to green tea did not significantly affect Ra or pH. A significant correlation was observed between surface pH and Ra change after exposure to the drinks. Optical surface analysis and micro pH sensor may be useful tools for non-damaging, quantitative assessment of soft drinks erosion on enamel.

  13. Simvastatin Results in a Dose-Dependent Toxic Effect on Spiral Ganglion Neurons in an In Vitro Organotypic Culture Assay

    Directory of Open Access Journals (Sweden)

    Katharina Leitmeyer

    2016-01-01

    Full Text Available Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme necessary for the production of mevalonate. They are widely used as cholesterol-lowering drugs. However, conflicting data about the effect of statins on neuronal cells has been published. To explore the effect of simvastatin on spiral ganglion neurons (SGNs, SG explants of 5-day-old rats were treated with increasing concentrations of simvastatin. In addition, SG explants were treated with mevalonate and with the combination of simvastatin and mevalonate. SGN number, length of the neurites, area of nonneuronal supporting cells, and neuronal survival were analyzed. Simvastatin treatment results in a significant dose-dependent decrease of SG neurite number, length of neurites, area of supporting cells, and SG neuronal survival compared to control. Interestingly, treatment with mevalonate in addition to simvastatin increased SG neuronal survival compared to simvastatin treatment only. However, treatment with mevalonate in addition to simvastatin did not influence SG neurite number, length of neurites, and area of supporting cells compared to simvastatin treatment only. Our results suggest a neurotoxic effect of simvastatin on SGNs in vitro. Neurotoxicity seems to be at least partially mediated by the mevalonate pathway. Therefore, caution is warranted to use simvastatin as a potential otoprotective drug.

  14. In-Vitro Studies on the Antioxidant Assay Profiling of Root of Withania somnifera L. (Ashwagandha Dunal: Part 2

    Directory of Open Access Journals (Sweden)

    Ajay Pal

    2012-06-01

    Full Text Available The anti-oxidative activities of six different extracts of Withania somnifera (Ashwagandha root, prepared in a sequential manner starting from non-polar (hexane to polar (water solvent, were investigated employing various established in-vitro systems that include total antioxidant activity (TAA, total reducing power (TRP, nitric oxide scavenging activity (NOSA and lipid peroxidation inhibition activity (LPIA. Among all the extracts, methanol extract was found the most potent and additionally, its DNA damage protective efficacy was tested using pRSET-A vector system. Positive correlations were established between total polyphenolic contents (TPC and various activities strongly suggesting that the observed activities of the extracts may be ascribed to their phenolic compounds that could be responsible, at least partly, for the observed antioxidant activities. Six main compounds viz. alkaloids, hydroxybenzene, terpene ansteroid, saponin, organic acids and flavone were identified in methanol extract using thin layer chromatography (TLC while by employing reverse-phase high pressure liquid chromatography (RP-HPLC four polyphenols namely epicatechin (3.21 μg/g, quercetin-3-rhamnoside (1.12 μg/g, gallic acid (0.05 μg/g and rutin hydrate (0.01 μg/g were identified and quantified in aforementioned extract. Overall, the results of study clearly demonstrated that methanolic extract of Ashwagandha root possesses a marked antioxidant activity.

  15. In-Vitro Studies on the Antioxidant Assay Profiling of Root of Withania somnifera L. (Ashwagandha Dunal: Part 2

    Directory of Open Access Journals (Sweden)

    Ajay Pal

    2014-02-01

    Full Text Available The anti-oxidative activities of six different extracts of Withania somnifera (Ashwagandha root, prepared in a sequential manner starting from non-polar (hexane to polar (water solvent, were investigated employing various established in-vitro systems that include total antioxidant activity (TAA, total reducing power (TRP, nitric oxide scavenging activity (NOSA and lipid peroxidation inhibition activity (LPIA. Among all the extracts, methanol extract was found the most potent and additionally, its DNA damage protective efficacy was tested using pRSET-A vector system. Positive correlations were established between total polyphenolic contents (TPC and various activities strongly suggesting that the observed activities of the extracts may be ascribed to their phenolic compounds that could be responsible, at least partly, for the observed antioxidant activities. Six main compounds viz. alkaloids, hydroxybenzene, terpene ansteroid, saponin, organic acids and flavone were identified in methanol extract using thin layer chromatography (TLC while by employing reverse-phase high pressure liquid chromatography (RP-HPLC four polyphenols namely epicatechin (3.21 μg/g, quercetin-3-rhamnoside (1.12 μg/g, gallic acid (0.05 μg/g and rutin hydrate (0.01 μg/g were identified and quantified in aforementioned extract. Overall, the results of study clearly demonstrated that methanolic extract of Ashwagandha root possesses a marked antioxidant activity.

  16. 3D printed bionic ears.

    Science.gov (United States)

    Mannoor, Manu S; Jiang, Ziwen; James, Teena; Kong, Yong Lin; Malatesta, Karen A; Soboyejo, Winston O; Verma, Naveen; Gracias, David H; McAlpine, Michael C

    2013-06-12

    The ability to three-dimensionally interweave biological tissue with functional electronics could enable the creation of bionic organs possessing enhanced functionalities over their human counterparts. Conventional electronic devices are inherently two-dimensional, preventing seamless multidimensional integration with synthetic biology, as the processes and materials are very different. Here, we present a novel strategy for overcoming these difficulties via additive manufacturing of biological cells with structural and nanoparticle derived electronic elements. As a proof of concept, we generated a bionic ear via 3D printing of a cell-seeded hydrogel matrix in the anatomic geometry of a human ear, along with an intertwined conducting polymer consisting of infused silver nanoparticles. This allowed for in vitro culturing of cartilage tissue around an inductive coil antenna in the ear, which subsequently enables readout of inductively-coupled signals from cochlea-shaped electrodes. The printed ear exhibits enhanced auditory sensing for radio frequency reception, and complementary left and right ears can listen to stereo audio music. Overall, our approach suggests a means to intricately merge biologic and nanoelectronic functionalities via 3D printing.

  17. Radiochromic 3D Detectors

    Science.gov (United States)

    Oldham, Mark

    2015-01-01

    Radiochromic materials exhibit a colour change when exposed to ionising radiation. Radiochromic film has been used for clinical dosimetry for many years and increasingly so recently, as films of higher sensitivities have become available. The two principle advantages of radiochromic dosimetry include greater tissue equivalence (radiologically) and the lack of requirement for development of the colour change. In a radiochromic material, the colour change arises direct from ionising interactions affecting dye molecules, without requiring any latent chemical, optical or thermal development, with important implications for increased accuracy and convenience. It is only relatively recently however, that 3D radiochromic dosimetry has become possible. In this article we review recent developments and the current state-of-the-art of 3D radiochromic dosimetry, and the potential for a more comprehensive solution for the verification of complex radiation therapy treatments, and 3D dose measurement in general.

  18. 3D Spectroscopic Instrumentation

    CERN Document Server

    Bershady, Matthew A

    2009-01-01

    In this Chapter we review the challenges of, and opportunities for, 3D spectroscopy, and how these have lead to new and different approaches to sampling astronomical information. We describe and categorize existing instruments on 4m and 10m telescopes. Our primary focus is on grating-dispersed spectrographs. We discuss how to optimize dispersive elements, such as VPH gratings, to achieve adequate spectral resolution, high throughput, and efficient data packing to maximize spatial sampling for 3D spectroscopy. We review and compare the various coupling methods that make these spectrographs ``3D,'' including fibers, lenslets, slicers, and filtered multi-slits. We also describe Fabry-Perot and spatial-heterodyne interferometers, pointing out their advantages as field-widened systems relative to conventional, grating-dispersed spectrographs. We explore the parameter space all these instruments sample, highlighting regimes open for exploitation. Present instruments provide a foil for future development. We give an...

  19. 3D Projection Installations

    DEFF Research Database (Denmark)

    Halskov, Kim; Johansen, Stine Liv; Bach Mikkelsen, Michelle

    2014-01-01

    Three-dimensional projection installations are particular kinds of augmented spaces in which a digital 3-D model is projected onto a physical three-dimensional object, thereby fusing the digital content and the physical object. Based on interaction design research and media studies, this article...... contributes to the understanding of the distinctive characteristics of such a new medium, and identifies three strategies for designing 3-D projection installations: establishing space; interplay between the digital and the physical; and transformation of materiality. The principal empirical case, From...... Fingerplan to Loop City, is a 3-D projection installation presenting the history and future of city planning for the Copenhagen area in Denmark. The installation was presented as part of the 12th Architecture Biennale in Venice in 2010....

  20. Assessment of relative bioavailability of heavy metals in soil using in vivo mouse model and its implication for risk assessment compared with bioaccessibility using in vitro assay.

    Science.gov (United States)

    Kang, Yuan; Pan, Weijian; Liang, Siyun; Li, Ning; Zeng, Lixuan; Zhang, Qiuyun; Luo, Jiwen

    2016-10-01

    There is limited study to simultaneously determine the relative bioavailability of heavy metals such as Cd, Pb, Cu, Cr(VI), and Ni in soil samples. In the present study, the bioaccessibility of heavy metals using in vitro assay was compared with the relative bioavailability of heavy metals using in vivo mouse model. The bioaccessibility of heavy metals ranged from 9.05 ± 0.97 % (Cr) to 42.8 ± 3.52 % (Cd). The uptake profile of heavy metals in soil and solution samples in mouse revealed that the uptake kinetics could be fitted to a two-compartment model. The relative bioavailability of heavy meals ranged from 34.8 ± 7.0 % (Ni) to 131 ± 20.3 % (Cu). Poor correlation between bioaccessibility and relative bioavailability of heavy metals was observed (r (2) = 0.11, p > 0.05). The relative bioavailability of heavy metals was significantly higher than the bioaccessibility of heavy metals (p < 0.05). The present study indicated that the in vitro digestion method should be carefully employed in risk assessment.

  1. Combined use of in silico and in vitro splicing assays for interpretation of genomic variants of unknown significance in cardiomyopathies and channelopathies

    Directory of Open Access Journals (Sweden)

    Hervé Crehalet

    2012-06-01

    Full Text Available The identification of molecular anomalies in patients with inherited arrhythmias or cardiomyopathies is a multi challenge due to: i the number of genes involved; ii the number of polymorphisms and the fact that most mutations are private; and iii the variable degree of penetrance which complicates family segregation study. Consequently, a number of unclassified variants (UV are found in patients’ DNA and some (outside the canonical GT/AG may affect splicing. Mutational screening on the most prevalent genes involved in arrythmias syndromes or in cardiomyopathies was performed on a cohort made up of 740 unrelated French index probands. To identify splice variants among the identified UVs, a combination of available in silico and in vitro tools were used since transcript is not available. Using this approach, 10 previously identified UVs were reclassified as disease-causing mutations and, more precisely, as haploinsufficiency mutations rather than dominant-negative mutations. Most of them (7 of 10 were observed in MYBPC3. Our study highlighted the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional impact of identified genetic variants. The primary challenge now, with new sequencing technologies, is to distinguish between background polymorphisms and pathogenic mutations. Since splice site mutations can account for almost 50% of disease-causing mutations, recognizing them from among other variations is essential.

  2. Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays.

    Science.gov (United States)

    Bolle, P; Mastrangelo, S; Perrone, F; Evandri, M G

    2007-01-01

    Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity. This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays. In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor alpha, with a lower potency but comparable efficacy to that of 17 beta-estradiol. Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F(2alpha,) and bradykinin than tissues obtained from estradiol valerate treated rats. The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F(2alpha,) and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 beta-estradiol. Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.

  3. Simple in vitro migration assay for neural crest cells and the opposite effects of all-trans-retinoic acid on cephalic- and trunk-derived cells.

    Science.gov (United States)

    Usami, Makoto; Mitsunaga, Katsuyoshi; Irie, Tomohiko; Miyajima, Atsuko; Doi, Osamu

    2014-08-01

    Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all-trans-retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.

  4. The sensitivity of the in vitro cytokinesis-blocked micronucleus assay in lymphocytes for different and combined radiation qualities

    Energy Technology Data Exchange (ETDEWEB)

    Wuttke, K.; Mueller, W.U.; Streffer, C. [Universitaetsklinikum Essen (Germany). Inst. fuer Medizinische Strahlenbiologie

    1998-05-01

    Purpose: The dose-response relationship and the relative biological effectiveness (RBE) for the induction of micronuclei in lymphocytes was analyzed after irradiation in vitro with a 6-MeV neutron beam that was followed by 240-kV X-rays. The dose range of the combined exposure comprised 1 to 3 Gy. For reference, the dose-effect relationships found after X-ray (0.5 to 5 Gy)- and neutron (0.5 to 4 Gy) exposure applied separately are presented. The possibility of an interaction between the 2 radiation qualities is investigated by the method of isobole calculation termed `envelope of additivity`. Methods: Micronuclei were analyzed in PHA-stimulated, cytokinesis-blocked human lymphocytes. Results: The dose-response relationships for the micronucleus frequencies induced by the neutron irradiation, as well as by the mixed exposure, were linear. A saturation effect was indicated after neutron doses higher than 3 Gy. After low LET exposure the dose-response curves were describable by a linear-quadratic model. For neutron-induced micronucleus frequencies, RBE-values of 2 to 3 and for the combined exposure RBE values of 1.5 to 2 were calculated for a range of effect of 0.5 to 1.5 micronuclei/binucleated lymphocyte. No indication was found for an interaction between the damage induced by X-rays and that produced by neutrons under our experimental conditions. (orig./MG) [Deutsch] Fragestellung: Die Mikronukeusinduktion in Lymphozyten wurde nach In-vitro-Bestrahlung mit 6-MeV-Neutronen (0,5 bis 4 Gy), 240-kV-Roentgenstrahlung (0,5 bis 5 Gy) bzw. einer Kombination dieser Strahlenqualitaeten (1 bis 3 Gy Gesamtdosis) untersucht. Anhand der Dosis-Wirkungs-Beziehungen fuer die einzelne und kombinierte Anwendung beider Strahlenarten wurde die relative biologische Wirksamkeit (RBW) fuer Neutronen bzw. fuer die Kombination von Neutronen und Roentgenstrahlen ermittelt. Mit Hilfe einer Isobolenkalkulation (`envelope of additivity`) wurde die Moeglichkeit einer Interaktion zwischen den

  5. Interaktiv 3D design

    DEFF Research Database (Denmark)

    Villaume, René Domine; Ørstrup, Finn Rude

    2002-01-01

    Projektet undersøger potentialet for interaktiv 3D design via Internettet. Arkitekt Jørn Utzons projekt til Espansiva blev udviklet som et byggesystem med det mål, at kunne skabe mangfoldige planmuligheder og mangfoldige facade- og rumudformninger. Systemets bygningskomponenter er digitaliseret som...... 3D elementer og gjort tilgængelige. Via Internettet er det nu muligt at sammenstille og afprøve en uendelig  række bygningstyper som  systemet blev tænkt og udviklet til....

  6. Assessment of antimutagenic and genotoxic potential of senna (Cassia angustifolia Vahl.) aqueous extract using in vitro assays.

    Science.gov (United States)

    Silva, C R; Monteiro, M R; Rocha, H M; Ribeiro, A F; Caldeira-de-Araujo, A; Leitão, A C; Bezerra, R J A C; Pádula, M

    2008-02-01

    Senna (Cassia angustifolia Vahl.) is widely used as a laxative, although potential side effects, such as toxicity and genotoxicity, have been reported. This study evaluated genotoxic and mutagenic effects of senna aqueous extract (SAE) by means of four experimental assays: inactivation of Escherichia coli cultures; bacterial growth inhibition; reverse mutation test (Mutoxitest) and DNA strand break analysis in plasmid DNA. Our results demonstrated that SAE produces single and double strand breaks in plasmid DNA in a cell free system. On the other hand, SAE was not cytotoxic or mutagenic to Escherichia coli strains tested. In effect, SAE was able to avoid H(2)O(2)-induced mutagenesis and toxicity in Escherichia coli IC203 (uvrA oxyR) and IC205 (uvrA mutM) strains, pointing to a new antioxidant/antimutagenic action of SAE.

  7. Assessment of two medicinal plants, Psidium guajava L. and Achillea millefolium L., in in vitro and in vivo assays

    Directory of Open Access Journals (Sweden)

    Teixeira Rosangela de Oliveira

    2003-01-01

    Full Text Available The use of medicinal plants by the general population is an old and still widespread practice, which makes studies of their genotoxicity essential. Psidium guajava L. and Achillea millefolium L. are examples of plants commonly used in popular medicine. P. guajava L. is indicated for diarrhea and also as an antiseptic, while A. millefolium L. is indicated as an analgesic, antispasmodic, digestive, diuretic, antiseptic, astringent, emollient, wound healer and hemorrhoid medication. The aim of this study was to determine the effects of the infusions of these two plant species on chromosomes and the cell cycle. Leaves from the plants were used to prepare infusions, in the same manner as teas, but at two different concentrations. Allium cepa L. root-tip cells (P. guajava L. - 2.62 and 26.2 mg/mL, and A. millefolium L. - 3.5 and 35.0 mg/mL and Wistar rat bone marrow cells (P. guajava L. - 2.62 and 26.2 mg/100g body weight, and A. millefolium L. - 3.5 and 35.0 mg/100g body weight were used as in vivo plant and animal test systems, respectively. Human peripheral blood lymphocytes (P. guajava L. - 0.262 and 2.62 mg/mL culture medium, and A. millefolium L. - 0.35 and 3.5 mg/mL culture medium were used as in vitro test system. The P. guajava L. infusion at the higher concentration caused a statistically significant inhibition of cellular division in the onion root-tip cells, not observed in onion root-tip cells treated with A. millefolium L. No statistically significant alterations were found, as compared to untreated controls, in either the cell cycle or the number of chromosome alterations, after treatments with either plant, in rat cells or in cultured human lymphocytes. These results regarding the cytotoxicity and mutagenicity of these plants provide valuable information about the safety of using them as therapeutic agents.

  8. A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM).

    Science.gov (United States)

    Pessina, Augusto; Croera, Cristina; Bayo, Maria; Malerba, Ilaria; Passardi, Laura; Cavicchini, Loredana; Neri, Maria G; Gribaldo, Laura

    2004-03-01

    In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a

  9. Tangible 3D Modelling

    DEFF Research Database (Denmark)

    Hejlesen, Aske K.; Ovesen, Nis

    2012-01-01

    This paper presents an experimental approach to teaching 3D modelling techniques in an Industrial Design programme. The approach includes the use of tangible free form models as tools for improving the overall learning. The paper is based on lecturer and student experiences obtained through...

  10. Shaping 3-D boxes

    DEFF Research Database (Denmark)

    Stenholt, Rasmus; Madsen, Claus B.

    2011-01-01

    Enabling users to shape 3-D boxes in immersive virtual environments is a non-trivial problem. In this paper, a new family of techniques for creating rectangular boxes of arbitrary position, orientation, and size is presented and evaluated. These new techniques are based solely on position data...

  11. 3D Wire 2015

    DEFF Research Database (Denmark)

    Jordi, Moréton; F, Escribano; J. L., Farias

    This document is a general report on the implementation of gamification in 3D Wire 2015 event. As the second gamification experience in this event, we have delved deeply in the previous objectives (attracting public areas less frequented exhibition in previous years and enhance networking) and ha......, improves socialization and networking, improves media impact, improves fun factor and improves encouragement of the production team....

  12. 3D photoacoustic imaging

    Science.gov (United States)

    Carson, Jeffrey J. L.; Roumeliotis, Michael; Chaudhary, Govind; Stodilka, Robert Z.; Anastasio, Mark A.

    2010-06-01

    Our group has concentrated on development of a 3D photoacoustic imaging system for biomedical imaging research. The technology employs a sparse parallel detection scheme and specialized reconstruction software to obtain 3D optical images using a single laser pulse. With the technology we have been able to capture 3D movies of translating point targets and rotating line targets. The current limitation of our 3D photoacoustic imaging approach is its inability ability to reconstruct complex objects in the field of view. This is primarily due to the relatively small number of projections used to reconstruct objects. However, in many photoacoustic imaging situations, only a few objects may be present in the field of view and these objects may have very high contrast compared to background. That is, the objects have sparse properties. Therefore, our work had two objectives: (i) to utilize mathematical tools to evaluate 3D photoacoustic imaging performance, and (ii) to test image reconstruction algorithms that prefer sparseness in the reconstructed images. Our approach was to utilize singular value decomposition techniques to study the imaging operator of the system and evaluate the complexity of objects that could potentially be reconstructed. We also compared the performance of two image reconstruction algorithms (algebraic reconstruction and l1-norm techniques) at reconstructing objects of increasing sparseness. We observed that for a 15-element detection scheme, the number of measureable singular vectors representative of the imaging operator was consistent with the demonstrated ability to reconstruct point and line targets in the field of view. We also observed that the l1-norm reconstruction technique, which is known to prefer sparseness in reconstructed images, was superior to the algebraic reconstruction technique. Based on these findings, we concluded (i) that singular value decomposition of the imaging operator provides valuable insight into the capabilities of

  13. Colorimetric Assay to Determine In Vitro Antibacterial Activity Against Clinical Isolates: Enhanced Activity in Damaged Chinese Masson Pine Needles

    Institute of Scientific and Technical Information of China (English)

    Guijun Dong; Weidong Pan; Tao Zheng; Xianghui Liu; Gongshe Liu

    2006-01-01

    A colorimetric assay for antibacterial susceptibility testing of clinical isolates (Escherichia coil, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Bacillus cereus, and Streptococcus pneumoniae) is described based on the reduction of a novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), in the presence of phenazine methosulfate (PMS) as an electron-coupling agent. The combination of 200μg/mL MTS with 25μmol/L PMS resulted in production of large amounts of formazan within 1 h of exposure. In this setting, fractions extracted from Chinese Masson pine (Pinus massoniana Lamb.) needles damaged by the pine caterpillar Dendrolimus punctatus Walker were found to have enhanced levels of antibacterial activity. These fractions, which were designated "Master", "Technique", and "Strength", were isolated and identified by reverse-phase C18cartridge concentration, gel filtration, and affinity chromatography. Two fractions purified from healthy and undamaged needles were designated H1 and H2, respectively. For all test bacteria species. Technique produced the lowest minimal inhibitory concentrations(MICs), ranging from 2 to 32μg/mL, and H2 produced the highest values, with four of the six MICs being higher than 128 μg/mL. We found that the Rmax model fitted the data well in that the r2 ranged between 0.87 and 0.96 (median, 0.92) and no statistically significant deviations from the model were found (P= 0.23). The median coefficient of variation of the log RC50 values and the slope m of the fitted model for all six strains among the replicates were 38 and 41%, respectively. In the course of the investigation, the physiological and functional factors involved in pest damage to plants were also explored.In summary, the MTS-PMS colorimetric assay has advantages over existing methods for the examination of antibacterial activity, and could be developed further such that it would be suitable

  14. Evaluation of the estrogenic potential of river and treated waters in the Paris area (France) using in vivo and in vitro assays.

    Science.gov (United States)

    Cargouët, Maëlle; Perdiz, Daniel; Levi, Yves

    2007-05-01

    For many years, surface waters have been shown to be contaminated by endocrine-disrupting compounds (EDCs), which can cause adverse effects on human and wildlife growth, development, and reproduction. It is therefore of primary importance to determine if drinking water could be contaminated by EDCs when produced from polluted surface waters. It is also essential to determine if disinfection by-products can account for estrogenic activity in treated waters. The estrogenic potential of river and treated waters was investigated using an in vivo assay. Adult male zebrafish were placed in three drinking water treatment plants (DWTPs) in the Paris area and exposed for 1 month to the two types of waters. After exposure, vitellogenin (VTG) was measured in the plasma of fish using a competitive ELISA. In addition, an in vitro assay (MELN cells) was used to assess the estrogenic potential of 10 major chlorination by-products. No significant induction of VTG was observed in fish exposed to river or treated waters. Among the 10 chlorination by-products tested, only 2-chlorophenol was found to be weakly estrogenic at concentrations up to 1mg/L. Therefore, the risk for the three DWTPs studied to produce drinking water with significant level of estrogenic substances appears to be low.

  15. Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

    Science.gov (United States)

    van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

    2014-01-15

    Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.

  16. Induction of a Tumor-Metastasis-Receptive Microenvironment as an Unwanted Side Effect After Radio/Chemotherapy and In Vitro and In Vivo Assays to Study this Phenomenon.

    Science.gov (United States)

    Schneider, Gabriela; Sellers, Zachariah Payne; Ratajczak, Mariusz Z

    2016-01-01

    Besides surgical removal of tumor tissue, chemotherapy and radiotherapy are the most important and efficient treatment modalities employed to treat therapy-susceptible malignancies. The main aim of this treatment-to destroy tumor cells-is unfortunately usually associated with toxicity to nontumor cells and different degrees of tissue and organ damage. In damaged tissues several chemoattractants are upregulated and released that may attract tumor cells. Moreover, highly migratory radio/chemotherapy treatment may endow cells with several properties of cancer stem cells which survive and respond to these chemoattractants upregulated in collateral tissues. Based on this, one of the unwanted and underappreciated side effects of chemotherapy or radiotherapy is the creation of a metastasis-receptive microenvironment in bones as well as in other organs of the body. Herein we describe methods and assays that can be employed to study migratory properties of cancer cells in in vitro (chemotaxis) and in vivo (seeding efficiency assay) conditions in response to the induction of pro-metastatic microenvironments in various organs and tissues.

  17. An exploration of the estrogen receptor transcription activity of capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays.

    Science.gov (United States)

    Li, Juan; Ma, Duo; Lin, Yuan; Fu, Jianjie; Zhang, Aiqian

    2014-06-16

    Capsaicin has been considered as an alternative template of dichlorodiphenyl trichloroethane (DDT) in antifouling paint. However, information regarding the estrogenic activity of capsaicin analogues is rather limited in comparison to that of DDT analogues and their metabolites. We here explore the ER transcription activity of selected capsaicin analogues via an integrated approach based on in silico prediction and in vitro assays. Molecular simulation and the agonist/antagonist differential-docking screening identified 6-iodonordihydrocapsaicin (6-I-CPS) as a weak ERα agonist, while anti-estrogenicity was expected for N-arachidonoyldopamine, capsazepine, dihydrocapsaicin, trichostatin A, and capsaicin. On the contrary, the large volume of analogues, such as phorbol 12-phenylacetate 13-acetate 20-homovanillate and phorbol 12,13-dinonanoate 20-homovanillate, cannot fit well with the ER cavity. The result of MVLN assay was in accord with the in silico prediction. 6-I-CPS was demonstrated to induce luciferase gene expression, while the other analogues of relatively small molecular volume reduced luciferase gene expression in MVLN cells, both in the absence and presence of estradiol. This finding suggested that the ER transcription activity of capsaicin analogues is generated at least partly through the ERα-mediated pathway. Moreover, receptor polymorphism analysis indicated that capsaicin analogues may exhibit diverse species selectivity for human beings and marine species.

  18. Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

    Directory of Open Access Journals (Sweden)

    Lo SH

    2016-08-01

    Full Text Available Shih-Hsiang Lo,1,2 Kai-Chung Cheng,3 Ying-Xiao Li,3,4 Chin-Hong Chang,4,5 Juei-Tang Cheng,4,6 Kung-Shing Lee7,8 1Division of Cardiology, Department of Internal Medicine, Zhongxing Branch of Taipei City Hospital, 2Department of History and Geography, University of Taipei, Taipei, Taiwan; 3Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; 4Department of Medical Research, 5Department of Neurosurgery, Chi-Mei Medical Center, Yong Kang, 6Institute of Medical Science, College of Health Science, Chang Jung Christian University, Tainan, 7Department of Surgery, Pingtung Hospital, 8Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Background: G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported. Methods: Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1 cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits. Results: Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also

  19. Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease

    Institute of Scientific and Technical Information of China (English)

    Gui-Xin Du; Li-Hua Hou; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2002-01-01

    AIM: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coliwas induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease.RESULTS: The single-chain recombinant protease was overexpressed as soluble protein when the E. coliwas induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15℃). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %).The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not.CONCLUSION: A simple

  20. In-vitro Quantitative Assay of Interferon Gamma in Serum of Nigerian Indigenous and Exotic Breeds of Chickens

    Directory of Open Access Journals (Sweden)

    Esan Oluwaseun and Oladele Omolade

    2014-12-01

    Full Text Available The Nigerian Indigenous breeds of Chicken (NIC have thrived in harsh tropical environment with little veterinary care and poor nutrition compared with the introduced exotic breeds which performs sub-optimally in the tropics. However, they receive little attention for commercial production in spite of low input required. A comparative assessment of cellular immune response of the indigenous and exotic breeds was carried out to provide scientific explanation for their hardy nature and justify production for economic purposes. Fifteen chickens from each of three indigenous breeds i.e. Frizzled- feathered, Naked-neck and Smooth-feathered, and 8 Isa Brown pullets were 10 weeks old and reared in separate cages. The chickens were stabilized and administered Newcastle Disease Vaccine (NDV, LaSota strain. At 14 and 16 weeks old, all breeds were administered NDV Komarov strain in Freund’s adjuvant and in PBS intramuscularly as sensitizing and challenge inoculants, respectively. They were bled for serum 5 days later and concentrations of Interferon-gamma (IFN-gamma were determined using competitive Enzyme-linked immunosorbent assay. Results showed that the Frizzled-feathered chickens had the highest concentration of IFN-gamma (58±2.8 pg/ml which was significantly higher than 49±3.2 pg/ml and 44±2.5 pg/ml recorded for Smooth-feathered and Isa brown breeds respectively. Also, concentration in Naked-neck breed was 54±2.9 pg/ml, which was significantly higher than Isa Brown. Isa Brown had the significantly lowest concentration. It was concluded that the three NIC studied, have inherent capacity to mount higher levels of cellular immune response compared with the exotic Isa brown, when challenged.

  1. Rapid prototyping for tissue-engineered bone scaffold by 3D printing and biocompatibility study.

    Science.gov (United States)

    He, Hui-Yu; Zhang, Jia-Yu; Mi, Xue; Hu, Yang; Gu, Xiao-Yu

    2015-01-01

    The prototyping of tissue-engineered bone scaffold (calcined goat spongy bone-biphasic ceramic composite/PVA gel) by 3D printing was performed, and the biocompatibility of the fabricated bone scaffold was studied. Pre-designed STL file was imported into the GXYZ303010-XYLE 3D printing system, and the tissue-engineered bone scaffold was fabricated by 3D printing using gel extrusion. Rabbit bone marrow stromal cells (BMSCs) were cultured in vitro and then inoculated to the sterilized bone scaffold obtained by 3D printing. The growth of rabbit BMSCs on the bone scaffold was observed under the scanning electron microscope (SEM). The effect of the tissue-engineered bone scaffold on the proliferation and differentiation of rabbit BMSCs using MTT assay. Universal testing machine was adopted to test the tensile strength of the bone scaffold. The leachate of the bone scaffold was prepared and injected into the New Zealand rabbits. Cytotoxicity test, acute toxicity test, pyrogenic test and intracutaneous stimulation test were performed to assess the biocompatibility of the bone scaffold. Bone scaffold manufactured by 3D printing had uniform pore size with the porosity of about 68.3%. The pores were well interconnected, and the bone scaffold showed excellent mechanical property. Rabbit BMSCs grew and proliferated on the surface of the bone scaffold after adherence. MTT assay indicated that the proliferation and differentiation of rabbit BMSCs on the bone scaffold did not differ significantly from that of the cells in the control. In vivo experiments proved that the bone scaffold fabricated by 3D printing had no acute toxicity, pyrogenic reaction or stimulation. Bone scaffold manufactured by 3D printing allows the rabbit BMSCs to adhere, grow and proliferate and exhibits excellent biomechanical property and high biocompatibility. 3D printing has a good application prospect in the prototyping of tissue-engineered bone scaffold.

  2. High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Andrew V Oleinikov

    2009-04-01

    Full Text Available Plasmodium falciparum-infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLbetaC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLbetaC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2betaC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2betaC2(PF11_0521 best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLbetaC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLbetaC2 domain. DBL2betaC2(PF11_0521 binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521 and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

  3. Unoriented 3d TFTs

    CERN Document Server

    Bhardwaj, Lakshya

    2016-01-01

    This paper generalizes two facts about oriented 3d TFTs to the unoriented case. On one hand, it is known that oriented 3d TFTs having a topological boundary condition admit a state-sum construction known as the Turaev-Viro construction. This is related to the string-net construction of fermionic phases of matter. We show how Turaev-Viro construction can be generalized to unoriented 3d TFTs. On the other hand, it is known that the "fermionic" versions of oriented TFTs, known as Spin-TFTs, can be constructed in terms of "shadow" TFTs which are ordinary oriented TFTs with an anomalous Z_2 1-form symmetry. We generalize this correspondence to Pin+ TFTs by showing that they can be constructed in terms of ordinary unoriented TFTs with anomalous Z_2 1-form symmetry having a mixed anomaly with time-reversal symmetry. The corresponding Pin+ TFT does not have any anomaly for time-reversal symmetry however and hence it can be unambiguously defined on a non-orientable manifold. In case a Pin+ TFT admits a topological bou...

  4. Estrogenic effects of nonylphenol and octylphenol isomers in vitro by recombinant yeast assay (RYA) and in vivo with early life stages of zebrafish.

    Science.gov (United States)

    Puy-Azurmendi, E; Olivares, A; Vallejo, A; Ortiz-Zarragoitia, M; Piña, B; Zuloaga, O; Cajaraville, M P

    2014-01-01

    Commercial OP and NP are complex isomer mixtures that can be individually present in the environment, showing different estrogenic potencies. The aims of this study were to establish the estrogenic potency of some AP isomers in comparison to the commercial NP (cNP) mixture in vitro and to investigate in vivo their possible effects during the embryo and larval development of zebrafish. An in vitro estrogen receptor-based recombinant yeast assay was used to test the estrogenicity of specific AP isomers (22-OP, 33-OP, 22-NP, 33-NP and 363-NP) and cNP. The EC₅₀ was in the range of 0.6-7.7 mg/L. Both OP isomers and 363-NP exhibited higher estrogenic activity than cNP. For in vivo experiments, one-day postfertilisation (dpf) embryos were exposed to cNP (50, 250 and 500 μg/L), 363-NP and 33-OP (50 μg/L), 17β-estradiol (100 ng/L) and DMSO (0.01% v/v) for 4weeks. After exposure fish were maintained for 2 weeks in clean water in order to evaluate a possible recovery. Fish of groups exposed to cNP and 363-NP were the last to hatch. Histological alterations were not observed after 7, 28 or 42 dpf. Exposure to 33-OP increased transcriptional levels of erα, vtg and cyp19a1b genes. However, transcriptional response in E2 exposure was observed at later stages and with higher fold induction levels. Exposure to cNP decreased levels of erα whereas increased levels of rxrγ and cyp19a1b. Exposure to 363-NP did not cause changes in transcriptional levels of studied genes. The differences in response of the OP isomer compared to the NP isomer in zebrafish could be related to the rapid decay in concentration of the latter.

  5. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  6. 3D-printed silicate porous bioceramics using a non-sacrificial preceramic polymer binder.

    Science.gov (United States)

    Zocca, A; Elsayed, H; Bernardo, E; Gomes, C M; Lopez-Heredia, M A; Knabe, C; Colombo, P; Günster, J

    2015-05-22

    Silicate bioceramics possess an excellent bioactivity; however, shaping them into complex geometries is still challenging. Therefore, this paper aims to present a new strategy for the shaping of a bioglass-ceramic with controlled geometry and properties starting from a glass powder combined with a preceramic polymer, i.e. a silicon resin, and reactive fillers. The powder-based three-dimensional (3D)-printing of wollastonite (CaSiO3)-based silicate bioceramic parts was demonstrated in this work. The resin plays a dual role, as it not only acts as a non-sacrificial binder for the filler powders in the printing process but it also reacts with the fillers to generate the desired bioceramic phases. The mechanical and physical properties, i.e. ball-on-three-balls test, density, porosity and morphology, were evaluated in 3D-printed discs. These samples possessed a total porosity around 64 vol% and a biaxial flexural strength around 6 MPa. The raw materials used in this work also enabled the 3D-printing of scaffolds possessing a designed multi-scale porosity, suitable bioceramic phase assemblage and a compressive strength of 1 MPa (for cylindrical scaffolds with total porosity ~80 vol%). Solubility in TRIS/HCl and in vitro assays, i.e. viability, cytotoxicity and apoptosis assays, were also performed. In vitro tests indicated good cell viability and no cytotoxicity effect on the cells.

  7. Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals

    OpenAIRE

    ALEPEE Nathalie; VIEGAS BARROSO JOAO FILIPE; De Smedt, Ann; Wever, Bart De; Hibatallah, Jalila; Klaric, Martina; MEWES Karsten R.; Millet, Marion; Pfannenbecker, Uwe; Tailhardat, Magalie; TEMPLIER Marie; McNamee, Pauline

    2015-01-01

    A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLCsp...

  8. 3D and beyond

    Science.gov (United States)

    Fung, Y. C.

    1995-05-01

    This conference on physiology and function covers a wide range of subjects, including the vasculature and blood flow, the flow of gas, water, and blood in the lung, the neurological structure and function, the modeling, and the motion and mechanics of organs. Many technologies are discussed. I believe that the list would include a robotic photographer, to hold the optical equipment in a precisely controlled way to obtain the images for the user. Why are 3D images needed? They are to achieve certain objectives through measurements of some objects. For example, in order to improve performance in sports or beauty of a person, we measure the form, dimensions, appearance, and movements.

  9. Effect of two homeopathic remedies at different degrees of dilutions on the wound closure of 3T3 fibroblasts in in vitro scratch assay

    Directory of Open Access Journals (Sweden)

    Reinhard Saller

    2012-09-01

    :100 dilutions (p0.05. Positive control 2 ng/ml EGF increased migratory activity of cells by 49.8%. Preparation (0712-2 at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p<0.001 from succussed solvent (0712-1, which caused 22.1% wound closure. Medium diluted remedy (1101-4 exerted accelerating effect on wound closure after 14h of treatment. Wounded area was closed by 20% with (1101-4 and 13% by (1101-3 compared to untreated control. Succussed solvent (1101-3 caused about 23% and the remedy (1101-4 about 30% wound closure after 24h. Remedy (1101-4 and succussed solvent (1101-3 modestly stimulated cell growth at dilutions 1:100 and 1:1000 by about 25% and 15%, respectively. No statistically significant differences between preparations 1101-3 and 1101-4 could be detected. Conclusions: Our results demonstrate that the Similasan® Arnica plus low dilution homeopathic remedy exerted wound healing potential, which is a result of increased ability of fibroblasts to migrate without affecting cell proliferation. Medium diluted preparation SIM WuS exerted stimulating effect on the wound closure accompanied by a cell proliferating effect. Used in vitro wound closure test was sensitive enough for low dilutions preparation, however for medium diluted preparation despite of a trend, no significant differences could be detected.

  10. 3D Surgical Simulation

    Science.gov (United States)

    Cevidanes, Lucia; Tucker, Scott; Styner, Martin; Kim, Hyungmin; Chapuis, Jonas; Reyes, Mauricio; Proffit, William; Turvey, Timothy; Jaskolka, Michael

    2009-01-01

    This paper discusses the development of methods for computer-aided jaw surgery. Computer-aided jaw surgery allows us to incorporate the high level of precision necessary for transferring virtual plans into the operating room. We also present a complete computer-aided surgery (CAS) system developed in close collaboration with surgeons. Surgery planning and simulation include construction of 3D surface models from Cone-beam CT (CBCT), dynamic cephalometry, semi-automatic mirroring, interactive cutting of bone and bony segment repositioning. A virtual setup can be used to manufacture positioning splints for intra-operative guidance. The system provides further intra-operative assistance with the help of a computer display showing jaw positions and 3D positioning guides updated in real-time during the surgical procedure. The CAS system aids in dealing with complex cases with benefits for the patient, with surgical practice, and for orthodontic finishing. Advanced software tools for diagnosis and treatment planning allow preparation of detailed operative plans, osteotomy repositioning, bone reconstructions, surgical resident training and assessing the difficulties of the surgical procedures prior to the surgery. CAS has the potential to make the elaboration of the surgical plan a more flexible process, increase the level of detail and accuracy of the plan, yield higher operative precision and control, and enhance documentation of cases. Supported by NIDCR DE017727, and DE018962 PMID:20816308

  11. TOWARDS: 3D INTERNET

    Directory of Open Access Journals (Sweden)

    Ms. Swapnali R. Ghadge

    2013-08-01

    Full Text Available In today’s ever-shifting media landscape, it can be a complex task to find effective ways to reach your desired audience. As traditional media such as television continue to lose audience share, one venue in particular stands out for its ability to attract highly motivated audiences and for its tremendous growth potential the 3D Internet. The concept of '3D Internet' has recently come into the spotlight in the R&D arena, catching the attention of many people, and leading to a lot of discussions. Basically, one can look into this matter from a few different perspectives: visualization and representation of information, and creation and transportation of information, among others. All of them still constitute research challenges, as no products or services are yet available or foreseen for the near future. Nevertheless, one can try to envisage the directions that can be taken towards achieving this goal. People who take part in virtual worlds stay online longer with a heightened level of interest. To take advantage of that interest, diverse businesses and organizations have claimed an early stake in this fast-growing market. They include technology leaders such as IBM, Microsoft, and Cisco, companies such as BMW, Toyota, Circuit City, Coca Cola, and Calvin Klein, and scores of universities, including Harvard, Stanford and Penn State.

  12. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    -I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol, 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide...

  13. Analysis of in vitro bioactivity data extracted from drug discovery literature and patents: Ranking 1654 human protein targets by assayed compounds and molecular scaffolds

    Directory of Open Access Journals (Sweden)

    Southan Christopher

    2011-05-01

    Full Text Available Abstract Background Since the classic Hopkins and Groom druggable genome review in 2002, there have been a number of publications updating both the hypothetical and successful human drug target statistics. However, listings of research targets that define the area between these two extremes are sparse because of the challenges of collating published information at the necessary scale. We have addressed this by interrogating databases, populated by expert curation, of bioactivity data extracted from patents and journal papers over the last 30 years. Results From a subset of just over 27,000 documents we have extracted a set of compound-to-target relationships for biochemical in vitro binding-type assay data for 1,736 human proteins and 1,654 gene identifiers. These are linked to 1,671,951 compound records derived from 823,179 unique chemical structures. The distribution showed a compounds-per-target average of 964 with a maximum of 42,869 (Factor Xa. The list includes non-targets, failed targets and cross-screening targets. The top-278 most actively pursued targets cover 90% of the compounds. We further investigated target ranking by determining the number of molecular frameworks and scaffolds. These were compared to the compound counts as alternative measures of chemical diversity on a per-target basis. Conclusions The compounds-per-protein listing generated in this work (provided as a supplementary file represents the major proportion of the human drug target landscape defined by published data. We supplemented the simple ranking by the number of compounds assayed with additional rankings by molecular topology. These showed significant differences and provide complementary assessments of chemical tractability.

  14. Optimization, validation and application of an assay for the activity of HMG-CoA reductase in vitro by LC-MS/MS

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Ji-Ye Sun; Chun-Jie Sha; Yu-Feng Shao; Yan-Hong Liu; You-Xin Li; Zhen-Wen Duan; Wan-Hui Liu

    2015-01-01

    A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. The optimized enzyme reac-tion condition contained 1.5μg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra-and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D7, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005–1.000μg/mL for MVAL and 0.010–0.500μg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005μg/mL for MVAL and 0.010μg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95%to 2.39%and 2.26%to 3.38%for MVAL, 1.46%to 3.89%and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.

  15. Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

    Directory of Open Access Journals (Sweden)

    Chan Chee

    2011-08-01

    Full Text Available Abstract Background Many SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as cis- rather than trans-orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted. Results A series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch and to optimise annealing conditions (~18.38°C below Tm of the mismatched oligonucleotide sets. Conclusions The in vitro 'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative

  16. Filling the concept with data: integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing.

    Science.gov (United States)

    Natsch, Andreas; Emter, Roger; Ellis, Graham

    2009-01-01

    Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients. Significant efforts are made to replace the current animal tests. It is widely recognized that this cannot be accomplished with a single in vitro test, but that rather the integration of results from different in vitro and in silico assays will be needed for the prediction of the skin sensitization potential of chemicals. This has been proposed as a theoretical scheme so far, but no attempts have been made to use experimental data to prove the validity of this concept. Here we thus try for the first time to fill this widely cited concept with data. To this aim, we integrate and report both novel and literature data on 116 chemicals of known skin sensitization potential on the following parameters: (1) peptide reactivity as a surrogate for protein binding, (2) induction of antioxidant/electrophile responsive element dependent luciferase activity as a cell-based assay; (3) Tissue Metabolism Simulator skin sensitization model in silico prediction; and (4) calculated octanol-water partition coefficient. The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score and compared to the local lymph node assay (LLNA) data, which were also scaled from 0 to 4 (nonsensitizer/weak/moderate/strong/extreme). Different ways of evaluating these data have been assessed to rate the hazard of chemicals (Cooper statistics) and to also scale their potency. With the optimized model an overall accuracy for predicting sensitizers of 87.9% was obtained. There is a linear correlation between the LLNA score and the in vitro score. However, the correlation needs further improvement as there is still a relatively high variation in the in vitro score between chemicals belonging to the same sensitization potency class.

  17. The model of amyloid aggregation of Escherichia coli RNA polymerase σ70 subunit based on AFM data and in vitro assays.

    Science.gov (United States)

    Koroleva, Olga N; Dubrovin, Evgeniy V; Khodak, Yu A; Kuzmina, Natalia V; Yaminsky, Igor V; Drutsa, Valeriy L

    2013-07-01

    To propose a model for recently described amyloid aggregation of E.coli RNA polymerase σ(70) subunit, we have investigated the role of its N-terminal region. For this purpose, three mutant variants of protein with deletions Δ1-73, Δ1-100 and Δ74-100 were constructed and studied in a series of in vitro assays and using atomic force microscopy (AFM). Specifically, all RNA polymerase holoenzymes, reconstituted with the use of mutant σ subunits, have shown reduced affinity for promoter-containing DNA and reduced activity in run-off transcription experiments (compared to that of WT species), thus substantiating the modern concept on the modulatory role of N-terminus in formation of open complex and transcription initiation. The ability of mutant proteins to form amyloid-like structures has been investigated using AFM, which revealed the increased propensity of mutant proteins to form rodlike aggregates with the effect being more pronounced for the mutant with the deletion Δ1-73 (10 fold increase). σ(70) subunit aggregation ability has shown complex dependence on the ionic surrounding, which we explain by Debye screening effect and the change of the internal state of the protein. Basing on the obtained data, we propose the model of amyloid fibril formation by σ(70) subunit, implying the involvement of N-terminal region according to the domain swapping mechanism.

  18. Characterization of Listeria monocytogenes isolates of food and human origins from Brazil using molecular typing procedures and in vitro cell culture assays.

    Science.gov (United States)

    Bueno, Valter F; Banerjee, Pratik; Banada, Padmapriya P; José de Mesquita, Albenones; Lemes-Marques, Eneida G; Bhunia, Arun K

    2010-02-01

    The spreading of diseases through foods is a worldwide concern. Here, molecular and in vitro cell-culture assays were employed to characterize 63 Brazilian Listeria monocytogenes isolates (food, 47; clinical, 16). Serotype 4b was the most predominant (49%) followed by (1/2)b (30%), (1/2)a (10%), (1/2)c (6%), 3c (3%) and 3b (2%). Ribotyping yielded 17 ribopatterns, which were grouped into four phylogenetic clusters. Cluster A comprised of 39/63 isolates primarily of food origin, and clusters B, C and D contained both food and clinical isolates. Isolates were positive for virulence determinants prfA, hlyA and inlA: clinical isolates were more invasive to Caco-2 cells and expressed high levels of inlA transcripts than the food isolates. Highly invasive isolates also provoked more Ped-2E9 cells to die by apoptosis than the weakly-invasive strains. These data demonstrate a strong genetic relatedness among clinical and food isolates and suggest transmission of a subset of L. monocytogenes strains from food to humans.

  19. Salvia somalensis essential oil as a potential cosmetic ingredient: solvent-free microwave extraction, hydrodistillation, GC-MS analysis, odour evaluation and in vitro cytotoxicity assays.

    Science.gov (United States)

    Villa, C; Trucchi, B; Bertoli, A; Pistelli, L; Parodi, A; Bassi, A M; Ruffoni, B

    2009-02-01

    Salvia somalensis Vatke, a wild sage native of Somalia, has been studied with the aim of assessing the potential cosmetic application of its essential oil, recovered from fresh aerial parts by solvent-free microwave extraction - SFME. To evaluate the efficiency and reliability of this eco-friendly procedure, the recovery of the essential oil was also processed by conventional hydrodistillation (HD) and the results compared. The essential oils obtained by both SFME and HD were analysed by gas chromatography-mass spectrometry using apolar and polar capillary columns. The essential oil recovered by SFME was submitted to an odour evaluation that revealed peculiar olfactive characteristics interesting in alcoholic male perfumery and body detergents.In vitro cytotoxicity assays were carried out using NCTC 2544 human keratinocytes as target cells. The oil displayed slight cytotoxic effects, which were three orders of magnitude lower than those found for sodium dodecyl sulphate positive control. The promising results in terms of chemical composition, scent and safety seem to indicate this essential oil as an interesting potential functional ingredient useful in a cosmetic context.

  20. Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis.

    Directory of Open Access Journals (Sweden)

    Benoit Stijlemans

    2015-03-01

    Full Text Available Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.

  1. In vitro CFU-E and BFU-E responses to androgen in bone marrow from children with primary hypoproliferative anaemia: a possible therapeutic assay.

    Science.gov (United States)

    Claustres, M; Margueritte, G; Sultan, C

    1986-02-01

    The effects of natural and synthetic androgens on erythroid colony formation in children's bone marrow cultures were studied using a methylcellulose microculture assay. In an attempt to predict the clinical response to androgens in two children with Fanconi anaemia (FA) and two children with Diamond-Blackfan syndrome (DB), we tested the hormonal stimulation of testosterone, nortestosterone and etiocholanolone on CFU-E, BFU-E and uroporphyrinogen I synthase activity (UROS). We observed that colony formation and UROS activity were reduced when compared to values obtained with normal children's bone marrow cultures. The addition of steroids to the cultures significantly enhanced the numbers of CFU-E and BFU-E derived colonies and their UROS activity in marrow from patients with FA and one patient with DB. The strong depletion of marrow progenitor cells in the unresponsive marrow from child 4 with DB could explain the absence of hormonal response. Whereas the responsiveness to steroids varied according to the individual, the in vitro testing of erythroid differentiation in the presence of androgens theoretically may lead to an effective prediction of response to therapy in children with hypoplastic anaemia.

  2. In vitro killing assays of antisera antibody sheep post-infected with Fasciola gigantica with the presence of macrophages cells against homologous and heterologous liver flukes

    Directory of Open Access Journals (Sweden)

    S.E Estuningsih

    1999-10-01

    Full Text Available The previous artificial infection known that the Indonesian Thin Tail (ITT sheep was resistance against the liver fluke of Fasciola gigantica, the resistances occurred in the early infection. In order to observe the immune resistance, some in vitro studies were undertaken in the laboratory, to assay the ability of the antisera antibody of ITT sheep post-infected with F. gigantica, with the presence of macrophages cells in killing the homologous and heterologous liver flukes. The viability of liver flukes were observed within 24-72 hours of incubation period by observing their motility (motile flukes were designated live and non-motile once were death. The results showed that after 72 hours incubation, the motilities of the Newly Excysted Juvenile (NEJ of F. gigantica incubated with the presence of post-infected sera and macrophages cells solution were significantly lower (P0.05. It seems that the occurrence of homologous antibody to the antigens is very important in the development of killing mechanism. The absence of homologous antibody did not reduce the number of flukes or the ability of macrophages cells in killing F. hepatica was not apparent.

  3. The DDX3 subfamily of the DEAD box helicases: divergent roles as unveiled by studying different organisms and in vitro assays.

    Science.gov (United States)

    Rosner, A; Rinkevich, B

    2007-01-01

    DDX3 (or Ded1p), the highly conserved subfamily of the DEAD-box RNA helicase family (40 members in humans), plays important roles in RNA metabolism. DDX3X and DDX3Y, the two human paralogous genes of this subfamily of proteins, have orthologous candidates in a diverse range of eukaryotes, from yeast and plants to animals. While DDX3Y, which is essential for normal spermatogenesis, is translated only in the testes, DDX3X protein is ubiquitously expressed, involved in RNA transcription, RNA splicing, mRNA transport, translation initiation and cell cycle regulation. Studies of recent years have revealed that DDX3X participates in HIV and hepatitis C viral infections, and in hepatocellular carcinoma, a complication of hepatitis B and hepatitis C infections. In the urochordates (i.e., Botryllus schlosseri) and in diverse invertebrate phyla (represented by model organisms such as: Drosophila, Hydra, Planaria), DDX3 proteins (termed also PL10) are involved in developmental pathways, highly expressed in adult undifferentiated soma and germ cells and in some adult and embryo's differentiating tissues. As the mechanistic and functional knowledge of DDX3 proteins is limited, we suggest assembling the available data on DDX3 proteins, from all studied organisms and in vitro assays, depicting a unified mechanistic scheme for DDX3 proteins' functions. Understanding the diverse functions of DDX3 in multicellular organisms may be particularly important for effective strategies of drug design.

  4. Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

    Science.gov (United States)

    Serena, Michela; Giorgetti, Alejandro; Busato, Mirko; Gasparini, Francesca; Diani, Erica; Romanelli, Maria Grazia; Zipeto, Donato

    2016-03-01

    HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.

  5. Xanthium strumarium L. Extracts Produce DNA Damage Mediated by Cytotoxicity in In Vitro Assays but Does Not Induce Micronucleus in Mice

    Science.gov (United States)

    Piloto Ferrer, Janet; Cozzi, Renata; Cornetta, Tommaso; Stano, Pasquale; Fiore, Mario; Degrassi, Francesca; De Salvia, Rosella; Remigio, Antonia; Francisco, Marbelis; Quiñones, Olga; Valdivia, Dayana; González, Maria L.; Pérez, Carlos; Sánchez-Lamar, Angel

    2014-01-01

    Xanthium strumarium L. is a member of the Asteraceae commonly used in Cuba, mainly as diuretic. Some toxic properties of this plant have also been reported and, to date, very little is known about its genotoxic properties. The present work aims was to evaluate the potential cytotoxic and genotoxic risk of whole extract from Xanthium strumarium L. whole extract of aerial parts. No positive response was observed in a battery of four Salmonella typhimurium strains, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation (liver microsomal S9 fraction from Wistar rats). In CHO cells, high concentrations (25–100 μg/mL) revealed significant reduction in cell viability. Results from sister chromatid exchanges, chromosome aberrations, and comet assay showed that X. strumarium extract is genotoxic at the highest concentration used, when clear cytotoxic effects were also observed. On the contrary, no increase in micronuclei frequency in bone marrow cells was observed when the extract was orally administered to mice (100, 500, and 2000 mg/Kg doses). The data presented here constitute the most complete study on the genotoxic potential of X. strumarium L. and show that the extract can induce in vitro DNA damage at cytotoxic concentrations. PMID:25025061

  6. Zinc substituted ferrite nanoparticles with Zn0.9Fe2.1O4 formula used as heating agents for in vitro hyperthermia assay on glioma cells

    Science.gov (United States)

    Hanini, Amel; Lartigue, Lenaic; Gavard, Julie; Kacem, Kamel; Wilhelm, Claire; Gazeau, Florence; Chau, François; Ammar, Souad

    2016-10-01

    In this paper we investigate the ability of zinc rich ferrite nanoparticles to induce hyperthermia on cancer cells using an alternating magnetic field (AMF). First, we synthesized ferrites and then we analyzed their physico-chemical properties by transmission electron microscopy, X-ray diffraction and magnetic and magnetocalorimetric measurements. We found that the polyol-made magnetically diluted particles are of 11 nm in size. They are superparamagnetic at body temperature (310 K) with a low but non-negligible magnetization. Interestingly, as nano-ferrimagnets they exhibit a Curie temperature of 366 K, close to the therapeutic temperature range. Their effect on human healthy endothelial (HUVEC) and malignant glioma (U87-MG) cells was also evaluated using MTT viability assays. Incubated with the two cell lines, at doses ≤100 μg mL-1 and contact times ≤4 h, they exhibit a mild in vitro toxicity. In these same operating biological conditions and coupled to AMF (700 kHz and 34.4 Oe) for 1 h, they rapidly induce a net temperature increase. In the case of tumor cells it reaches 4 K, making the produced particles particularly promising for self-regulated magnetically-induced heating in local glioma therapy.

  7. Xanthium strumarium L. Extracts Produce DNA Damage Mediated by Cytotoxicity in In Vitro Assays but Does Not Induce Micronucleus in Mice

    Directory of Open Access Journals (Sweden)

    Janet Piloto Ferrer

    2014-01-01

    Full Text Available Xanthium strumarium L. is a member of the Asteraceae commonly used in Cuba, mainly as diuretic. Some toxic properties of this plant have also been reported and, to date, very little is known about its genotoxic properties. The present work aims was to evaluate the potential cytotoxic and genotoxic risk of whole extract from Xanthium strumarium L. whole extract of aerial parts. No positive response was observed in a battery of four Salmonella typhimurium strains, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation (liver microsomal S9 fraction from Wistar rats. In CHO cells, high concentrations (25–100 μg/mL revealed significant reduction in cell viability. Results from sister chromatid exchanges, chromosome aberrations, and comet assay showed that X. strumarium extract is genotoxic at the highest concentration used, when clear cytotoxic effects were also observed. On the contrary, no increase in micronuclei frequency in bone marrow cells was observed when the extract was orally administered to mice (100, 500, and 2000 mg/Kg doses. The data presented here constitute the most complete study on the genotoxic potential of X. strumarium L. and show that the extract can induce in vitro DNA damage at cytotoxic concentrations.

  8. 3D in vitro cell culture models of tube formation

    NARCIS (Netherlands)

    Zegers, M.M.P.

    2014-01-01

    Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesench

  9. Spiroimidazolidinone NPC1L1 inhibitors. 1: Discovery by 3D-similarity-based virtual screening.

    Science.gov (United States)

    McMasters, Daniel R; Garcia-Calvo, Margarita; Maiorov, Vladimir; McCann, Margaret E; Meurer, Roger D; Bull, Herbert G; Lisnock, Jeanmarie; Howell, Kobporn L; Devita, Robert J

    2009-06-01

    A series of spiroimidazolidinone NPC1L1 inhibitors was discovered by virtual screening of the Merck corporate sample repository using 3D-similarity-based screening. Selection of 330 compounds for testing in an in vitro NPC1L1 binding assay yielded six hits in six distinct chemical series. Follow-up 2D similarity searching yielded several sub- to low-micromolar leads; among these was spiroimidazolidinone 10, with an IC(50) of 2.5 microM. Compound 10 provided a useful scaffold to initiate a medicinal chemistry campaign.

  10. Two zinc-aminoclays' in-vitro cytotoxicity assessment in HeLa cells and in-vivo embryotoxicity assay in zebrafish.

    Science.gov (United States)

    Chun, Hang-Suk; Park, Duckshin; Eun Lim, Song; Jeong, Kwang-Hun; Park, Ji-Seon; Park, Han-Jin; Kang, Shinyoung; Kang, Kyoung Suk; Park, Hyun Gyu; An, Ha-Rim; Huh, Yun Suk; Lee, Young-Chul

    2017-03-01

    Two zinc-aminoclays [ZnACs] with functionalized primary amines [(-CH2)3NH2] were prepared by a simple sol-gel reaction using cationic metal precursors of ZnCl2 and Zn(NO3)2 with 3-aminopropyl triethoxysilane [APTES] under ambient conditions. Due to the facile interaction of heavy metals with primary amine sites and Zn-related intrinsic antimicrobial activity, toxicity assays of ZnACs nanoparticles (NPs) prior to their environmental and human-health applications are essential. However, such reports remain rare. Thus, in the present study, a cell viability assay of in-vitro HeLa cells comparing ZnCl2, Zn(NO3)2 salts, and ZnO (~50nm average diameter) NPs was performed. Interestingly, compared with the ZnCl2, and Zn(NO3)2 salts, and ZnO NPs (18.73/18.12/51.49µg/mL and 18.12/15.19/46.10µg/mL of IC50 values for 24 and 48h), the two ZnACs NPs exhibited the highest toxicity (IC50 values of 21.18/18.36µg/mL and 18.37/17.09µg/mL for 24 and 48h, respectively), whose concentrations were calculated on Zn elemental composition. This might be due to the enhanced bioavailability and uptake into cells of ZnAC NPs themselves and their positively charged hydrophilicity by reactive oxygen species (ROS) generation, particularly as ZnACs exist in cationic NP's form, not in released Zn(2+) ionic form (i.e., dissolved nanometal). However, in an in-vivo embryotoxicity assay in zebrafish, ZnACs and ZnO NPs showed toxic effects at 50-100µg/mL (corresponding to 37.88-75.76 of Zn wt% µg/mL). The hatching rate (%) of zebrafish was lowest for the ZnO NPs, particularly where ZnAC-[(NO3)2] is slightly more toxic than ZnAC-[Cl2]. These results are all very pertinent to the issue of ZnACs' potential applications in the environmental and biomedical fields.

  11. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  12. A database of IC50 values and principal component analysis of results from six basal cytotoxicity assays, for use in the modelling of the in vivo and in vitro data of the EU ACuteTox project.

    Science.gov (United States)

    Clothier, Richard; Dierickx, Paul; Lakhanisky, Thaly; Fabre, Myriam; Betanzos, Monica; Curren, Rodger; Sjöström, Michael; Raabe, Hans; Bourne, Nicola; Hernandez, Vanessa; Mainez, Jessica; Owen, Monika; Watts, Sarah; Anthonissen, Roel

    2008-11-01

    The main aim of the ACuteTox project (part of the EU 6th Framework programme) is to demonstrate that animal tests for acute systemic toxicity can be replaced by alternative in vitro assays. In this project, data for 97 reference chemicals were collected in the AcuBase database, designed to handle deposited in vitro and in vivo (human and animal) data. To demonstrate the applicability of in vitro basal cytotoxicity tests and in vitro-in vivo modelling, it was deemed necessary to obtain data that were generated via defined standard operating procedures. The molar basal cytotoxicity IC50 values (the 50% inhibitory concentrations for the endpoint measured) for a mouse fibroblast cell line (3T3), a human hepatic cell line (HepG2), a rat hepatic cell line (Fa32), and a human neutrophil cell line (HL-60), were compared, and gave an R(2) correlation of 0.83. To identify chemicals that showed differential cytotoxicity to the various cell types involved, principal component analysis (PCA) was undertaken independently, once all the results had been returned. This showed that colchicine, cycloheximide, digoxin, 5-fluorouracil and hexachlorobenzene gave the lowest correlations with the first score vector of the PCA. The results presented are to be used to identify outliers that need to be further studied via the use of tissue-specific in vitro assays.

  13. Bioactive polymeric–ceramic hybrid 3D scaffold for application in bone tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Torres, A.L.; Gaspar, V.M.; Serra, I.R.; Diogo, G.S.; Fradique, R. [CICS-UBI — Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã (Portugal); Silva, A.P. [CAST-UBI — Centre for Aerospace Science and Technologies, University of Beira Interior, Calçada Fonte do Lameiro, 6201-001 Covilhã (Portugal); Correia, I.J., E-mail: icorreia@ubi.pt [CICS-UBI — Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã (Portugal)

    2013-10-01

    The regeneration of large bone defects remains a challenging scenario from a therapeutic point of view. In fact, the currently available bone substitutes are often limited by poor tissue integration and severe host inflammatory responses, which eventually lead to surgical removal. In an attempt to address these issues, herein we evaluated the importance of alginate incorporation in the production of improved and tunable β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) three-dimensional (3D) porous scaffolds to be used as temporary templates for bone regeneration. Different bioceramic combinations were tested in order to investigate optimal scaffold architectures. Additionally, 3D β-TCP/HA vacuum-coated with alginate, presented improved compressive strength, fracture toughness and Young's modulus, to values similar to those of native bone. The hybrid 3D polymeric–bioceramic scaffolds also supported osteoblast adhesion, maturation and proliferation, as demonstrated by fluorescence microscopy. To the best of our knowledge this is the first time that a 3D scaffold produced with this combination of biomaterials is described. Altogether, our results emphasize that this hybrid scaffold presents promising characteristics for its future application in bone regeneration. - Graphical abstract: B-TCP:HA–alginate hybrid 3D porous scaffolds for application in bone regeneration. - Highlights: • The produced hybrid 3D scaffolds are prone to be applied in bone tissue engineering. • Alginate coated 3D scaffolds present high mechanical and biological properties. • In vitro assays for evaluation of human osteoblast cell attachment in the presence of the scaffolds • The hybrid 3D scaffolds present suitable mechanical and biological properties for use in bone regenerative medicine.

  14. Predicting in vivo gene expression in macrophages after exposure to benzo(a)pyrene based on in vitro assays and toxicokinetic/toxicodynamic models

    OpenAIRE

    Péry, Alexandre R R; Brochot, Céline; Desmots, Sophie; Boize, Magali; Sparfel, Lydie; Fardel, Olivier

    2011-01-01

    International audience; Predictive toxicology aims at developing methodologies to relate the results obtained from in vitro experiments to in vivo exposure. In the case of polycyclic aromatic hydrocarbons (PAHs), a substantial amount of knowledge on effects and modes of action has been recently obtained from in vitro studies of gene expression. In the current study, we built a physiologically based toxicokinetic (PBTK) model to relate in vivo and in vitro gene expression in case of exposure t...

  15. 3D printing for dummies

    CERN Document Server

    Hausman, Kalani Kirk

    2014-01-01

    Get started printing out 3D objects quickly and inexpensively! 3D printing is no longer just a figment of your imagination. This remarkable technology is coming to the masses with the growing availability of 3D printers. 3D printers create 3-dimensional layered models and they allow users to create prototypes that use multiple materials and colors.  This friendly-but-straightforward guide examines each type of 3D printing technology available today and gives artists, entrepreneurs, engineers, and hobbyists insight into the amazing things 3D printing has to offer. You'll discover methods for

  16. Intraoral 3D scanner

    Science.gov (United States)

    Kühmstedt, Peter; Bräuer-Burchardt, Christian; Munkelt, Christoph; Heinze, Matthias; Palme, Martin; Schmidt, Ingo; Hintersehr, Josef; Notni, Gunther

    2007-09-01

    Here a new set-up of a 3D-scanning system for CAD/CAM in dental industry is proposed. The system is designed for direct scanning of the dental preparations within the mouth. The measuring process is based on phase correlation technique in combination with fast fringe projection in a stereo arrangement. The novelty in the approach is characterized by the following features: A phase correlation between the phase values of the images of two cameras is used for the co-ordinate calculation. This works contrary to the usage of only phase values (phasogrammetry) or classical triangulation (phase values and camera image co-ordinate values) for the determination of the co-ordinates. The main advantage of the method is that the absolute value of the phase at each point does not directly determine the coordinate. Thus errors in the determination of the co-ordinates are prevented. Furthermore, using the epipolar geometry of the stereo-like arrangement the phase unwrapping problem of fringe analysis can be solved. The endoscope like measurement system contains one projection and two camera channels for illumination and observation of the object, respectively. The new system has a measurement field of nearly 25mm × 15mm. The user can measure two or three teeth at one time. So the system can by used for scanning of single tooth up to bridges preparations. In the paper the first realization of the intraoral scanner is described.

  17. Martian terrain - 3D

    Science.gov (United States)

    1997-01-01

    This area of terrain near the Sagan Memorial Station was taken on Sol 3 by the Imager for Mars Pathfinder (IMP). 3D glasses are necessary to identify surface detail.The IMP is a stereo imaging system with color capability provided by 24 selectable filters -- twelve filters per 'eye.' It stands 1.8 meters above the Martian surface, and has a resolution of two millimeters at a range of two meters.Mars Pathfinder is the second in NASA's Discovery program of low-cost spacecraft with highly focused science goals. The Jet Propulsion Laboratory, Pasadena, CA, developed and manages the Mars Pathfinder mission for NASA's Office of Space Science, Washington, D.C. JPL is an operating division of the California Institute of Technology (Caltech). The Imager for Mars Pathfinder (IMP) was developed by the University of Arizona Lunar and Planetary Laboratory under contract to JPL. Peter Smith is the Principal Investigator.Click below to see the left and right views individually. [figure removed for brevity, see original site] Left [figure removed for brevity, see original site] Right

  18. Pharmaceutical evaluation of naftopidil enantiomers: Rat functional assays in vitro and estrogen/androgen induced rat benign prostatic hyperplasia model in vivo.

    Science.gov (United States)

    Huang, Jun-Jun; Cai, Yi; Huang, Min-Yi; Zhu, Liu; He, Fei; Liu, Xia-Wen; Huang, Bi-Yun; Yi, Yan-Zhen; Yuan, Mu

    2016-11-15

    Naftopidil (NAF) is a α1D/1A adrenoceptor selective drug used for the treatment of both benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS). However, NAF is used as a racemate in clinic. To compare the differences and similarities among two enantiomers and racemate, pharmacological activities were evaluated through rat functional assays in vitro and estrogen/androgen (E/T) induced rat BPH model in vivo. NAF and the two enantiomers showed similar blocking activity on α1 receptor. S-NAF exhibited more α1D/1A adrenoceptor subtype selectivity than R-NAF and the racemate. The selectivity ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 40.7- and 16.2-fold, respectively. NAF and its enantiomers effectively prevented the development of rat prostatic hyperplasia via suppressing the increase of the prostatic wet weight, visually. The quantitative analysis of the relative acinus volume, relative stroma volume, relative epithelial volume, epithelial height and expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were carried out. S-NAF showed an advantage on the effect of inhibiting prostate wet weight and stroma volume over R-NAF and racemate NAF (P<0.05). Nevertheless, no other significant difference was observed between these two enantiomers. In conclusion, both R-NAF and S-NAF not only relax prostate muscle but also inhibit the prostate growth, thus relieve BPH.

  19. Endocrine disrupting activities in sewage effluent and river water determined by chemical analysis and in vitro assay in the context of granular activated carbon upgrade.

    Science.gov (United States)

    Grover, D P; Balaam, J; Pacitto, S; Readman, J W; White, S; Zhou, J L

    2011-09-01

    As part of endocrine disruption in catchments (EDCAT) programme, this work aims to assess the temporal and spatial variations of endocrine disrupting chemicals (EDCs) in River Ray, before and after the commissioning of a full-scale granular activated carbon (GAC) plant at a sewage treatment works (STW). Through spot and passive sampling from effluent and river sites, estrogenic and anti-androgenic activities were determined by chemical analysis and in vitro bio-assay. A correlation was found between chemical analyses of the most potent estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2)) and yeast estrogen screen (YES) measurement, both showing clearly a reduction in estrogenic activity after the commissioning of the GAC plant at the STW. During the study period, the annual average concentrations of E1, E2 and EE2 had decreased from 3.5 ng L(-1), 3.1 ng L(-1) and 0.5 ng L(-1) to below their limit of detection (LOD), respectively, with a concentration reduction of at least 91%, 81% and 60%. Annual mean estrogenic activity measured by YES of spot samples varied from 1.9 ng L(-1) to 0.4 ng L(-1) E2 equivalent between 2006 and 2008 representing a 79% reduction. Similarly, anti-androgenic activity measured by yeast anti-androgen screen (anti-YAS) of spot samples was reduced from 148.8 to 22.4 μg flutamide L(-1), or by 85%. YES and anti-YAS values were related to each other, suggesting co-existence of both types of activities from chemical mixtures in environmental samples. The findings confirm the effectiveness of a full-scale GAC in removing both estrogenic and anti-androgenic activities from sewage effluent.

  20. Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s bearing functionalizable carbonate building blocks: II. Enzymatic biodegradation and in vitro biocompatibility assay.

    Science.gov (United States)

    Yang, Jing; Tian, Weisheng; Li, Qiaobo; Li, Yang; Cao, Amin

    2004-01-01

    In a previous study, we have reported chemical synthesis of novel aliphatic poly(butylene succinate-co-cyclic carbonate) P(BS-co-CC)s bearing various functionalizable carbonate building blocks, and this work will continue to present our new studies on their enzymatic degradation and in vitro cell biocompatibility assay. First, enzymatic degradation of the novel P(BS-co-CC) film samples was investigated with two enzymes of lipase B Candida Antartic (Novozyme 435) and lipase Porcine Pancreas PPL, and it was revealed that copolymerizing linear poly(butylene succinate) PBS with a functionalizable carbonate building block could remarkably accelerate the enzymatic degradation of a synthesized product P(BS-co-CC), and its biodegradation behavior was found to strongly depend on the overall impacts of several important factors as the cyclic carbonate (CC) comonomer structure and molar content, molar mass, thermal characteristics, morphology, the enzyme-substrate specificity, and so forth. Further, the biodegraded residual film samples and water-soluble enzymatic degradation products were allowed to be analyzed by means of proton nuclear magnetic resonance (1H NMR), gel permeation chromatograph (GPC), differential scanning calorimeter (DSC), attenuated total reflection FTIR (ATR-FTIR), scanning electron microscope (SEM), and liquid chromatograph-mass spectrometry (LC-MS). On the experimental evidences, an exo-type mechanism of enzymatic chain hydrolysis preferentially occurring in the noncrystalline domains was suggested for the synthesized new P(BS-co-CC) film samples. With regard to their cell biocompatibilities, an assay with NIH 3T3 mouse fibroblast cell was conducted using the novel synthesized P(BS-co-CC) films as substrates with respect to the cell adhesion and proliferation, and these new biodegradable P(BS-co-CC) samples were found to exhibit as low cell toxicity as the PLLA control, particularly the two samples of poly(butylene succinate-co-18.7 mol % dimethyl

  1. Study of Low-intensity 2450-MHz Microwave Exposure Enhancing the Genotoxic Effects of Mitomycin C Using Micronucleus Test and Comet Assay in vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To determine the interaction between 2450-MHz microwaves (MW) radiation and mitomycin C (MMC). Methods The synergistic genotoxic effects of low-intensity 2450-MHz microwave and MMC on human lymphocytes were studied using single cell gel electrophoresis (SCGE) assay (comet assay) and cytokinesis-blocked micronucleus (CBMN) test in vitro. The whole blood cells from a male donor and a female donor were either only exposed to 2450-MHz microwaves (5.0 mW/cm2) for 2 h or only exposed to MMC (0.0125 μg/mL, 0.025 μg/mL, 0.05 μg/mL and 0.1 μg/mL) for 24 h; and the samples were exposed to MMC for 24 h after exposure to MW for 2 h. Results In the comet assay, the comet lengths ( 29.1 μm and 25.9 μm) of MW were not significantly longer than those (26.3 μm and 24.1 μm) of controls (P>0.05). The comet lengths (57.4 μm, 68.9 μm, 91.4 μam, 150.6 μm and 50.6 μm, 71.7 μm, 100.1 μm, 145.1 μm) of 4 MMC groups were significantly longer than those of controls (P<0.01). The comet lengths (59.1 μm, 92.3 μm, 124.5 μm, 182.7 μm and 57.4 μm, 85.5 μm, 137.5 μm, 178.3 μm) of 4 MW plus MMC groups were significantly longer than those of controls too (P<0.01). The comet lengths of MW plus MMC groups were significantly longer than those of the corresponding MMC doses (P<0.05 or P<0.01) when the doses of MMC were ≥0.025 μg/mL. In the CBMN, the micronucleated cell (MNC) rates of MW were 5‰ and 6‰,which showed no difference compared with those (4‰ and 4‰) of controls (P>0.05). The MNC rates of 4 MMC groups were 8‰, 9‰, 14‰, 23‰ and 8‰, 8‰, 16‰, 30‰ respectively. When the doses of MMC were ≥0.05 μg/mL, MNC rates of MMC were higher than those of controls (P<0.05).MNC rates of 4 MW plus MMC groups were 12‰, 13‰, 20‰, 32‰ and 8‰, 9‰, 23‰, 40‰.When the doses of MMC were ≥0.05 μg/mL, MNC rates of MW plus MMC groups were much higher than those of controls (P<0.01). MNC rates of 4 MW plus MMC groups were not

  2. Establishment of flow cytometric in micronucleus assay in vitro%流式细胞术检测体外微核方法的建立

    Institute of Scientific and Technical Information of China (English)

    欧红梅; 周长慧; 涂宏刚; 黄鹏程; 常艳

    2015-01-01

    OBJECTIVE:Establish the flow cytometric 96-well microplate-basedin vitro micronucleus assay in CHO-K1 cells,and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS:The test included treatment with and without metabolic activation. For the treatment with metabolic activation,CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mixmedium for 4 h,then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation,cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases,after a total of 24 h since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate,and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy.RESULTS:Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000.CONCLUSION:Similar to literature published,mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was estab