Tilmicosin reduces lipopolysaccharide-stimulated bovine alveolar macrophage prostaglandin E(2) production via a mechanism involving phospholipases.

PubMed

Lakritz, Jeffrey; Tyler, Jeff W; Marsh, Antoinette E; Romesburg-Cockrell, Mary; Smith, Kathy; Holle, Julie M

2002-01-01

Tilmicosin is a potent antimicrobial with broad-spectrum activity against the bacterial agents involved in the bovine respiratory disease complex. Recent studies indicate that in addition to being bactericidal, tilmicosin is capable of modulating inflammation in the lung. A series of experiments were designed to determine whether tilmicosin alters alveolar macrophage-prostaglandin E(2) (PGE(2)) production induced by Escherichia coli (O55:B5) lipopolysaccharide (LPS). Twenty-two healthy Holstein bull calves were used to study the effects of LPS-induced PGE(2) production of alveolar macrophages after in vivo or in vitro treatment with tilmicosin. In Experiment 1, tilmicosin was given by subcutaneous injection (15 mg/kg) twice, 48 hours apart, to four calves; four control calves received no treatment. Twenty-four hours after the second treatment, alveolar macrophages were stimulated with LPS in vitro. In Experiment 2, alveolar macrophages from five untreated calves were harvested and treated in vitro with tilmicosin, followed by LPS stimulation. In Experiment 3, the ability of in vitro tilmicosin treatment to alter the expression of LPS-induced cyclooxygenase-2 (COX-2) mRNA was evaluated. In Experiments 4 and 5, secretory phospholipase A(2) activity was examined in untreated calves. Treatment of calves with tilmicosin resulted in reduced LPS-induced alveolar macrophage PGE(2) production. Similar reductions in PGE(2) by LPS-stimulated alveolar macrophages after in vitro tilmicosin treatment were noted. This in vitro tilmicosin treatment was not associated with reduction of the expression of LPS-induced COX-2. Alveolar macrophage phospholipase A(2) activity induced by LPS was significantly reduced by prior tilmicosin treatment in vitro. Tilmicosin (in vivo and in vitro) appears to reduce the PGE(2) eicosanoid response of LPS-stimulated alveolar macrophages by reducing the in vitro substrate availability without altering in vitro COX-2 mRNA expression.

In vitro cell and tissue models for studying host-microbe interactions: a review.

PubMed

Bermudez-Brito, Miriam; Plaza-Díaz, Julio; Fontana, Luis; Muñoz-Quezada, Sergio; Gil, Angel

2013-01-01

Ideally, cell models should resemble the in vivo conditions; however, in most in vitro experimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that exist in vivo between the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details the in vitro models used to study host-microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.

Colorectal tissue engineering: A comparative study between porcine small intestinal submucosa (SIS) and chitosan hydrogel patches.

PubMed

Denost, Quentin; Adam, Jean-Philippe; Pontallier, Arnaud; Montembault, Alexandra; Bareille, Reine; Siadous, Robin; Delmond, Samantha; Rullier, Eric; David, Laurent; Bordenave, Laurence

2015-12-01

Tissue engineering may provide new operative tools for colorectal surgery in elective indications. The aim of this study was to define a suitable bioscaffold for colorectal tissue engineering. We compared 2 bioscaffolds with in vitro and in vivo experiments: porcine small intestinal submucosa (SIS) versus chitosan hydrogel matrix. We assessed nontoxicity of the scaffold in vitro by using human adipose-derived stem cells (hADSC). In vivo, a 1 × 2-cm colonic wall defect was created in 16 rabbits. Animals were divided randomly into 2 groups according to the graft used, SIS or chitosan hydrogel. Graft area was explanted at 4 and 8 weeks. The end points of in vivo experiments were technical feasibility, behavior of the scaffold, in situ putative inflammatory effect, and the quality of tissue regeneration, in particular smooth muscle layer regeneration. In vitro, hADSC attachment and proliferation occurred on both scaffolds without a substantial difference. After proliferation, hADSCs kept their mesenchymal stem cell characteristics. In vivo, one animal died in each group. Eight weeks after implantation, the chitosan scaffold allowed better wound healing compared with the SIS scaffold, with more effective control of inflammatory activity and an integral regeneration of the colonic wall including the smooth muscle cell layer. The outcomes of in vitro experiments did not differ greatly between the 2 groups. Macroscopic and histologic findings, however, revealed better wound healing of the colonic wall in the chitosan group suggesting that the chitosan hydrogel could serve as a better scaffold for colorectal tissue engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative analysis of Fe ion-induced mutations in murine tissue and cells

NASA Astrophysics Data System (ADS)

Kronenberg, A.; Gauny, S.; Kwoh, E.; Dan, C.; Connolly, L.; Turker, M.

Space flight exposes astronauts to densely ionizing heavy ions including Fe ions This study is designed to assess the impact of the tissue microenvironment on the cytotoxic and mutagenic effects of 1 GeV amu Fe ions in kidney epithelial cells from one mouse strain irradiated either in vitro or in vivo Three to five month old Aprt heterozygous mice are used from a C57BL6 DBA2 cross B6D2F1 or kidney cells are used that were established from these mice Cells and animals were exposed in the plateau portion of the Bragg peak 159 keV mu m at the NASA Space Radiation Laboratories NSRL at Brookhaven National Laboratory Approximately equal numbers of male and female animals were used for the in vivo studies In vitro experiments demonstrated exponential cell killing with a D 0 of 92 cGy Three Aprt mutation experiments have been performed in kidney cells exposed to graded doses of Fe ions in vitro 0-2 Gy Studies to date indicate that Fe ions are mutagenic to kidney epithelial cells irradiated in vitro with a linear induction of mutants as a function of dose In vivo experiments have been completed on two thirds of the animals planned for the study Kidney cells were retrieved from the animals at two time points 2-3 months post-irradiation or 8-9 months post-irradiation Fe ion exposure in vivo led to exponential killing of kidney epithelial cells that was still evident 8-9 months post-exposure In vivo irradiation also results

In-Vitro Immunology - Skylab Student Experiment ED-31

NASA Technical Reports Server (NTRS)

1973-01-01

This chart describes the Skylab student experiment In-Vitro Immunology, proposed by Todd A. Meister of Jackson Heights, New York. He suggested an in-vitro observation of the effects of zero-gravity on a presipitin-type antigen-antibody reaction, as compared with the same reaction carried out in an Earth-based laboratory. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

In vitro and in situ degradation of alkali treated sorghum wet distillers grains alone or in combination with corn stalks to increase their nutritive value

USDA-ARS?s Scientific Manuscript database

This experiment was conducted to evaluate the effect of alkali treatment on in vitro and in situ digestibility of fiber sources. An in vitro and in situ experiment were conducted to determine the effects of treating sorghum WDG with solubles (SWDG) and corn stalks (CS) with calcium hydroxide on in ...

Selection of animal models for pre-clinical strategies in evaluating the fracture healing, bone graft substitutes and bone tissue regeneration and engineering.

PubMed

Bigham-Sadegh, Amin; Oryan, Ahmad

2015-06-01

In vitro assays can be useful in determining biological mechanism and optimizing scaffold parameters, however translation of the in vitro results to clinics is generally hard. Animal experimentation is a better approximation than in vitro tests, and usage of animal models is often essential in extrapolating the experimental results and translating the information in a human clinical setting. In addition, usage of animal models to study fracture healing is useful to answer questions related to the most effective method to treat humans. There are several factors that should be considered when selecting an animal model. These include availability of the animal, cost, ease of handling and care, size of the animal, acceptability to society, resistance to surgery, infection and disease, biological properties analogous to humans, bone structure and composition, as well as bone modeling and remodeling characteristics. Animal experiments on bone healing have been conducted on small and large animals, including mice, rats, rabbits, dogs, pigs, goats and sheep. This review also describes the molecular events during various steps of fracture healing and explains different means of fracture healing evaluation including biomechanical, histopathological and radiological assessments.

Pursuing conception: a physician's experience with in-vitro fertilization.

PubMed Central

McCall, M

1996-01-01

Infertility is a common problem. Approximately one in seven North American couples will experience it, either by being unable to conceive after a year of trying or by experiencing recurrent miscarriages. A family physician outlines her experiences when being treated for infertility by in-vitro fertilization and embryo transfer. PMID:8625028

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

PubMed

Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

Hydrophobic interaction mediated coating of Pluronics on mesoporous silica nanoparticle with stimuli responsiveness for cancer therapy.

PubMed

Sha, Luping; Wang, Da; Mao, Yuling; Shi, Wei; Gao, Tianbin; Zhao, Qinfu; Wang, Si-Ling

2018-05-22

In this research, a novel method was used to successfully make Pluronic P123 stably coated on mesoporous silica nanoparticles (MSN). That P123 and MSN co-constructed a drug delivery system (DDS) had not been reported. In this DDS, the coating of P123 was realized through hydrophobic interaction with octadecyl chain modified MSN. Experiments found only Pluronic with an appropriate ratio of hydrophilic and lipophilic segment could keep the nanoassemblies stable. For comparison, nanoassemblies consisted of P123 and octadecyl chain modified MSN with or without disulfide bond were prepared, which were denoted as PSMSN and PMSN respectively. Disulfide bond was expected to endow the system with redox-responsiveness to enhance the therapeutic effect meanwhile decrease toxicity. A series of experiments including characterization of the nanoparticles, in vitro drug release, cell uptake and cellular drug release, in vitro cytotoxicity, cell migration and biodistribution of the nanoparticles were carried out. Compared with PMSN, PSMSN displayed redox-responsive drug release property not only in in vitro release text, but also on the cellular level. In addition, cell migration experiments proved that the coating of P123 endowed the system with the ability of anti-metastasis. The accumulation of P123 in tumor was enhanced after coating on MSN by virtue of the "EPR" effect of nanoparticles compared with the solution form. . © 2018 IOP Publishing Ltd.

Improved In Vitro and In Vivo Biocompatibility of Graphene Oxide through Surface Modification: Poly(Acrylic Acid)-Functionalization is Superior to PEGylation.

PubMed

Xu, Ming; Zhu, Jianqiang; Wang, Fanfan; Xiong, Yunjing; Wu, Yakun; Wang, Qiuquan; Weng, Jian; Zhang, Zhihong; Chen, Wei; Liu, Sijin

2016-03-22

The unique physicochemical properties of two-dimensional (2D) graphene oxide (GO) could greatly benefit the biomedical field; however, recent research demonstrated that GO could induce in vitro and in vivo toxicity. We determined the mechanism of GO induced toxicity, and our in vitro experiments revealed that pristine GO could impair cell membrane integrity and functions including regulation of membrane- and cytoskeleton-associated genes, membrane permeability, fluidity and ion channels. Furthermore, GO induced platelet depletion, pro-inflammatory response and pathological changes of lung and liver in mice. To improve the biocompatibility of pristine GO, we prepared a series of GO derivatives including aminated GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and poly(ethylene glycol)-functionalized GO (GO-PEG), and compared their toxicity with pristine GO in vitro and in vivo. Among these GO derivatives, GO-PEG and GO-PAA induced less toxicity than pristine GO, and GO-PAA was the most biocompatible one in vitro and in vivo. The differences in biocompatibility were due to the differential compositions of protein corona, especially immunoglobulin G (IgG), formed on their surfaces that determine their cell membrane interaction and cellular uptake, the extent of platelet depletion in blood, thrombus formation under short-term exposure and the pro-inflammatory effects under long-term exposure. Overall, our combined data delineated the key molecular mechanisms underlying the in vivo and in vitro biological behaviors and toxicity of pristine GO, and identified a safer GO derivative that could be used for future applications.

[Formulation and in vitro examination of furosemide-containing suppositories and preliminary experiences of their clinical use].

PubMed

Regdon, G; Fazekas, T; Regdon, G; Selmeczi, B

1997-01-01

Rectal suppositories containing Furosemide (4-Chloro-N-furfuryl-5-sulfamoylanthranilic acid) and Furosemide Sodium were formulated with various suppository bases. The in vitro drug release of Massa Estarinum 299 proved to be the best from among the vehicles having various physical-chemical properties. The diuretic effect of the two suppositories was compared in a prospective, cross-over clinical trial including 8 patients. Both preparations have induced and increase of urine flow, which was comparable to the diuretic effect of the tablet. Thus the possibility of rectal use has been added to the modalities of therapeutic Furosemide administration.

Concentration dependence of biotransformation in fish liver S9: Optimizing substrate concentrations to estimate hepatic clearance for bioaccumulation assessment.

PubMed

Lo, Justin C; Allard, Gayatri N; Otton, S Victoria; Campbell, David A; Gobas, Frank A P C

2015-12-01

In vitro bioassays to estimate biotransformation rate constants of contaminants in fish are currently being investigated to improve bioaccumulation assessments of hydrophobic contaminants. The present study investigates the relationship between chemical substrate concentration and in vitro biotransformation rate of 4 environmental contaminants (9-methylanthracene, pyrene, chrysene, and benzo[a]pyrene) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions and methods to determine maximum first-order biotransformation rate constants. Substrate depletion experiments using a series of initial substrate concentrations showed that in vitro biotransformation rates exhibit strong concentration dependence, consistent with a Michaelis-Menten kinetic model. The results indicate that depletion rate constants measured at initial substrate concentrations of 1 μM (a current convention) could underestimate the in vitro biotransformation potential and may cause bioconcentration factors to be overestimated if in vitro biotransformation rates are used to assess bioconcentration factors in fish. Depletion rate constants measured using thin-film sorbent dosing experiments were not statistically different from the maximum depletion rate constants derived using a series of solvent delivery-based depletion experiments for 3 of the 4 test chemicals. Multiple solvent delivery-based depletion experiments at a range of initial concentrations are recommended for determining the concentration dependence of in vitro biotransformation rates in fish liver fractions, whereas a single sorbent phase dosing experiment may be able to provide reasonable approximations of maximum depletion rates of very hydrophobic substances. © 2015 SETAC.

Assessment and characterisation of yeast-based products intended to mitigate ochratoxin exposure using in vitro and in vivo models.

PubMed

Pfohl-Leszkowicz, A; Hadjeba-Medjdoub, K; Ballet, N; Schrickx, J; Fink-Gremmels, J

2015-01-01

The aim of this paper was to evaluate the capacity of several yeast-based products, derived from baker's and brewer's yeasts, to sequester the mycotoxin ochratoxin A (OTA) and to decrease its rate of absorption and DNA adduct formation in vivo. The experimental protocol included in vitro binding studies using isotherm models, in vivo chicken experiments, in which the serum and tissue concentrations of OTA were analysed in the absence and presence of the test compounds, and the profile of OTA-derived metabolites and their associated DNA adducts were determined. Additionally in vitro cell culture studies (HK2 cells) were applied to assess further the effects for yeast cell product enriched with glutathione (GSH) or selenium. Results of the in vitro binding assay in a buffer system indicated the ability of the yeast-based products, as sequester of OTA, albeit at a different level. In the in vitro experiments in chickens, decreased serum and tissue concentrations of treated animals confirmed that yeast-based products are able to prevent the absorption of OTA. A comparison of the binding affinity in a standard in vitro binding assay with the results obtained in an in vivo chicken experiment, however, showed a poor correlation and resulted in a different ranking of the products. More importantly, we could show that yeast-based products actively modulate the biotransformation of OTA in vivo as well as in vitro in a cell culture model. This effect seems to be attributable to residual enzymatic activities in the yeast-based products. An enrichment of yeast cell wall products with GSH or selenium further modulated the profile of the generated OTA metabolites and the associated pattern of OTA-induced DNA adducts by increasing the conversion of OTA into less toxic metabolites such as OTA, OTB and 4-OH-OTA. A reduced absorption and DNA adduct formation was particularly observed with GSH-enriched yeast, whereas selenium-enriched yeasts could counteract the OTA-induced decrease in cell viability, but at the same time increased the OTA-DNA adducts formation. These findings indicate the need for an in-depth characterisation of yeast-based products used as mycotoxin-mitigating feed additives, in in vivo models with target animal species taking into account not only their ability to sequester toxins in the gastrointestinal tract but also their potential effects on the biotransformation of mycotoxins.

Experiences of physical activity during pregnancy resulting from in vitro fertilisation: an interpretative phenomenological analysis.

PubMed

Walker, Chloe; Mills, Hayley; Gilchrist, Angela

2017-09-01

To explore the qualitative experiences and decision-making processes surrounding physical activity (PA) for women who have undergone IVF treatment. PA during pregnancy is safe for both mother and fetus in the majority of cases, including for women who have undergone in vitro fertilisation (IVF) treatment; however, there is a paucity of research into decision-making and PA in this population. Eight women, who had undergone successful IVF treatment and were currently pregnant or had given birth within the last two years, participated in semi-structured interviews about their experiences of infertility and PA during pregnancy. Interview transcripts were analysed using interpretative phenomenological analysis. Three superordinate themes emerged from the data: 'navigating away from childlessness and towards motherhood', 'negotiating a safe passage' and 'balancing the challenges of pregnancy with the needs of the self'. Ten subthemes indicated the processes adopted to navigate experiences of infertility, the IVF process, and subsequent decision-making about PA during pregnancy. PA during pregnancy was experienced as a way to soothe the self and control the experience of pregnancy; however, this was mediated by concerns about safety and physical limitations on PA. Limitations of the study are considered, as well as implications for clinical practice and directions for future research.

Analysis of the Effects of Five Factors Relevant to In Vitro Chondrogenesis of Human Mesenchymal Stem Cells Using Factorial Design and High Throughput mRNA-Profiling

PubMed Central

Jakobsen, Rune B.; Østrup, Esben; Zhang, Xiaolan; Mikkelsen, Tarjei S.; Brinchmann, Jan E.

2014-01-01

The in vitro process of chondrogenic differentiation of mesenchymal stem cells for tissue engineering has been shown to require three-dimensional culture along with the addition of differentiation factors to the culture medium. In general, this leads to a phenotype lacking some of the cardinal features of native articular chondrocytes and their extracellular matrix. The factors used vary, but regularly include members of the transforming growth factor β superfamily and dexamethasone, sometimes in conjunction with fibroblast growth factor 2 and insulin-like growth factor 1, however the use of soluble factors to induce chondrogenesis has largely been studied on a single factor basis. In the present study we combined a factorial quality-by-design experiment with high-throughput mRNA profiling of a customized chondrogenesis related gene set as a tool to study in vitro chondrogenesis of human bone marrow derived mesenchymal stem cells in alginate. 48 different conditions of transforming growth factor β 1, 2 and 3, bone morphogenetic protein 2, 4 and 6, dexamethasone, insulin-like growth factor 1, fibroblast growth factor 2 and cell seeding density were included in the experiment. The analysis revealed that the best of the tested differentiation cocktails included transforming growth factor β 1 and dexamethasone. Dexamethasone acted in synergy with transforming growth factor β 1 by increasing many chondrogenic markers while directly downregulating expression of the pro-osteogenic gene osteocalcin. However, all factors beneficial to the expression of desirable hyaline cartilage markers also induced undesirable molecules, indicating that perfect chondrogenic differentiation is not achievable with the current differentiation protocols. PMID:24816923

Evaluation of polymerization shrinkage of resin cements through in vitro and in situ experiments

NASA Astrophysics Data System (ADS)

Franco, A. P. G. O.; Karam, L. Z.; Pulido, C. A.; Gomes, O. M. M.; Kalinowski, H. J.

2014-08-01

The aim of this study was to evaluate the behavior of two types of resin cements , conventional dual and dual self adhesive, through in vitro and in situ experiments. For the in vitro assay were selected two resin cements that were handled and dispensed over a mylar strip supported by a glass plate. The Bragg grating sensors were positioned and another portion of cement. was placed, covered by another mylar strip. For the in situ experiment 16 single-rooted teeth were selected who were divided into 2 groups: group 1 - conventional dual resin cement Relyx ARC and group 2 - dual self adhesive resin cement Relyx U200 ( 3M/ESPE ). The teeth were treated and prepared to receive the intracanal posts. Two Bragg grating sensors were recorded and introduced into the root canal at different apical and coronal positions. The results showed that the in vitro experiment presented similar values of polymerization shrinkage that the in situ experiment made in cervical position; whereas Relyx ARC resulted lower values compared to Relyx U200; and cervical position showed higher shrinkage than the apical.

Impact of Humidity on In Vitro Human Skin Permeation Experiments for Predicting In Vivo Permeability.

PubMed

Ishida, Masahiro; Takeuchi, Hiroyuki; Endo, Hiromi; Yamaguchi, Jun-Ichi

2015-12-01

In vitro skin permeation studies have been commonly conducted to predict in vivo permeability for the development of transdermal therapeutic systems (TTSs). We clarified the impact of humidity on in vitro human skin permeation of two TTSs having different breathability and then elucidated the predictability of in vivo permeability based on in vitro experimental data. Nicotinell(®) TTS(®) 20 and Frandol(®) tape 40mg were used as model TTSs in this study. The in vitro human skin permeation experiments were conducted under humidity levels similar to those used in clinical trials (approximately 50%) as well as under higher humidity levels (approximately 95%). The skin permeability values of drugs at 95% humidity were higher than those at 50% humidity. The time profiles of the human plasma concentrations after TTS application fitted well with the clinical data when predicted based on the in vitro permeation parameters at 50% humidity. On the other hand, those profiles predicted based on the parameters at 95% humidity were overestimated. The impact of humidity was higher for the more breathable TTS; Frandol(®) tape 40mg. These results show that in vitro human skin permeation experiments should be investigated under realistic clinical humidity levels especially for breathable TTSs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

PubMed

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.

Influence of dental materials on cells of the equine periodontium.

PubMed

Ringeisen, H; Pöschke, A; Krähling, B; Schröck, C; Stoll, M; Vogelsberg, J; Failing, K; Staszyk, C

2018-05-01

Therapy for equine periodontal disease can include filling of the periodontal pockets and widened interproximal spaces. Recommended dental materials are generally adopted from human dentistry. To evaluate the biocompatibility of dental materials for equine periodontal fillings in vitro. In vitro experiments. Four different dental materials (PeriCare ® , Provicol ® , Calxyl ® and Honigum) were tested on equine periodontal fibroblasts. Possible cytotoxic effects were assessed microscopically and by MTT assay, and the expression of inflammatory marker genes was measured by qRT-PCR. PeriCare ® and Provicol ® had no effects on the cells, whereas Honigum and Calxyl ® were associated with severe cytotoxic effects. The results of this in vitro study need to be confirmed by clinical studies. Before adapting dental materials from human dentistry, it is crucial to initially test them in a specific equine model. © 2017 EVJ Ltd.

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

PubMed

Torres, V; Hamdi, M; Millán de la Blanca, M G; Urrego, R; Echeverri, J; López-Herrera, A; Rizos, D; Gutiérrez-Adán, A; Sánchez-Calabuig, M J

2018-03-26

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl β-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control - ) and a group with cyclodextrin (control + ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p < .05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p < .05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts. © 2018 Blackwell Verlag GmbH.

Development of an integrated semi-automated system for in vitro pharmacodynamic modelling.

PubMed

Wang, Liangsu; Wismer, Michael K; Racine, Fred; Conway, Donald; Giacobbe, Robert A; Berejnaia, Olga; Kath, Gary S

2008-11-01

The aim of this study was to develop an integrated system for in vitro pharmacodynamic modelling of antimicrobials with greater flexibility, easier control and better accuracy than existing in vitro models. Custom-made bottle caps, fittings, valve controllers and a modified bench-top shaking incubator were used. A temperature-controlled automated sample collector was built. Computer software was developed to manage experiments and to control the entire system including solenoid pinch valves, peristaltic pumps and the sample collector. The system was validated by pharmacokinetic simulations of linezolid 600 mg infusion. The antibacterial effect of linezolid against multiple Staphylococcus aureus strains was also studied in this system. An integrated semi-automated bench-top system was built and validated. The temperature-controlled automated sample collector allowed unattended collection and temporary storage of samples. The system software reduced the labour necessary for many tasks and also improved the timing accuracy for performing simultaneous actions in multiple parallel experiments. The system was able to simulate human pharmacokinetics of linezolid 600 mg intravenous infusion accurately. A pharmacodynamic study of linezolid against multiple S. aureus strains with a range of MICs showed that the required 24 h free drug AUC/MIC ratio was approximately 30 in order to keep the organism counts at the same level as their initial inoculum and was about > or = 68 in order to achieve > 2 log(10) cfu/mL reduction in the in vitro model. The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.

Effects of brevetoxin exposure on the immune system of loggerhead sea turtles.

PubMed

Walsh, Catherine J; Leggett, Stephanie R; Carter, Barbara J; Colle, Clarence

2010-05-10

Blooms of the toxic dinoflagellate, Karenia brevis, occur almost annually off the Florida coast. These blooms, commonly called "red tides", produce a group of neurotoxins collectively termed brevetoxins. Many species of sealife, including sea turtles, are severely impacted by brevetoxin exposure. Effects of brevetoxins on immune cells were investigated in rescued loggerhead sea turtles, Caretta caretta, as well as through in vitro experiments using peripheral blood leukocytes (PBL) collected from captive sea turtles. In rescued animals, plasma brevetoxin concentrations were measured using a competitive ELISA. Plasma lysozyme activity was measured using a turbidity assay. Lysozyme activity correlated positively with plasma brevetoxin concentrations. Differential expression of genes affected by brevetoxin exposure was determined using two separate suppression subtractive hybridization experiments. In one experiment, genes from PBL collected from sea turtles rescued from red tide toxin exposure were compared to genes from PBL collected from healthy captive loggerhead sea turtles. In the second experiment, PBL from healthy captive loggerhead sea turtles were exposed to brevetoxin (500 ng PbTx-2/ml) in vitro for 18 h and compared to unexposed PBL. Results from the subtraction hybridization experiment conducted with red tide rescued sea turtle PBL indicated that genes involved in oxidative stress or xenobiotic metabolism were up-regulated. Using quantitative real-time PCR, a greater than 2-fold increase in superoxide dismutase and thioredoxin and greater than 10-fold increase in expression of thiopurine S-methyltransferase were observed. Results from the in vitro subtraction hybridization experiment indicated that genes coding for cytochrome c oxidases were the major up-regulated genes. Using quantitative real-time PCR, a greater than 8-fold increase in expression of beta-tubulin and greater than 3-fold increase in expression of ubiquinol were observed. Brevetoxin exposure may have significant implications for immune function in loggerhead sea turtles. 2009 Elsevier B.V. All rights reserved.

Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

PubMed

Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

2016-06-01

Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

Isolation of Nicotinic Acid (Vitamin B3) and N-Propylamine after Myosmine Peroxidation.

PubMed

Zwickenpflug, Wolfgang; Högg, Christof; Feierfeil, Johannes; Dachs, Manuel; Gudermann, Thomas

2016-01-13

The alkaloid myosmine (3-(1-pyrroline-2-yl)pyridine) is widespread in biological matrixes including foodstuffs and tobacco products. Some in vitro tests in cellular systems showed mutagenic activity for myosmine. Myosmine activation including peroxidation mechanism employs unstable oxazirane intermediates. The formation of minor metabolite 3-hydroxymethyl-pyridine in rat metabolism experiments as well as in in vitro peroxidation assays suggests its further oxidation to nicotinic acid and possible concomitant formation of n-propylamine. A sensitive high-performance liquid chromatography-ultraviolet (HPLC-UV) method was developed for the direct analysis of n-propylamine in the peroxidation assay solution of myosmine employing derivatization with 3,5-dinitrobenzoyl chloride. Additionally, during peroxidation procedures, formation of 3-pyridylmethanol to nicotinic acid, the essential vitamin B3, was observed and characterized using HPLC-UV and gas chromatography/mass spectrometry. This new reaction pathway may present further contribution to our knowledge of myosmine's significance in human food including its activation in human organism, foodstuffs, and biological systems.

Design of experiments (DOE) - history, concepts, and relevance to in vitro culture

USDA-ARS?s Scientific Manuscript database

Design of experiments (DOE) is a large and well-developed field for understanding and improving the performance of complex systems. Because in vitro culture systems are complex, but easily manipulated in controlled conditions, they are particularly well-suited for the application of DOE principle...

Dissolution and dissolution/permeation experiments for predicting systemic exposure following oral administration of the BCS class II drug clarithromycin.

PubMed

Kristin, Forner; René, Holm; Boontida, Morakul; Buraphacheep, Junyaprasert Varaporn; Maximilian, Ackermann; Johanna, Mazur; Peter, Langguth

2017-04-01

In order to save time and resources in early drug development, in vitro methods that correctly predict the formulation effect on oral drug absorption are necessary. The aim of this study was to 1) evaluate various BCS class II drug formulations with in vitro methods and in vivo in order to 2) determine which in vitro method best correlates with the in vivo results. Clarithromycin served as model compound in formulations with different particle sizes and content of excipients. The performed in vitro experiments were dissolution and dissolution/permeation experiments across two types of membrane, Caco-2 cells and excised rat intestinal sheets. The in vivo study was performed in rats. The oral absorption was enhanced by downsizing drug particles and by increasing the excipient concentration. This correlated strongly with the flux across Caco-2 cells but not with the other in vitro experiments. The insufficient correlation with the dissolution experiments can be partly explained by excipient caused problems during the filtration step. The very poor correlation of the in vivo data with the flux across excised rat intestinal sheets might be due to an artificially enlarged mucus layer ex vivo. In conclusion, downsizing BCS class II drug particles and the addition of surfactants enhanced the in vivo absorption, which was best depicted by dissolution/permeation experiments across Caco-2 cells. This setup is proposed as best model to predict the in vivo formulation effect. Also, this is the first study to evaluate the impact of the nature of the permeation membrane in dissolution/permeation experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

PubMed

Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

2013-10-01

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Delayed in vitro development of Up states but normal network plasticity in Fragile X circuits.

PubMed

Motanis, Helen; Buonomano, Dean

2015-09-01

A broad range of neurophysiological phenotypes have been reported since the generation of the first mouse model of Fragile X syndrome (FXS). However, it remains unclear which phenotypes are causally related to the cognitive deficits associated with FXS. Indeed, because many of these phenotypes are known to be modulated by experience, a confounding factor in the interpretation of many studies is whether some phenotypes are an indirect consequence of abnormal development and experience. To help diminish this confound we first conducted an in vitro developmental study of spontaneous neural dynamics in cortical organotypic cultures. A significant developmental increase in network activity and Up states was observed in both wild-type and Fmr1(-/y) circuits, along with a specific developmental delay in the emergence of Up states in knockout circuits. To determine whether Up state regulation is generally impaired in FXS circuits, we examined Up state plasticity using chronic optogenetic stimulation. Wild-type and Fmr1(-/y) stimulated circuits exhibited a significant decrease in overall spontaneous activity including Up state frequency; however, no significant effect of genotype was observed. These results demonstrate that developmental delays characteristic of FXS are recapitulated during in vitro development, and that Up state abnormalities are probably a direct consequence of the disease, and not an indirect consequence of abnormal experience. However, the fact that Fmr1(-/y) circuits exhibited normal homeostatic modulation of Up states suggests that these plasticity mechanisms are largely intact, and that some of the previously reported plasticity deficits could reflect abnormal experience or the engagement of compensatory mechanisms. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Non-animal approaches for toxicokinetics in risk evaluations of food chemicals.

PubMed

Punt, Ans; Peijnenburg, Ad A C M; Hoogenboom, Ron L A P; Bouwmeester, Hans

2017-01-01

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data.

Harnessing the landscape of microbial culture media to predict new organism–media pairings

PubMed Central

Oberhardt, Matthew A.; Zarecki, Raphy; Gronow, Sabine; Lang, Elke; Klenk, Hans-Peter; Gophna, Uri; Ruppin, Eytan

2015-01-01

Culturing microorganisms is a critical step in understanding and utilizing microbial life. Here we map the landscape of existing culture media by extracting natural-language media recipes into a Known Media Database (KOMODO), which includes >18,000 strain–media combinations, >3300 media variants and compound concentrations (the entire collection of the Leibniz Institute DSMZ repository). Using KOMODO, we show that although media are usually tuned for individual strains using biologically common salts, trace metals and vitamins/cofactors are the most differentiating components between defined media of strains within a genus. We leverage KOMODO to predict new organism–media pairings using a transitivity property (74% growth in new in vitro experiments) and a phylogeny-based collaborative filtering tool (83% growth in new in vitro experiments and stronger growth on predicted well-scored versus poorly scored media). These resources are integrated into a web-based platform that predicts media given an organism's 16S rDNA sequence, facilitating future cultivation efforts. PMID:26460590

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

PubMed

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. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Exploiting Inhibitory Siglecs to Combat Food Allergies

DTIC Science & Technology

2017-10-01

project including mouse handling, liposome preparation, and in vitro assays, such as ELISAs . Shiteng and Kevin Worrell participated in groups...h 2. She also contributed by running some ELISA experiments. Funding Support: N/A Name: Kelly Orgel Project Role: Graduate Student (UNC...flow cytometry, and ELISA . Kelly performed the human CD33 basophil assays as well. Funding Support: N/A Name: Johanna Smeekens, PhD Project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voigt, Christopher

SEED2014 focused on advances in the science and technology emerging from the field of synthetic biology. We broadly define this as technologies that accelerate the process of genetic engineering. It highlighted new tool development, as well as the application of these tools to diverse problems in biotechnology, including therapeutics, industrial chemicals and fuels, natural products, and agriculture. Systems spanned from in vitro experiments and viruses, through diverse bacteria, to eukaryotes (yeast, mammalian cells, plants).

MICRONUCLEI IN BINUCLEATED LYMPHOCYTES OF MICE FOLLOWING EXPOSURE TO GAMMA RADIATION

EPA Science Inventory

Experiments were designed to investigate the induction of micronuclei (MN) in mouse peripheral blood lymphocytes (PBLs) after in vitro or in vivo exposure to 60Co gamma radiation. or the in vitro experiments, 4 ml of blood from male C57BL/6J mice were either irradiated in 6 ml Fa...

"In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

ERIC Educational Resources Information Center

Higgins, Pamela J.

2005-01-01

This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

Treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates Ebola virus in vitro.

PubMed

Cap, Andrew P; Pidcoke, Heather F; Keil, Shawn D; Staples, Hilary M; Anantpadma, Manu; Carrion, Ricardo; Davey, Robert A; Frazer-Abel, Ashley; Taylor, Audra L; Gonzales, Richard; Patterson, Jean L; Goodrich, Raymond P

2016-03-01

Transfusion of plasma from recovered patients after Ebolavirus (EBOV) infection, typically called "convalescent plasma," is an effective treatment for active disease available in endemic areas, but carries the risk of introducing other pathogens, including other strains of EBOV. A pathogen reduction technology using ultraviolet light and riboflavin (UV+RB) is effective against multiple enveloped, negative-sense, single-stranded RNA viruses that are similar in structure to EBOV. We hypothesized that UV+RB is effective against EBOV in blood products without activating complement or reducing protective immunoglobulin titers that are important for the treatment of Ebola virus disease (EVD). Four in vitro experiments were conducted to evaluate effects of UV+RB on green fluorescent protein EBOV (EBOV-GFP), wild-type EBOV in serum, and whole blood, respectively, and on immunoglobulins and complement in plasma. Initial titers for Experiments 1 to 3 were 4.21 log GFP units/mL, 4.96 log infectious units/mL, and 4.23 log plaque-forming units/mL. Conditions tested in the first three experiments included the following: 1-EBOV-GFP plus UV+RB; 2-EBOV-GFP plus RB only; 3-EBOV-GFP plus UV only; 4-EBOV-GFP without RB or UV; 5-virus-free control plus UV only; and 6-virus-free control without RB or UV. UV+RB reduced EBOV titers to nondetectable levels in both nonhuman primate serum (≥2.8- to 3.2-log reduction) and human whole blood (≥3.0-log reduction) without decreasing protective antibody titers in human plasma. Our in vitro results demonstrate that the UV+RB treatment efficiently reduces EBOV titers to below limits of detection in both serum and whole blood. In vivo testing to determine whether UV+RB can improve convalescent blood product safety is indicated. © 2016 AABB.

Treatment of blood with a pathogen reduction technology using UV light and riboflavin inactivates Ebola virus in vitro

PubMed Central

Cap, Andrew P.; Pidcoke, Heather F.; Keil, Shawn D.; Staples, Hilary M.; Anantpadma, Manu; Carrion, Ricardo; Davey, Robert A.; Frazer-Abel, Ashley; Taylor, Audra L.; Gonzales, Richard; Patterson, Jean L.; Goodrich, Raymond P.

2018-01-01

BACKGROUND Transfusion of plasma from recovered patients after Ebolavirus (EBOV) infection, typically called ‘convalescent plasma,’ is an effective treatment for active disease available in endemic areas, but carries the risk of introducing other pathogens, including other strains of EBOV. A pathogen reduction technology using ultraviolet light and riboflavin (UV + RB) is effective against multiple enveloped, negative-sense, single-stranded RNA viruses that are similar in structure to EBOV. We hypothesized that UV + RB is effective against EBOV in blood products without activating complement or reducing protective immunoglobulin titers that are important for the treatment of ebolavirus disease (EVD). STUDY DESIGN AND METHODS Four in vitro experiments were conducted to evaluate effects of UV + RB on green fluorescent protein EBOV (EBOV-GFP), wild-type EBOV in serum and whole blood, respectively, and on immunoglobulins and complement in plasma. Initial titers for Experiments 1–3 were: 4.21 log10 GFP units/mL, 4.96 log10 infectious units per mL, and 4.23 log10 plaque forming units per mL (PFU/mL). Conditions tested in the first three experiments included: 1. EBOV-GFP + UV + RB; 2. EBOV-GFP + RB only; 3 EBOV-GFP + UV only; 4. EBOV-GFP without RB or UV; 5. Virus-free control + UV only; and 6. Virus-free control without RB or UV. RESULTS UV + RB reduced EBOV titers to non-detectable levels in both non-human primate serum (≥ 2.8 to 3.2 log reduction) and human whole blood (≥ 3.0 log reduction) without decreasing protective antibody titers in human plasma. CONCLUSION Our in vitro results demonstrate that the UV + RB treatment efficiently reduces EBOV titers to below limits of detection in both serum and whole blood. In vivo testing to determine whether UV + RB can improve convalescent blood product safety is indicated. PMID:27001363

Implementation and enforcement of the 3Rs principle in the field of transgenic animals used for scientific purposes. Report and recommendations of the BfR expert workshop, May 18-20, 2009, Berlin, Germany.

PubMed

Kretlow, Ariane; Butzke, Daniel; Goetz, Mario E; Grune, Barbara; Halder, Marlies; Henkler, Frank; Liebsch, Manfred; Nobiling, Rainer; Oelgeschlaeger, Michael; Reifenberg, Kurt; Schaefer, Bernd; Seiler, Andrea; Luch, Andreas

2010-01-01

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals. These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or three-dimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified ("humanised") animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with "negative" outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use. The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.

Seeking conception: experiences of urban Indian women with in vitro fertilisation.

PubMed

Widge, Anjali

2005-12-01

This paper reports on a study of involuntarily childless Indian women/couples seeking in vitro fertilisation (IVF). The focus is on the social context of infertility and on women's perceptions of and experiences with IVF. Twenty-two childless women/couples who sought IVF. The sample was drawn from consenting clients of clinics in two major Indian cities, viz. New Delhi and Mumbai. In-depth interviews revealed that infertility is deeply feared, women's status and security are affected, and they experience stigmatisation and isolation. IVF was pursued after less intrusive avenues had been exhausted. Inadequate information/counselling is provided, success rates are low, IVF is commercialised and the process is physiologically, emotionally and financially stressful. In Indian society fertility defines womanhood and motherhood, and infertility is stigmatised. Women faced a lot of pressures to produce a biological child, and go through all kinds of treatments, including the expensive ARTs, to have a child. Integration of infertility services into the state's reproductive health programme and disseminate information on infertility and to offer other appropriate choices, such as adoption. Effective counselling on coping with psychosocial/sexual problems. Monitoring of the prevalence of sex preselection.

[Effects of sulfomethylation on the mechanism of action of colimycin towards "Escherichia coli" B (author's transl)].

PubMed

Cornu, J

1980-01-01

Colimycin is sulfomethylated in order to inactivate it temporarily in vivo. In vitro, this process may have unexpected results because the experiment is carried out within a closed system. The time required to activate the antibiotic varies according to incubation conditions. The freed sulfomethyl groups are probably eliminated in vivo. In vitro, these residues may interact with the outer membrane of Escherichia coli B which is charged with badly positioned molecules of colimycin, thus creating a barrier for the active molecules. As the expected mechanism of polymyxin B or E action include first electrostatic interactions, it is not surprising that a modification of the ionic balance of the medium should be of such importance.

Continuous "in vitro" Evolution of a Ribozyme Ligase: A Model Experiment for the Evolution of a Biomolecule

ERIC Educational Resources Information Center

Ledbetter, Michael P.; Hwang, Tony W.; Stovall, Gwendolyn M.; Ellington, Andrew D.

2013-01-01

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. "In vitro" directed evolution (IVDE) frequently serves as a model system for the study of Darwinian…

MEDIA SERUM LEVELS AND IN VITRO HEPATIC ABSORPTION OF LINDANE

EPA Science Inventory

High plasma protein binding is known to reduce the tissue uptake of chemicals in vivo, but the extent of its importance in vitro is less clear. Experiments were conducted to determine the cellular uptake of lindane in vitro under different conditions. Lindane was selected because...

In vitro and in situ characterization of arthroscopic loop security and knot security of braided polyblend sutures: a biomechanical study.

PubMed

Armstrong, Lucas C; Chong, Alexander; Livermore, Ryan W; Prohaska, Daniel J; Doyon, Amanda N; Wooley, Paul H

2015-04-01

We conducted a study to evaluate biomechanical performance during destructive testing of several different suture materials in various arthroscopic knot configurations under both in vitro and in situ conditions. Surgeons of different levels of experience tied the knots. Three different arthroscopic knots (static surgeon's, Weston, Tennessee slider) with 3 reverse half-hitches on alternating posts were tested using Fiberwire, ForceFiber, Orthocord, and Ultrabraid suture materials under both in vitro and in situ (blood plasma at 37°C) conditions. Three surgeons of different experience levels tied the knots on a post 30 mm in circumference. A single load-to-failure test was performed. There were no significant in vitro-in situ differences for Ultrabraid in the different knot configurations or with the different experience levels. Surgeon B (intermediate experience) showed no significant differences between test conditions for any knot configuration or suture material. With Tennessee slider knots, surgeon C (least experience) showed significantly lower clinical failure load under both test conditions and had a higher percentage of complete knot slippage. Surgeon B had no knot slippage with use of Fiberwire. Both the aqueous environment and the surgeon's familiarity with certain knots have an effect on knot security.

Active Curved Polymers Form Vortex Patterns on Membranes.

PubMed

Denk, Jonas; Huber, Lorenz; Reithmann, Emanuel; Frey, Erwin

2016-04-29

Recent in vitro experiments with FtsZ polymers show self-organization into different dynamic patterns, including structures reminiscent of the bacterial Z ring. We model FtsZ polymers as active particles moving along chiral, circular paths by Brownian dynamics simulations and a Boltzmann approach. Our two conceptually different methods point to a generic phase behavior. At intermediate particle densities, we find self-organization into vortex structures including closed rings. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation.

Synthetic thrombus model for in vitro studies of laser thrombolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hermes, R.E.; Trajkovska, K.

1998-07-01

Laser thrombolysis is the controlled ablation of a thrombus (blood clot) blockage in a living arterial system. Theoretical modeling of the interaction of laser light with thrombi relies on the ability to perform in vitro experiments with well characterized surrogate materials. A synthetic thrombus formulation may offer more accurate results when compared to in vivo clinical experiments. The authors describe the development of new surrogate materials based on formulations incorporating chick egg, guar gum, modified food starch, and a laser light absorbing dye. The sound speed and physical consistency of the materials were very close to porcine (arterial) and humanmore » (venous) thrombi. Photographic and videotape recordings of pulsed dye laser ablation experiments under various experimental conditions were used to evaluate the new material as compared to in vitro tests with human (venous) thrombus. The characteristics of ablation and mass removal were similar to that of real thrombi, and therefore provide a more realistic model for in vitro laser thrombolysis when compared to gelatin.« less

In vitro selection of functional nucleic acids

NASA Technical Reports Server (NTRS)

Wilson, D. S.; Szostak, J. W.

1999-01-01

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

In vitro validation of a novel mechanical model for testing the anchorage capacity of pedicle screws using physiological load application.

PubMed

Liebsch, Christian; Zimmermann, Julia; Graf, Nicolas; Schilling, Christoph; Wilke, Hans-Joachim; Kienle, Annette

2018-01-01

Biomechanical in vitro tests analysing screw loosening often include high standard deviations caused by high variabilities in bone mineral density and pedicle geometry, whereas standardized mechanical models made of PU foam often do not integrate anatomical or physiological boundary conditions. The purpose of this study was to develop a most realistic mechanical model for the standardized and reproducible testing of pedicle screws regarding the resistance against screw loosening and the holding force as well as to validate this model by in vitro experiments. The novel mechanical testing model represents all anatomical structures of a human vertebra and is consisting of PU foam to simulate cancellous bone, as well as a novel pedicle model made of short carbon fibre filled epoxy. Six monoaxial cannulated pedicle screws (Ø6.5 × 45mm) were tested using the mechanical testing model as well as human vertebra specimens by applying complex physiological cyclic loading (shear, tension, and bending; 5Hz testing frequency; sinusoidal pulsating forces) in a dynamic materials testing machine with stepwise increasing load after each 50.000 cycles (100.0N shear force + 20.0N per step, 51.0N tension force + 10.2N per step, 4.2Nm bending moment + 0.8Nm per step) until screw loosening was detected. The pedicle screw head was fixed on a firmly clamped rod while the load was applied in the vertebral body. For the in vitro experiments, six human lumbar vertebrae (L1-3, BMD 75.4 ± 4.0mg/cc HA, pedicle width 9.8 ± 0.6mm) were tested after implanting pedicle screws under X-ray control. Relative motions of pedicle screw, specimen fixture, and rod fixture were detected using an optical motion tracking system. Translational motions of the mechanical testing model experiments in the point of load introduction (0.9-2.2mm at 240N shear force) were reproducible within the variation range of the in vitro experiments (0.6-3.5mm at 240N shear force). Screw loosening occurred continuously in each case between 140N and 280N, while abrupt failures of the specimen were observed only in vitro. In the mechanical testing model, no translational motion was detected in the screw entry point, while in vitro, translational motions of up to 2.5mm in inferior direction were found, leading to a slight shift of the centre of rotation towards the screw tip. Translational motions of the screw tip of about 5mm in superior direction were observed both in vitro and in the mechanical testing model, while they were continuous in the mechanical testing model and rapidly increasing after screw loosening initiation in vitro. The overall pedicle screw loosening characteristics were qualitatively and quantitatively similar between the mechanical testing model and the human vertebral specimens as long as there was no translation of the screw at the screw entrance point. Therefore, the novel mechanical testing model represents a promising method for the standardized testing of pedicle screws regarding screw loosening for cases where the screw rotates around a point close to the screw entry point. Copyright © 2017 Elsevier Ltd. All rights reserved.

Silibinin and indocyanine green-loaded nanoparticles inhibit the growth and metastasis of mammalian breast cancer cells in vitro.

PubMed

Sun, Hui-Ping; Su, Jing-Han; Meng, Qing-Shuo; Yin, Qi; Zhang, Zhi-Wen; Yu, Hai-Jun; Zhang, Peng-Cheng; Wang, Si-Ling; Li, Ya-Ping

2016-07-01

To improve the therapeutic efficacy of cancer treatments, combinational therapies based on nanosized drug delivery system (NDDS) has been developed recently. In this study we designed a new NDDS loaded with an anti-metastatic drug silibinin and a photothermal agent indocyanine green (ICG), and investigated its effects on the growth and metastasis of breast cancer cells in vitro. Silibinin and ICG were self-assembled into PCL lipid nanoparticles (SIPNs). Their physical characteristics including the particle size, zeta potential, morphology and in vitro drug release were examined. 4T1 mammalian breast cancer cells were used to evaluate their cellular internalization, cytotoxicity, and their influences on wound healing, in vitro cell migration and invasion. SIPNs showed a well-defined spherical shape with averaged size of 126.3±0.4 nm and zeta potential of -10.3±0.2 mV. NIR laser irradiation substantially increased the in vitro release of silibinin from the SIPNs (58.3% at the first 8 h, and 97.8% for the total release). Furthermore, NIR laser irradiation markedly increased the uptake of SIPNs into 4T1 cells. Under the NIR laser irradiation, both SIPNs and IPNs (PCL lipid nanoparticles loaded with ICG alone) caused dose-dependent ablation of 4T1 cells. The wound healing, migration and invasion experiments showed that SIPNs exposed to NIR laser irradiation exhibited dramatic in vitro anti-metastasis effects. SIPNs show temperature-sensitive drug release following NIR laser irradiation, which can inhibit the growth and metastasis of breast cancer cells in vitro.

Does a kampo medicine containing schisandra fruit affect pharmacokinetics of nifedipine like grapefruit juice?

PubMed

Makino, Toshiaki; Mizuno, Fumika; Mizukami, Hajime

2006-10-01

Herb-drug interaction has attracted attention as medicinal topics recently. However, the drug information is sometimes confusing. Previous in vitro studies revealed that schisandra fruit had strong inhibitory effect on CYP3A4 and claimed the possibilities of its herb-drug interaction. In the present study, we evaluated the inhibitory effects of schisandra fruit and shoseiryuto, an herbal formula in Japanese traditional kampo medicine containing eight herbal medicines including schisandra fruit, on rat CYP3A activity in vitro, and the effect of shoseiryuto on pharmacokinetics of nifedipine in rats, in comparison with those of grapefruit juice, a well-characterized natural CYP3A inhibitor. Shoseiryuto and its herbal constituents, schisandra fruit, ephedra herb and cinnamon bark exhibited in vitro inhibitory effect of CYP3A. Although shoseiryuto inhibited rat CYP3A activity in vitro with a degree comparable to grapefruit juice, shoseiryuto did not significantly affect a plasma concentration profile of nifedipine in rats as grapefruit juice did. These results indicate that in vivo experiments using the extract of herbal medicine prepared with the same dosage form as patients take are necessary to provide proper information about herb-drug interaction.

Methods and options in vitro dialyzability; benefits and limitations.

PubMed

Sandberg, Ann-Sofie

2005-11-01

In vitro dialyzability methods involve a two-step digestion process simulating the gastric and intestinal phase, and dialysis through a semi-permeable membrane with a selected molecular weight cut-off. Dialyzable iron/zinc is used as an estimation of available mineral. Final pH adjustment and use of a strict time schedule were found to be critical factors for standardization. In addition the selected cut-off of the dialysis membrane and the method used for iron and zinc determination influence the results. For screening purposes, simple solubility or dialyzability methods seem preferable to the more sophisticated computer-controlled gastrointestinal model. This is likely more valuable in studies of different transit times and sites of dialyzability. In vitro solubility/dialyzability methods correlate in most cases with human absorption studies in ranking iron and zinc availability from different meals. Exceptions may be that effects of milk, certain proteins, tea, and organic acids cannot be predicted. The dialyzability methods exclude iron bound to large molecules, which in some cases is available and include iron bound to small molecules, which is not always available. In vitro experiments based on solubility/dialyzability are tools to understand factors that may affect subsequent mineral absorption.

Effects of feeding condensed distillers solubles and crude glycerin alone or in combination on finishing beef cattle performance, carcass characteristics, and in vitro fermentation.

USDA-ARS?s Scientific Manuscript database

Two experiments were conducted to evaluate the effects of feeding condensed distillers solubles (DS) and crude glycerin alone or in combination on performance of finishing beef cattle and in vitro fermentation. In both experiments, dietary treatments consisted of a steam flaked corn (SFC) based diet...

In vivo diagnosis of skin cancer using polarized and multiple scattered light spectroscopy

NASA Astrophysics Data System (ADS)

Bartlett, Matthew Allen

This thesis research presents the development of a non-invasive diagnostic technique for distinguishing between skin cancer, moles, and normal skin using polarized and multiple scattered light spectroscopy. Polarized light incident on the skin is single scattered by the epidermal layer and multiple scattered by the dermal layer. The epidermal light maintains its initial polarization while the light from the dermal layer becomes randomized and multiple scattered. Mie theory was used to model the epidermal light as the scattering from the intercellular organelles. The dermal signal was modeled as the diffusion of light through a localized semi-homogeneous volume. These models were confirmed using skin phantom experiments, studied with in vitro cell cultures, and applied to human skin for in vivo testing. A CCD-based spectroscopy system was developed to perform all these experiments. The probe and the theory were tested on skin phantoms of latex spheres on top of a solid phantom. We next extended our phantom study to include in vitro cells on top of the solid phantom. Optical fluorescent microscope images revealed at least four distinct scatterers including mitochondria, nucleoli, nuclei, and cell membranes. Single scattering measurements on the mammalian cells consistently produced PSD's in the size range of the mitochondria. The clinical portion of the study consisted of in vivo measurements on cancer, mole, and normal skin spots. The clinical study combined the single scattering model from the phantom and in vitro cell studies with the diffusion model for multiple scattered light. When parameters from both layers were combined, we found that a sensitivity of 100% and 77% can be obtained for detecting cancers and moles, respectively, given the number of lesions examined.

Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success.

PubMed

Hagedorn, Mary; Carter, Virginia L

2011-01-01

Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 10(6) cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 10(6) motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization.

Zebrafish Reproduction: Revisiting In Vitro Fertilization to Increase Sperm Cryopreservation Success

PubMed Central

Hagedorn, Mary; Carter, Virginia L.

2011-01-01

Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 106 cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 106 motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization. PMID:21698162

Multifunctional biodegradable polymer nanoparticles with uniform sizes: generation and in vitro anti-melanoma activity

NASA Astrophysics Data System (ADS)

Liang, Ruijing; Wang, Jing; Wu, Xian; Dong, Liyun; Deng, Renhua; Wang, Ke; Sullivan, Martin; Liu, Shanqin; Wu, Min; Tao, Juan; Yang, Xiangliang; Zhu, Jintao

2013-11-01

We present a simple, yet versatile strategy for the fabrication of uniform biodegradable polymer nanoparticles (NPs) with controllable sizes by a hand-driven membrane-extrusion emulsification approach. The size and size distribution of the NPs can be easily tuned by varying the experimental parameters, including initial polymer concentration, surfactant concentration, number of extrusion passes, membrane pore size, and polymer molecular weight. Moreover, hydrophobic drugs (e.g., paclitaxel (PTX)) and inorganic NPs (e.g., quantum dots (QDs) and magnetic NPs (MNPs)) can be effectively and simultaneously encapsulated into the polymer NPs to form the multifunctional hybrid NPs through this facile route. These PTX-loaded NPs exhibit high encapsulation efficiency and drug loading density as well as excellent drug sustained release performance. As a proof of concept, the A875 cell (melanoma cell line) experiment in vitro, including cellular uptake analysis by fluorescence microscope, cytotoxicity analysis of NPs, and magnetic resonance imaging (MRI) studies, indicates that the PTX-loaded hybrid NPs produced by this technique could be potentially applied as a multifunctional delivery system for drug delivery, bio-imaging, and tumor therapy, including malignant melanoma therapy.

The Genome-Wide Expression Profile of Saussurea lappa Extract on House Dust Mite-Induced Atopic Dermatitis in Nc/Nga Mice.

PubMed

Lim, Hye-Sun; Ha, Hyekyung; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

2015-09-01

Saussurea lappa has been reported to possess anti-atopic properties. In this study, we have confirmed the S. lappa's anti-atopic properties in Nc/Nga mice and investigated the candidate gene related with its properties using microarray. We determined the target gene using real time PCR in in vitro experiment. S. lappa showed the significant reduction in atopic dermatitis (AD) score and immunoglobulin E compared with the AD induced Nc/Nga mice. In the results of microarray using back skin obtained from animals, we found that S. lappa's properties are closely associated with cytokine-cytokine receptor interaction and the JAK-STAT signaling pathway. Consistent with the microarray data, real-time RT-PCR confirmed these modulation at the mRNA level in skin tissues from S. lappa-treated mice. Among these genes, PI3Kca and IL20Rβ were significantly downregulated by S. lappa treatment in Nc/Nga mouse model. In in vitro experiment using HaCaT cells, we found that the S. lappa components, including alantolactone, caryophyllene, costic acid, costunolide and dehydrocostus lactone significantly decreased the expression of PI3Kca but not IL20Rβ in vitro. Therefore, our study suggests that PI3Kca-related signaling is closely related with the protective effects of S. lappa against the development of atopic-dermatitis.

An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.

PubMed

Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun

2016-01-01

Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p < 0.05) by 5.0 and 2.6 percentage unit, respectively. The IVTTD of DM for corn increased (p < 0.05) by 3.1 percentage unit with enzyme complex addition. As the effect of enzyme complex was the greatest in corn digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p < 0.05) by 16.0, 2.8 and 1.2 percentage unit, respectively. The IVTTD of DM for corn starch and hull also increased (p < 0.05) by 8.6 and 0.9 percentage unit, respectively, with enzyme complex addition. In conclusion, the enzyme complex increases in vitro DM digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch.

Predicting mutant selection in competition experiments with ciprofloxacin-exposed Escherichia coli.

PubMed

Khan, David D; Lagerbäck, Pernilla; Malmberg, Christer; Kristoffersson, Anders N; Wistrand-Yuen, Erik; Sha, Cao; Cars, Otto; Andersson, Dan I; Hughes, Diarmaid; Nielsen, Elisabet I; Friberg, Lena E

2018-03-01

Predicting competition between antibiotic-susceptible wild-type (WT) and less susceptible mutant (MT) bacteria is valuable for understanding how drug concentrations influence the emergence of resistance. Pharmacokinetic/pharmacodynamic (PK/PD) models predicting the rate and extent of takeover of resistant bacteria during different antibiotic pressures can thus be a valuable tool in improving treatment regimens. The aim of this study was to evaluate a previously developed mechanism-based PK/PD model for its ability to predict in vitro mixed-population experiments with competition between Escherichia coli (E. coli) WT and three well-defined E. coli resistant MTs when exposed to ciprofloxacin. Model predictions for each bacterial strain and ciprofloxacin concentration were made for in vitro static and dynamic time-kill experiments measuring CFU (colony forming units)/mL up to 24 h with concentrations close to or below the minimum inhibitory concentration (MIC), as well as for serial passage experiments with concentrations well below the MIC measuring ratios between the two strains with flow cytometry. The model was found to reasonably well predict the initial bacterial growth and killing of most static and dynamic time-kill competition experiments without need for parameter re-estimation. With parameter re-estimation of growth rates, an adequate fit was also obtained for the 6-day serial passage competition experiments. No bacterial interaction in growth was observed. This study demonstrates the predictive capacity of a PK/PD model and further supports the application of PK/PD modelling for prediction of bacterial kill in different settings, including resistance selection. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Osteogenic Potential of Dental Mesenchymal Stem Cells in Preclinical Studies: A Systematic Review Using Modified ARRIVE and CONSORT Guidelines

PubMed Central

Ramamoorthi, Murali; Bakkar, Mohammed; Jordan, Jack; Tran, Simon D.

2015-01-01

Background and Objective. Dental stem cell-based tissue engineered constructs are emerging as a promising alternative to autologous bone transfer for treating bone defects. The purpose of this review is to systematically assess the preclinical in vivo and in vitro studies which have evaluated the efficacy of dental stem cells on bone regeneration. Methods. A literature search was conducted in Ovid Medline, Embase, PubMed, and Web of Science up to October 2014. Implantation of dental stem cells in animal models for evaluating bone regeneration and/or in vitro studies demonstrating osteogenic potential of dental stem cells were included. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were used to ensure the quality of the search. Modified ARRIVE (Animal research: reporting in invivo experiments) and CONSORT (Consolidated reporting of trials) were used to critically analyze the selected studies. Results. From 1914 citations, 207 full-text articles were screened and 137 studies were included in this review. Because of the heterogeneity observed in the studies selected, meta-analysis was not possible. Conclusion. Both in vivo and in vitro studies indicate the potential use of dental stem cells in bone regeneration. However well-designed randomized animal trials are needed before moving into clinical trials. PMID:26106427

Identification of the anti-tumor activity and mechanisms of nuciferine through a network pharmacology approach

PubMed Central

Qi, Quan; Li, Rui; Li, Hui-ying; Cao, Yu-bing; Bai, Ming; Fan, Xiao-jing; Wang, Shu-yan; Zhang, Bo; Li, Shao

2016-01-01

Aim: Nuciferine is an aporphine alkaloid extracted from lotus leaves, which is a raw material in Chinese medicinal herb for weight loss. In this study we used a network pharmacology approach to identify the anti-tumor activity of nuciferine and the underlying mechanisms. Methods: The pharmacological activities and mechanisms of nuciferine were identified through target profile prediction, clustering analysis and functional enrichment analysis using our traditional Chinese medicine (TCM) network pharmacology platform. The anti-tumor activity of nuciferine was validated by in vitro and in vivo experiments. The anti-tumor mechanisms of nuciferine were predicted through network target analysis and verified by in vitro experiments. Results: The nuciferine target profile was enriched with signaling pathways and biological functions, including “regulation of lipase activity”, “response to nicotine” and “regulation of cell proliferation”. Target profile clustering results suggested that nuciferine to exert anti-tumor effect. In experimental validation, nuciferine (0.8 mg/mL) markedly inhibited the viability of human neuroblastoma SY5Y cells and mouse colorectal cancer CT26 cells in vitro, and nuciferine (0.05 mg/mL) significantly suppressed the invasion of 6 cancer cell lines in vitro. Intraperitoneal injection of nuciferine (9.5 mg/mL, ip, 3 times a week for 3 weeks) significantly decreased the weight of SY5Y and CT26 tumor xenografts in nude mice. Network target analysis and experimental validation in SY5Y and CT26 cells showed that the anti-tumor effect of nuciferine was mediated through inhibiting the PI3K-AKT signaling pathway and IL-1 levels in SY5Y and CT26 cells. Conclusion: By using a TCM network pharmacology method, nuciferine is identified as an anti-tumor agent against human neuroblastoma and mouse colorectal cancer in vitro and in vivo, through inhibiting the PI3K-AKT signaling pathways and IL-1 levels. PMID:27180984

An Improved Method for Generating Consistent Soluble Amyloid-beta Oligomer Preparations for In Vitro Neurotoxicity Studies

PubMed Central

Ryan, Deborah A.; Narrow, Wade C.; Federoff, Howard J.; Bowers, William J.

2010-01-01

Soluble Aβ oligomers are recognized as playing a key role in Alzheimer’s disease (AD) pathophysiology. Despite their significance, many investigators encounter difficulty generating reliable preparations for in vitro and in vivo experiments. Solutions of Aβ are often unstable and soluble conformer profiles inconsistent. In this study we describe detailed methods for preparing Aβ oligomers that are stable for several weeks and are enriched for low and high molecular weight oligomeric forms, including the 56-kDa form, a conformer implicated in AD-related cognitive impairment. We characterize their structural and functional properties using Western blot, dot blot, atomic force microscopy, Thioflavine T fluorescence, and primary neuronal culture toxicity assays. These synthetic preparations should prove valuable to many studying Aβ-mediated mechanisms underlying AD. PMID:20452375

Active implantable medical device EMI assessment for wireless power transfer operating in LF and HF bands.

PubMed

Hikage, Takashi; Nojima, Toshio; Fujimoto, Hiroshi

2016-06-21

The electromagnetic interference (EMI) imposed on active implantable medical devices by wireless power transfer systems (WPTSs) is discussed based upon results of in vitro experiments. The purpose of this study is to present comprehensive EMI test results gathered from implantable-cardiac pacemakers and implantable cardioverter defibrillators exposed to the electromagnetic field generated by several WPTSs operating in low-frequency (70 kHz-460 kHz) and high-frequency (6.78 MHz) bands. The constructed in vitro experimental test system based upon an Irnich's flat torso phantom was applied. EMI test experiments are conducted on 14 types of WPTSs including Qi-compliant system and EV-charging WPT system mounted on current production EVs. In addition, a numerical simulation model for active implantable medical device (AIMD) EMI estimation based on the experimental test system is newly proposed. The experimental results demonstrate the risk of WPTSs emitting intermittent signal to affect the correct behavior of AIMDs when operating at very short distances. The proposed numerical simulation model is applicable to obtain basically the EMI characteristics of various types of WPTSs.

Active implantable medical device EMI assessment for wireless power transfer operating in LF and HF bands

NASA Astrophysics Data System (ADS)

Hikage, Takashi; Nojima, Toshio; Fujimoto, Hiroshi

2016-06-01

The electromagnetic interference (EMI) imposed on active implantable medical devices by wireless power transfer systems (WPTSs) is discussed based upon results of in vitro experiments. The purpose of this study is to present comprehensive EMI test results gathered from implantable-cardiac pacemakers and implantable cardioverter defibrillators exposed to the electromagnetic field generated by several WPTSs operating in low-frequency (70 kHz-460 kHz) and high-frequency (6.78 MHz) bands. The constructed in vitro experimental test system based upon an Irnich’s flat torso phantom was applied. EMI test experiments are conducted on 14 types of WPTSs including Qi-compliant system and EV-charging WPT system mounted on current production EVs. In addition, a numerical simulation model for active implantable medical device (AIMD) EMI estimation based on the experimental test system is newly proposed. The experimental results demonstrate the risk of WPTSs emitting intermittent signal to affect the correct behavior of AIMDs when operating at very short distances. The proposed numerical simulation model is applicable to obtain basically the EMI characteristics of various types of WPTSs.

Effect of seasonal and geographical differences on skin and effect of treatment with an osmoprotectant: Sorbitol.

PubMed

Muizzuddin, Neelam; Ingrassia, Michael; Marenus, Kenneth D; Maes, Daniel H; Mammone, Thomas

2013-01-01

Human skin maintains an optimal permeability barrier function in a terrestrial environment that varies considerably in humidity. Cells cultured under hyperosmotic stress accumulate osmolytes including sorbitol. Epidermal keratinocytes experience similar high osmolality under dry environmental conditions because of increased transepidermal water loss (TEWL) and concomitant drying of the skin. This study was designed to determine if epidermal keratinocytes, in vitro, could be protected from high osmotic stress, with the exogenous addition of sorbitol. In addition, we evaluated the effect of a formulation containing topical sorbitol on skin barrier and moisturization of subjects living in arid and humid regions in summer as well as in winter. Results from in vitro experiments showed that 50 mM sorbitol protected epidermal keratinocytes from osmotic toxicity induced by sodium chloride. Clinical studies indicated that skin chronically exposed to hot, dry environment appeared to exhibit stronger skin barrier and a lower baseline TEWL. In addition, skin barrier was stronger in summer than in winter. Sorbitol exhibited significant improvement in both barrier repair and moisturization, especially in individuals subjected to arid environmental conditions.

Human intravenous immunoglobulin (IVIG) preparations degranulate human neutrophils in vitro

PubMed Central

Teeling, J L; de Groot, E R; Eerenberg, A J M; Bleeker, W K; Van Mierlo, G; Aarden, L A; Hack, C E

1998-01-01

IVIG preparations have biological effects in vivo that are not fully understood. Possible effects include the property to stimulate Fc receptors on various cell types. To study whether IVIG may interact with neutrophils we developed an in vitro system, in which neutrophils, in whole blood or purified, were incubated with IVIG and assessed for degranulation by measuring the release of elastase and lactoferrin in culture medium. All commercially available IVIG preparations tested induced degranulation of neutrophils when incubated for 2 h at therapeutically relevant concentrations. In studies with blocking antibodies against Fc receptors (FcR), this degranulation was shown to be dependent on FcγRII, whereas FcγRIII had no effect. Experiments with purified neutrophils as well as binding experiments with labelled IVIG preparations indicated that neutrophil degranulation resulted from a direct interaction of IVIG with neutrophils. Using gel filtration fractions, it was found that polymeric and dimeric IgG present in IVIG was mainly responsible for the degranulation. We suggest that degranulation of neutrophils may contribute to the (side)effects of IVIG treatment in vivo. PMID:9822286

Cholesterol-PEG comodified poly (N-butyl) cyanoacrylate nanoparticles for brain delivery: in vitro and in vivo evaluations.

PubMed

Hu, Xiao; Yang, Feifei; Liao, Yonghong; Li, Lin; Zhang, Lan

2017-11-01

This study investigated cholesterol-polyethylene glycol (PEG) comodified poly (ethyleneglycol)-poly (lactide) nanoparticles (CLS-PEG NPs) as a novel, biodegradable brain drug delivery system and included an evaluation of its in vitro and in vivo properties. To this end, coumarin-6 (C6), a fluorescent probe, was encapsulated into CLS-PEG NPs by an emulsion polymerization method. We reported that the use of CLS-PEG NPs led to a sustained drug release in vitro. Additionally, cell viability experiments confirmed their safety. The uptake and transport of CLS-PEG NPs, by bEnd.3 cells (an immortalized mouse brain endothelial cell line), was significantly higher than that of a control C6 solution. An investigation of the uptake mechanisms of different NP formulations demonstrated that cholesterol modifications may be the primary way to improve the efficiency of cellular uptake, wherein macropinocytosis may be the most important endocytic pathway in this process. An investigation of the transport mechanisms of CLS-PEG NPs also implicated macropinocytosis, energy and cholesterol in bEnd.3 cells lines. Following an intravenous (IV) administration to rats, pharmacokinetic experiments indicated that C6-loaded CLS-PEG NPs achieved sustained release for up to 12 h. In addition, IV delivery of CLS-PEG NPs appeared to significantly improve the ability of C6 to pass through the blood-brain barrier: the concentration of C6 found in the brain increased nearly 14.2-fold when C6 CLS-PEG NPs were used rather than a C6 solution. These in vitro and in vivo results strongly suggest that CLS-PEG NPs are a promising drug delivery system for targeting the brain, with low toxicity.

Drug-drug interaction and doping: Effect of non-prohibited drugs on the urinary excretion profile of methandienone.

PubMed

Mazzarino, Monica; Khevenhüller-Metsch, Franziska L; Fiacco, Ilaria; Parr, Maria Kristina; de la Torre, Xavier; Botrè, Francesco

2018-05-15

The potential consequences of drug-drug interactions on the excretion profile of the anabolic androgenic steroid methandienone (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one) are discussed here. More specifically, we have evaluated by in vitro and in vivo experiments the effects of seven non-prohibited drugs (fluconazole, ketoconazole, itraconazole, miconazole, fluoxetine, paroxetine and nefazodone) on the main metabolic pathways of methandienone. These are selected among those most commonly used by the athletes. The in vitro assays were based on the use of human liver microsomes, specific recombinant enzyme isoforms of cytochrome P450 and uridine 5'-diphospho-glucuronosyl-transferase. The in vivo study was performed by analyzing urines collected after the oral administration of methandienone with and without the co-administration of ketoconazole. Methandienone and its metabolites were determined by liquid chromatography-mass spectrometry-based techniques after sample pre-treatment including an enzymatic hydrolysis step (performed only for the investigation on phase II metabolism) and liquid/liquid extraction with t-butyl methyl-ether. The results from the in vitro experiments showed that the formation of the hydroxylated and dehydrogenated metabolites was significantly reduced in the presence of itraconazole, ketoconazole, miconazole and nefazodone, whereas the production of the 18-nor-hydroxylated metabolites and glucuronidation reactions was reduced significantly only in the presence of ketoconazole and miconazole. The analysis of the post-administration samples confirmed the in vitro observations, validating the hypothesis that drug-drug interaction may cause significant alterations in the metabolic profile of banned drugs, making their detection during doping control tests more challenging. This article is protected by copyright. All rights reserved.

Tissue engineering of heart valves: in vitro experiences.

PubMed

Sodian, R; Hoerstrup, S P; Sperling, J S; Daebritz, S H; Martin, D P; Schoen, F J; Vacanti, J P; Mayer, J E

2000-07-01

Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Using acoustic cavitation to enhance chemotherapy of DOX liposomes: experiment in vitro and in vivo.

PubMed

Zhao, Ying-Zheng; Dai, Dan-Dan; Lu, Cui-Tao; Lv, Hai-Feng; Zhang, Yan; Li, Xing; Li, Wen-Feng; Wu, Yan; Jiang, Lei; Li, Xiao-Kun; Huang, Pin-Tong; Chen, Li-Juan; Lin, Min

2012-09-01

Experiments in vitro and in vivo were designed to investigate tumor growth inhibition of chemotherapeutics-loaded liposomes enhanced by acoustic cavitation. Doxorubicin-loaded liposomes (DOX liposomes) were used in experiments to investigate acoustic cavitation mediated effects on cell viability and chemotherapeutic function. The influence of lingering sensitive period after acoustic cavitation on tumor inhibition was also investigated. Animal experiment was carried out to verify the practicability of this technique in vivo. From experiment results, blank phospholipid-based microbubbles (PBM) combined with ultrasound (US) at intensity below 0.3 W/cm² could produce acoustic cavitation which maintained cell viability at high level. Compared with DOX solution, DOX liposomes combined with acoustic cavitation exerted effective tumor inhibition in vitro and in vivo. The lingering sensitive period after acoustic cavitation could also enhance the susceptibility of tumor to chemotherapeutic drugs. DOX liposomes could also exert certain tumor inhibition under preliminary acoustic cavitation. Acoustic cavitation could enhance the absorption efficiency of DOX liposomes, which could be used to reduce DOX adverse effect on normal organs in clinical chemotherapy.

'Unconventional' experiments in biology and medicine with optimized design based on quantum-like correlations.

PubMed

Beauvais, Francis

2017-02-01

In previous articles, a description of 'unconventional' experiments (e.g. in vitro or clinical studies based on high dilutions, 'memory of water' or homeopathy) using quantum-like probability was proposed. Because the mathematical formulations of quantum logic are frequently an obstacle for physicians and biologists, a modified modeling that rests on classical probability is described in the present article. This modeling is inspired from a relational interpretation of quantum physics that applies not only to microscopic objects, but also to macroscopic structures, including experimental devices and observers. In this framework, any outcome of an experiment is not an absolute property of the observed system as usually considered but is expressed relatively to an observer. A team of interacting observers is thus described from an external view point based on two principles: the outcomes of experiments are expressed relatively to each observer and the observers agree on outcomes when they interact with each other. If probability fluctuations are also taken into account, correlations between 'expected' and observed outcomes emerge. Moreover, quantum-like correlations are predicted in experiments with local blind design but not with centralized blind design. No assumption on 'memory' or other physical modification of water is necessary in the present description although such hypotheses cannot be formally discarded. In conclusion, a simple modeling of 'unconventional' experiments based on classical probability is now available and its predictions can be tested. The underlying concepts are sufficiently intuitive to be spread into the homeopathy community and beyond. It is hoped that this modeling will encourage new studies with optimized designs for in vitro experiments and clinical trials. Copyright © 2017 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

PubMed

Paes, V M; Vieira, L A; Correia, H H V; Sa, N A R; Moura, A A A; Sales, A D; Rodrigues, A P R; Magalhães-Padilha, D M; Santos, F W; Apgar, G A; Campello, C C; Camargo, L S A; Figueiredo, J R

2016-09-01

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC. Copyright © 2016 Elsevier Inc. All rights reserved.

The effect of in vivo and in vitro irradiation (25 Gy) on the subsequent in vitro growth of satellite cells

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Schultz, E.; Cassens, R. G.

1996-01-01

The effect of in vivo and in vitro irradiation on subsequent satellite cell growth, in vitro, was investigated to ascertain the ability of a 25 Gy dose to inhibit satellite cell proliferation. Satellite cells were isolated from the left (irradiated) and right (non-irradiated) Pectoralis thoracicus of two-week-old tom turkeys 16 h (n=3) and seven weeks (n=2) after the left Pectoralis thoracicus had been irradiated (25 Gy). Satellite cells isolated from the irradiated and non-irradiated muscles exhibited similar (P>0.10) in vitro proliferation indicating that a population of satellite cells survived an in vivo dose of 25 Gy. In additional experiments, satellite cell cultures derived from tom turkey Pectoralis thoracicus were irradiated (25 Gy) in vitro. The number of satellite cells did not (P>0.05) increase in irradiated cultures for 134 h following irradiation, while satellite cells in non-irradiated cultures proliferated (P<0.05) over this time. At later time periods, satellite cell number increased (P<0.05) in irradiated cultures indicating that a population of satellite cells survived irradiation. The results of these in vitro experiments suggest that a 25 Gy dose of irradiation does not abolish satellite cell divisions in the turkey Pectoralis thoracicus.

The anxiety of Taiwanese women with or without continuity treatment after previous in vitro fertilisation failure.

PubMed

Su, Tsann-Juu; Tzeng, Ya-Ling; Kuo, Pi-Chao

2011-08-01

To compare the anxiety levels of Taiwanese women who continued with in vitro fertilisation treatment and those who discontinued treatment post-in vitro fertilisation failure. In vitro fertilisation is perceived as the last resort of infertility treatment. The impact of unsuccessful in vitro fertilisation treatment on psychological function has been documented; however, research comparing the levels of anxiety of women who cease and those who continue in vitro fertilisation post-failure is scant. A cross-sectional comparative study design was used. Fifty-eight women in whom in vitro fertilisation had failed within the previous year were recruited to this study from a medical centre in northern Taiwan; 34 women continued treatment and 24 discontinued treatment. The State-Trait Anxiety Inventory was used to assess their levels of anxiety. Women in the group who continued treatment exhibited higher state and trait anxiety (TA) than women in the group who discontinued treatment (p < 0·005). The number and frequency of in vitro fertilisation cycles were significantly higher in the group who continued treatment than in those who did not. A strong positive correlation between state and TA (r = 0·8, p < 0·01) existed in both groups. Both groups exhibited considerable levels of anxiety; however, the women who continued in vitro fertilisation treatment had higher levels of anxiety than those who discontinued treatment. The level of anxiety of women who decide to continue in vitro fertilisation treatment should be assessed as early as possible and counselling services provided to women who experience in vitro fertilisation failure should concentrate more on relieving psychological distress. One year after discontinuing treatment, some women still experience considerable anxiety; therefore, the care and assistance provided to these women need to be continually evaluated. © 2011 Blackwell Publishing Ltd.

Influence of near null magnetic field on in vitro growth of potato and wild Solanum species.

PubMed

Rakosy-Tican, Lenuta; Aurori, C M; Morariu, V V

2005-10-01

The influence of near null magnetic field on in vitro growth of different cultures of potato and related Solanum species was investigated for various exposure times and dates. Potato (Solanum tuberosum L. cv. Désirée) in vitro cultures of shoot tips or nodal segments were used. Three different exposure periods revealed either stimulation or inhibition of root, stem, or leaf in vitro growth after 14 or 28 days of exposure. In one experiment the significant stimulation of leaf growth was also demonstrated at biochemical level, the quantity of chlorophyll a and b and carotenoids increasing more than two-fold. For the wild species Solanum chacoense, S. microdontum, and S. verrucosum, standardized in vitro cultures of nodal stem segments were used. Root and stem growth was either stimulated or slightly inhibited after 9 days exposure to near null magnetic field. Callus cultures obtained from potato dihaploid line 120/19 were maintained in near null magnetic field in 2 different months. For these experiments as well as for Solanum verrucosum, callus cultures recorded either slight inhibition or no effect on fresh weight. For all experiments significant growth variation was brought about only when geomagnetic activity (AP index) showed variations at the beginning of in vitro growth and when the explant had at least one meristematic tissue. Moreover longer maintenance in near null magnetic field, 28 days as compared to 14 days or the controls, can also make a difference in plant growth in response to geomagnetic field variations when static component was reduced to zero value. These results of in vitro plant growth stimulation by variable component of geomagnetic field also sustain the so-called seasonal "window" effect. (c) 2005 Wiley-Liss, Inc.

Geissoschizine methyl ether N-oxide, a new alkaloid with antiacetylcholinesterase activity from Uncaria rhynchophylla.

PubMed

Jiang, Wei-Wei; Su, Jia; Wu, Xing-De; He, Juan; Peng, Li-Yan; Cheng, Xiao; Zhao, Qin-Shi

2015-01-01

Geissoschizine methyl ether N-oxide, a new oxindole alkaloid, along with 14 known alkaloids, was isolated from the aerial part of Uncaria rhynchophylla. Their structures were identified by comprehensive spectral methods, including 2D NMR experiments, and confirmed by comparing with the literature data. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that the new compound exhibited anti-AChE activity with IC₅₀ value of 23.4 μM.

Does coffee consumption impact on heaviness of smoking?

PubMed Central

Ware, Jennifer J.; Tanner, Julie‐Anne; Taylor, Amy E.; Bin, Zhao; Haycock, Philip; Bowden, Jack; Rogers, Peter J.; Davey Smith, George; Tyndale, Rachel F.

2017-01-01

Abstract Background and Aims Coffee consumption and cigarette smoking are strongly associated, but whether this association is causal remains unclear. We sought to: (1) determine whether coffee consumption influences cigarette smoking causally, (2) estimate the magnitude of any association and (3) explore potential mechanisms. Design We used Mendelian randomization (MR) analyses of observational data, using publicly available summarized data from the Tobacco and Genetics (TAG) consortium, individual‐level data from the UK Biobank and in‐vitro experiments of candidate compounds. Setting The TAG consortium includes data from studies in several countries. The UK Biobank includes data from men and women recruited across England, Wales and Scotland. Participants The TAG consortium provided data on n ≤ 38 181 participants. The UK Biobank provided data on 8072 participants. Measurements In MR analyses, the exposure was coffee consumption (cups/day) and the outcome was heaviness of smoking (cigarettes/day). In our in‐vitro experiments we assessed the effect of caffeic acid, quercetin and p‐coumaric acid on the rate of nicotine metabolism in human liver microsomes and cDNA‐expressed human CYP2A6. Findings Two‐sample MR analyses of TAG consortium data indicated that heavier coffee consumption might lead to reduced heaviness of smoking [beta = −1.49, 95% confidence interval (CI) = −2.88 to −0.09]. However, in‐vitro experiments found that the compounds investigated are unlikely to inhibit significantly the rate of nicotine metabolism following coffee consumption. Further MR analyses in UK Biobank found no evidence of a causal relationship between coffee consumption and heaviness of smoking (beta = 0.20, 95% CI = –1.72 to 2.12). Conclusions Amount of coffee consumption is unlikely to have a major causal impact upon amount of cigarette smoking. If it does influence smoking, this is not likely to operate via effects of caffeic acid, quercetin or p‐coumaric acid on nicotine metabolism. The observational association between coffee consumption and cigarette smoking may be due to smoking impacting on coffee consumption or confounding. PMID:28556459

Does coffee consumption impact on heaviness of smoking?

PubMed

Ware, Jennifer J; Tanner, Julie-Anne; Taylor, Amy E; Bin, Zhao; Haycock, Philip; Bowden, Jack; Rogers, Peter J; Davey Smith, George; Tyndale, Rachel F; Munafò, Marcus R

2017-10-01

Coffee consumption and cigarette smoking are strongly associated, but whether this association is causal remains unclear. We sought to: (1) determine whether coffee consumption influences cigarette smoking causally, (2) estimate the magnitude of any association and (3) explore potential mechanisms. We used Mendelian randomization (MR) analyses of observational data, using publicly available summarized data from the Tobacco and Genetics (TAG) consortium, individual-level data from the UK Biobank and in-vitro experiments of candidate compounds. The TAG consortium includes data from studies in several countries. The UK Biobank includes data from men and women recruited across England, Wales and Scotland. The TAG consortium provided data on n ≤ 38 181 participants. The UK Biobank provided data on 8072 participants. In MR analyses, the exposure was coffee consumption (cups/day) and the outcome was heaviness of smoking (cigarettes/day). In our in-vitro experiments we assessed the effect of caffeic acid, quercetin and p-coumaric acid on the rate of nicotine metabolism in human liver microsomes and cDNA-expressed human CYP2A6. Two-sample MR analyses of TAG consortium data indicated that heavier coffee consumption might lead to reduced heaviness of smoking [beta = -1.49, 95% confidence interval (CI) = -2.88 to -0.09]. However, in-vitro experiments found that the compounds investigated are unlikely to inhibit significantly the rate of nicotine metabolism following coffee consumption. Further MR analyses in UK Biobank found no evidence of a causal relationship between coffee consumption and heaviness of smoking (beta = 0.20, 95% CI = -1.72 to 2.12). Amount of coffee consumption is unlikely to have a major causal impact upon amount of cigarette smoking. If it does influence smoking, this is not likely to operate via effects of caffeic acid, quercetin or p-coumaric acid on nicotine metabolism. The observational association between coffee consumption and cigarette smoking may be due to smoking impacting on coffee consumption or confounding. © 2017 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.

Gonadotropin-releasing hormone antagonist in in vitro fertilization superovulation.

PubMed

Seng, Shay Way; Ong, Kee Jiet; Ledger, W L

2006-11-01

The use of gonadotropin-releasing hormone (GnRH) antagonists in in vitro fertilization superovulation remains controversial. The GnRH agonist 'long protocol' has been seen as the gold standard for many years. Comparisons and meta-analyses of the efficacy of GnRH antagonists and agonists have been largely inconclusive, with the dataset being contaminated with outdated reports of poorer efficacy with GnRH antagonists, which have stemmed from studies of their use as a second-line drug in older women and women who were poor responders. This work cannot reflect the actual clinical effectiveness of GnRH antagonist and must be interpreted with care. The major advantages of GnRH antagonists use in superovulation include a gentler and more patient-friendly stimulation cycle with less hypoestrogenic side effects, with the potential to lower the risk of ovarian hyperstimulation and enhanced embryo growth. Our current clinical experience with GnRH antagonists in in vitro fertilization is limited, although there are a growing number of in vitro fertilization centers embracing this new technology. There is a clear need for a modern, suitably powered clinical trial to demonstrate the place of GnRH antagonist-based superovulation protocols and in subgroups of patients, such as polycystic ovary syndrome or poor responders.

In Vitro Simulation and Validation of the Circulation with Congenital Heart Defects

PubMed Central

Figliola, Richard S.; Giardini, Alessandro; Conover, Tim; Camp, Tiffany A.; Biglino, Giovanni; Chiulli, John; Hsia, Tain-Yen

2010-01-01

Despite the recent advances in computational modeling, experimental simulation of the circulation with congenital heart defect using mock flow circuits remains an important tool for device testing, and for detailing the probable flow consequences resulting from surgical and interventional corrections. Validated mock circuits can be applied to qualify the results from novel computational models. New mathematical tools, coupled with advanced clinical imaging methods, allow for improved assessment of experimental circuit performance relative to human function, as well as the potential for patient-specific adaptation. In this review, we address the development of three in vitro mock circuits specific for studies of congenital heart defects. Performance of an in vitro right heart circulation circuit through a series of verification and validation exercises is described, including correlations with animal studies, and quantifying the effects of circuit inertiance on test results. We present our experience in the design of mock circuits suitable for investigations of the characteristics of the Fontan circulation. We use one such mock circuit to evaluate the accuracy of Doppler predictions in the presence of aortic coarctation. PMID:21218147

Deriving an explicit hepatic clearance equation accounting for plasma protein binding and hepatocellular uptake.

PubMed

Yoon, Miyoung; Clewell, Harvey J; Andersen, Melvin E

2013-02-01

High throughput in vitro biochemical and cell-based assays have the promise to provide more mechanism-based assessments of the adverse effects of large numbers of chemicals. One of the most challenging hurdles for interpreting in vitro toxicity findings is the need for reverse dosimetry tools that estimate the exposures that will give concentrations in vivo similar to the active concentrations in vitro. Recent experience using IVIVE approaches to estimate in vivo pharmacokinetics (Wetmore et al., 2012) identified the need to develop a hepatic clearance equation that explicitly accounted for a broader set of protein binding and membrane transport processes and did not depend on a well-mixed description of the liver compartment. Here we derive an explicit steady-state hepatic clearance equation that includes these factors. In addition to the derivation, we provide simple computer code to calculate steady-state extraction for any combination of blood flow, membrane transport processes and plasma protein-chemical binding rates. This expanded equation provides a tool to estimate hepatic clearance for a more diverse array of compounds. Copyright © 2012 Elsevier Ltd. All rights reserved.

Infectivity of Theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing: evaluation by in vitro titration.

PubMed

Mbao, V; Berkvens, D; Dolan, T; Speybroeck, N; Brandt, J; Dorny, P; Van den Bossche, P; Marcotty, T

2006-09-01

Theileria parva sporozoite stabilates are used for immunizing cattle against East Coast fever and in in vitro sporozoite neutralization assays. In this study, we attempted to identify a cheaper freezing medium and quantified the infectivity loss of sporozoites due to refreezing of stabilates, using an in vitro technique. Pools of stabilates prepared using Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI 1640), foetal calf serum (FCS) and phosphate-buffered saline (PBS) were compared. All were supplemented with bovine serum albumin except the FCS. RPMI 1640 was as effective as MEM in maintaining sporozoite infectivity while the infectivity in PBS and FCS reached only 59% and 67%, respectively. In a second experiment, a stabiiate based on MEM was subjected to several freeze-thaw cycles including various holding times on ice between thawing and refreezing. Refrozen stabilate gave an average sporozoite infectivity loss of 35% per cycle. The results indicate that RPMI can be used as a cheaper freezing medium for T. parva stabilates and that refrozen stabilate doses need to be adjusted for the 35% loss of infectivity.

Protective Effect of Fucoxanthin Isolated from Laminaria japonica against Visible Light-Induced Retinal Damage Both in Vitro and in Vivo.

PubMed

Liu, Yixiang; Liu, Meng; Zhang, Xichun; Chen, Qingchou; Chen, Haixiu; Sun, Lechang; Liu, Guangming

2016-01-20

With increasingly serious eye exposure to light stresses, such as light-emitting diodes, computers, and widescreen mobile phones, efficient natural compounds for preventing visible light-induced retinal damages are becoming compelling needs in the modern society. Fucoxanthin, as the main light absorption system in marine algae, may possess an outstanding bioactivity in vision protection because of its filtration of blue light and excellent antioxidative activity. In this work, both in vitro and in vivo simulated visible light-induced retinal damage models were employed. The in vitro results revealed that fucoxanthin exhibited better bioactivities than lutein, zeaxanthin, and blueberry anthocyanins in inhibiting overexpression of vascular endothelial growth factor, resisting senescence, improving phagocytic function, and clearing intracellular reactive oxygen species in retinal pigment epithelium cells. The in vivo experiment also confirmed the superiority of fucoxanthin than lutein in protecting retina against photoinduced damage. This excellent bioactivity may be attributed to its unique structural features, including allenic, epoxide, and acetyl groups. Fucoxanthin is expected to be an important ocular nutrient in the future.

Development of biomimetic in vitro fatigue assessment for UHMWPE implant materials.

PubMed

Scholz, Ronja; Knyazeva, Marina; Porchetta, Dario; Wegner, Nils; Senatov, Fedor; Salimon, Alexey; Kaloshkin, Sergey; Walther, Frank

2018-05-26

An important research goal in the field of biomaterials lies in the progressive amendment of in vivo tests with suitable in vitro experiments. Such approaches are gaining more significance nowadays because of an increasing demand on life sciences and the ethical issues bound to the sacrifice of animals for the sake of scientific research. Another advantage of transferring the experiments to the in vitro field is the possibility of accurately control the boundary conditions and experimental parameters in order to reduce the need of validation tests involving animals. With the aim to reduce the amount of needed in vivo studies for this cause, a short-time in vitro test procedure using instrumented load increase tests with superimposed environmental loading has been developed at TUD to assess the mechanical long-term durability of ultra-high molecular weight polyethylene (UHMWPE) under fatigue loading in a biological environment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Anti-Toxoplasma activity of various molecular weights and concentrations of chitosan nanoparticles on tachyzoites of RH strain.

PubMed

Teimouri, Aref; Azami, Sanaz Jafarpour; Keshavarz, Hossein; Esmaeili, Fariba; Alimi, Rasoul; Mavi, Sara Ayazian; Shojaee, Saeedeh

2018-01-01

Natural polysaccharides such as chitosan (CS) are widely used as antimicrobial agents. In recent years, and considering that CS has a strong antimicrobial potential, interest has been focused on antimicrobial activity of chitosan nanoparticles (CS NPs). The main factors affecting the antibacterial activity of chitosan include molecular weight (MW) and concentration. In this regard, the aim of this study was to produce various MWs and concentrations of CS NPs, through the ionic gelation method, and investigate their potential anti-parasitic activity against tachyzoites of Toxoplasma gondii RH strain. The MWs and degree of deacetylation of the CS were characterized using viscometric and acid-base titration methods, respectively. The efficacy of various MWs and concentrations of NPs was assessed by performing in vitro experiments for tachyzoites of T. gondii RH strain, such as MTT assay, scanning electron microscopy, bioassay in mice and PCR. In vivo experiment was carried out in BALB/c mice which were inoculated with tachyzoites of T. gondii RH strain and treated with various MWs of CS NPs. The results of in vitro and in vivo experiments revealed that anti- Toxoplasma activity strengthened as the CS NPs concentration increased and the MW decreased. In vitro experiment showed 100% mortality of tachyzoites at 500 and 1,000 ppm concentrations of low molecular weight (LMW) CS NPs after 180 min and at 2,000 ppm after 120 min. Furthermore, a 100% mortality of tachyzoites was observed at 1,000 and 2,000 ppm concentrations of medium molecular weight (MMW) CS NPs and at 2,000 ppm concentration of high molecular weight (HMW) CS NPs after 180 min. Growth inhibition rates of tachyzoites in peritoneal exudates of mice receiving low, medium and high MWs of CS NPs were found to be 86%, 84% and 79% respectively, compared to those of mice in sulfadiazine treatment group (positive control). Various MWs of CS NPs exhibited great anti- Toxoplasma efficiency against tachyzoites of RH strain, with the greatest efficacy shown by LMW CS NPs in both experiments. It seems that CS NPs can be used as an alternative natural medicine in the treatment of toxoplasmosis.

A comprehensive training approach for biomedical engineers in biochemistry and in vitro diagnostics technology.

PubMed

Spyropoulos, Basile; Tzavaras, Aris

2007-01-01

The purpose of this paper is to review 20 years (1987-2007) of experience in training young Biomedical Engineers in Biochemistry and in vitro Diagnostics (IVD) Technology. This encountering has resulted in the gradual formation of a comprehensive training package that includes lectures and laboratory practicals, supported by both, traditional and on-line digital means, such as lecture-notes, slides, videos, demos and equipment simulations. Further, this course is maintained up to date by several research and development activities that offer partially feed back to the course and enrich its contents with custom developed devices, methods and application software. In this paper are presented, first, the structure and the components of this course, and second, the most important custom developed novelties, which have been integrated in the IVD Technology laboratory-practicals.

Docking simulations and in vitro assay unveil potent inhibitory action of papaverine against protein tyrosine phosphatase 1B.

PubMed

Bustanji, Yasser; Taha, Mutasem Omar; Al-Masri, Ihab Mustafa; Mohammad, Mohammad Khalil

2009-04-01

The structural similarity between papaverine and berberine, a known inhibitor of human protein tyrosine phosphatase 1B (h-PTP 1B), prompted us to investigate the potential of papaverine as h-PTP 1B inhibitor. The investigation included simulated docking experiments to fit papaverine into the binding pocket of h-PTP 1B. Papaverine was found to readily dock within the binding pocket of h-PTP 1B in a low energy orientation via an optimal set of attractive interactions. Experimentally, papaverine illustrated potent in vitro inhibitory effect against recombinant h-PTP 1B (IC(50)=1.20 microM). In vivo, papaverine significantly decreased fasting blood glucose level of Balb/c mice. Our findings should encourage screening of other natural alkaloids for possible anti-h-PTP 1B activities.

The Standardized Extract of Juniperus communis Alleviates Hyperpigmentation in Vivo HRM-2 Hairless Mice and in Vitro Murine B16 Melanoma Cells.

PubMed

Jegal, Jonghwan; Chung, Ki Wung; Chung, Hae Young; Jeong, Eun Ju; Yang, Min Hye

2017-01-01

In European folk medicine, the fruits of Juniperus communis are used in the treatment of skin-related disorders such as skin infection, itching, and psoriasis. Previously, we reported that the EtOAc fraction of J. communis (EAJC) contained tyrosinase inhibition properties in vitro non-cellular experiment. The aim of this study was to evaluate anti-melanogenic effect of standardized EAJC on a hyperpigmentation animal model. Therapeutic effects of EAJC toward skin hyperpigmentation were confirmed by both in vivo experiment and in vitro cell-based assay. Skin depigmenting effect was detected by topical treatment of EAJC for 11 d to HRM-2 melanin-possessing hairless mice. Histologic findings including significantly decreased melanin depositions could be observed in dorsal skin samples of EAJC-treated group. In addition, the EAJC (50 µg/mL) attenuated melanin production through down-regulation of tyrosinase activity and protein expression in B16 murine melanoma cells. According to the phytochemical analysis, EAJC was found to contain hypolaetin-7-O-β-D-xylopyranoside and isoscutellarein-7-O-β-D-xylopyranoside as main components. Hypolaetin-7-O-β-D-xylopyranoside was responsible for the skin-lightening effect of EAJC by reducing the number of melanocytes in dorsal skins of HRM-2 mice. The present study provided direct experimental evidence for skin-lightening effect of EAJC in UV-irradiated hairless mouse model. Therapeutic attempts with the J. communis might be useful in the management of skin pigmentation-related diseases.

In vitro microbial protein synthesis, ruminal degradation and post-ruminal digestibility of crude protein of dairy rations containing Quebracho tannin extract.

PubMed

Castro-Montoya, J; Westreicher-Kristen, E; Henke, A; Diaby, M; Susenbeth, A; Dickhoefer, U

2018-02-01

This study evaluated the effects of Quebracho tannin extract (QTE) on in vitro ruminal fermentation, chemical composition of rumen microbes, ruminal degradation and intestinal digestibility of crude protein (iCPd). Three treatments were tested, the control (basal diet without QTE), the basal diet with 15 g QTE/kg dry matter (DM) and the basal diet with 30 g QTE/kg DM. The basal diet contained (g/kg DM): 339 grass silage, 317 maize silage and 344 concentrate. In vitro gas production kinetic was determined using the Hohenheim gas test (Experiment 1). The Ankom RF technique, a batch system with automatic gas pressure recordings, was used to determine in vitro production of short-chain fatty acids (SCFA) and ammonia-nitrogen concentration (NH 3 -N), as well as nitrogen and purine bases content in liquid-associated microbes (LAM) and in a residue of undegraded feed and solid-associated microbes (Feed+SAM) (Experiment 2). Ruminal degradation and iCPd were determined using the nylon bag technique and the mobile nylon bag technique, respectively (Experiment 3). Gas production (Experiment 1), total SCFA and NH 3 -N (Experiment 2) decreased with increasing QTE levels. Microbial mass and composition of LAM were not affected by QTE, but total mass of Feed+SAM linearly increased, likely due to decreased substrate degradation with increasing QTE levels. The total amount of N in microbial mass and undegraded feed after the in vitro incubation increased with increasing QTE levels, suggesting a potential greater N flow from the rumen to the duodenum. In contrast to in vivo studies with the same QTE, no effects were detected on ruminal effective degradability and iCPd, when using the nylon bag techniques. Based on the in vitro procedures, QTE increased the supply of N post-rumen; however, some evidence of a decreased fibre degradation were also observed. Therefore, the benefit of adding QTE to diets of cattle is still questionable. © 2017 Blackwell Verlag GmbH.

Flux analysis of the human proximal colon using anaerobic digestion model 1.

PubMed

Motelica-Wagenaar, Anne Marieke; Nauta, Arjen; van den Heuvel, Ellen G H M; Kleerebezem, Robbert

2014-08-01

The colon can be regarded as an anaerobic digestive compartment within the gastro intestinal tract (GIT). An in silico model simulating the fluxes in the human proximal colon was developed on basis of the anaerobic digestion model 1 (ADM1), which is traditionally used to model waste conversion to biogas. Model calibration was conducted using data from in vitro fermentation of the proximal colon (TIM-2), and, amongst others, supplemented with the bio kinetics of prebiotic galactooligosaccharides (GOS) fermentation. The impact of water and solutes absorption by the host was also included. Hydrolysis constants of carbohydrates and proteins were estimated based on total short chain fatty acids (SCFA) and ammonia production in vitro. Model validation was established using an independent dataset of a different in vitro model: an in vitro three-stage continuous culture system. The in silico model was shown to provide quantitative insight in the microbial community structure in terms of functional groups, and the substrate and product fluxes between these groups as well as the host, as a function of the substrate composition, pH and the solids residence time (SRT). The model confirms the experimental observation that methanogens are washed out at low pH or low SRT-values. The in silico model is proposed as useful tool in the design of experimental setups for in vitro experiments by giving insight in fermentation processes in the proximal human colon. Copyright © 2014. Published by Elsevier Ltd.

Easy assessment of the biocompatibility of Ni-Ti alloys by in vitro cell culture experiments on a functionally graded Ni-NiTi-Ti material.

PubMed

Bogdanski, Denise; Köller, Manfred; Müller, Dietmar; Muhr, Gert; Bram, Martin; Buchkremer, Hans Peter; Stöver, Detlev; Choi, Jongsik; Epple, Matthias

2002-12-01

The biocompatibility of nickel-titanium alloys was investigated by single-culture experiments on functionally graded samples with a stepwise change in composition from pure nickel to pure titanium, including an Ni-Ti shape memory alloy for a 50:50 mixture. This approach permitted a considerable decrease of experimental resources by simultaneously studying a full variation of composition. The results indicate a good biocompatibility for a nickel content up to about 50%. The cells used in the biocompatibility studies comprised osteoblast-like osteosarcoma cells (SAOS-2, MG-63), primary human osteoblasts (HOB), and murine fibroblasts (3T3).

Bacterial cell-free expression technology to in vitro systems engineering and optimization.

PubMed

Caschera, Filippo

2017-06-01

Cell-free expression system is a technology for the synthesis of proteins in vitro . The system is a platform for several bioengineering projects, e.g. cell-free metabolic engineering, evolutionary design of experiments, and synthetic minimal cell construction. Bacterial cell-free protein synthesis system (CFPS) is a robust tool for synthetic biology. The bacteria lysate, the DNA, and the energy module, which are the three optimized sub-systems for in vitro protein synthesis, compose the integrated system. Currently, an optimized E. coli cell-free expression system can produce up to ∼2.3 mg/mL of a fluorescent reporter protein. Herein, I will describe the features of ATP-regeneration systems for in vitro protein synthesis, and I will present a machine-learning experiment for optimizing the protein yield of E. coli cell-free protein synthesis systems. Moreover, I will introduce experiments on the synthesis of a minimal cell using liposomes as dynamic containers, and E. coli cell-free expression system as biochemical platform for metabolism and gene expression. CFPS can be further integrated with other technologies for novel applications in environmental, medical and material science.

Evolution in vitro: analysis of a lineage of ribozymes

NASA Technical Reports Server (NTRS)

Lehman, N.; Joyce, G. F.

1993-01-01

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

An experiment to study the effects of space flight cells of mesenchymal origin in the new model 3D-graft in vitro

NASA Astrophysics Data System (ADS)

Volova, Larissa

One of the major health problems of the astronauts are disorders of the musculoskeletal system, which determines the relevance of studies of the effect of space flight factors on osteoblastic and hondroblastic cells in vitro. An experiment to study the viability and proliferative activity of cells of mesenchymal origin on culture: chondroblasts and dermal fibroblasts was performed on SC "BION -M" No. 1 with scientific equipment " BIOKONT -B ." To study the effect of space flight conditions in vitro at the cellular level has developed a new model with 3D- graft as allogeneic demineralized spongiosa obtained on technology Lioplast ®. For space and simultaneous experiments in the laboratory of the Institute of Experimental Medicine and Biotechnology Samara State Medical University were obtained from the cell culture of hyaline cartilage and human skin, which have previously been grown, and then identified by morphological and immunohistochemical methods. In the experiment, they were seeded on the porous 3D- graft (controlled by means of scanning electron and confocal microscopy) and cultured in full growth medium. After completion of the flight of spacecraft "BION -M" No. 1 conducted studies of biological objects using a scanning electron microscope (JEOL JSM-6390A Analysis Station, Japan), confocal microscopy and LDH - test. According to the results of the experiment revealed that after a 30- day flight of the cells not only retained vitality, but also during the flight actively proliferate, and their number has increased by almost 8 times. In synchronous experiment, all the cells died by this date. The experimentally confirmed the adequacy of the proposed model 3D- graft in studying the effect of space flight on the morphological and functional characteristics of cells in vitro.

Effects of plants containing secondary compounds and plant oils on rumen fermentation and ecology.

PubMed

Wanapat, Metha; Kongmun, Pongthon; Poungchompu, Onanong; Cherdthong, Anusorn; Khejornsart, Pichad; Pilajun, Ruangyote; Kaenpakdee, Sujittra

2012-03-01

A number of experiments have been conducted to investigate effects of tropical plants containing condensed tannins and/or saponins present in tropical plants and some plant oils on rumen fermentation and ecology in ruminants. Based on both in vitro and in vivo trials, the results revealed important effects on rumen microorganisms and fermentation including methane production. Incorporation and/or supplementation of these plants containing secondary metabolites have potential for improving rumen ecology and subsequently productivity in ruminants.

[Effect of rat intestinal flora on in vitro metabolic transformation of pumiloside].

PubMed

Fang, Hui; Li, Meng-Xuan; Li, Hai-Bo; Liu, Wen-Jun; Meng, Zhao-Qing; Huang, Wen-Zhe; Wang, Zhen-Zhong; Xiao, Wei

2016-05-01

To study the metabolic transformation of pumiloside by rat intestinal flora in vitro and identify its metabolites. Pumiloside was incubated in the rat intestinal flora in vitro. HPLC was used to monitor the metabolic process, and HPLC-Q-TOF-MS was used to identify the structures of biotransformation products. In vitro, pumiloside was easily metabolized by rat intestinal flora, and with the prolongation of metabolic time, pumiloside was transformed into several metabolites. Three metabolites were initially identified in this experiment. The study indicated that pumiloside could be extensively metabolized in the rat intestinal flora in vitro. Copyright© by the Chinese Pharmaceutical Association.

Use of in vitro dry matter digestibility and gas production to predict apparent total tract digestibility of total dietary fiber for growing pigs.

PubMed

Huang, Z; Urriola, P E; Shurson, G C

2017-12-01

In vitro DM disappearance (IVDMD) and gas production methods have been developed and used to measure in vivo nutrient digestibility of feed ingredients, but further validation is needed for ingredients containing high concentrations of insoluble fiber such as corn distiller's dried grains with solubles (DDGS). A 3-step in vitro procedure and resulting gas production were used to predict in vivo apparent total tract digestibility (ATTD) of total dietary fiber (TDF) among 3 sources each of wheat straw (WS), soybean hulls (SBH), and DDGS. A total of 34 barrows and 2 gilts (84 ± 7 kg BW) were used in a changeover design to determine the ATTD of 9 dietary treatments. The WS, SBH, or DDGS sources were the only ingredients containing fiber in each diet, and all diets were formulated to contain the same TDF concentration (22.3%). The in vivo experiment was conducted in 2 consecutive 13-d periods, each including a 10-d adaptation and a 3-d collection period to provide 8 replications/dietary treatment, and 0.5% TiO was added to each diet as an indigestible marker. Pigs had ad libitum access to water and were fed an amount of feed equivalent to 2.5% of initial BW in each period. The in vitro experiment was used to determine IVDMD and gas production of the 9 ingredients (5 to 8 replicates/ingredient) fed during the in vivo experiment. Gas production kinetics were fitted using a nonlinear model and analyzed using a mixed model, and predictions were evaluated using correlations and regression models. There were differences ( < 0.01) in ATTD of TDF among WS (26.7%), SBH (78.9%), and DDGS (43.0%) and among sources of DDGS (36.0 to 49.8%). Differences ( < 0.05) in IVDMD from simulated gastric and small intestinal hydrolysis were observed among WS (13.3%), SBH (18.9%), and DDGS (53.7%) and among sources of WS (12.8 to 13.8%), SBH (17.0 to 20.5%), and DDGS (52.0 to 56.9%). Differences ( < 0.05) in IVDMD from simulated large intestine fermentation (IVDMDf) were also observed among WS (23.3%), SBH (84.6%), and DDGS (69.6%) and among sources of WS (18.7 vs. 26.8%). In vitro DM disappearance from simulated total tract digestion of SBH (88.9%) and DDGS (86.1%) were greater ( < 0.01) than that of WS (33.5%). Differences ( < 0.01) in asymptotic gas production (A; mL/g DM substrate) were observed among WS (121), SBH (412), and DDGS (317), and ATTD of TDF was highly correlated with IVDMDf and A. In conclusion, low variability in ATTD of TDF and IVDMD among sources of WS and SBH evaluated in the current study may not justify the use of in vitro measurements, but in vitro fermentation accurately predicts ATTD of TDF among sources of corn DDGS.

Evaluation of cholesterol- treated dromedary camel sperm function by heterologous IVF and AI.

PubMed

Crichton, Elizabeth G; Malo, Clara; Pukazhenthi, Budhan S; Nagy, Peter; Skidmore, Julian A

2016-11-01

Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7-9 per treatment) and examining embryo production (Experiment 4: n=4-5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested. Copyright © 2016 Elsevier B.V. All rights reserved.

From laboratory to field studies - The assessment of Biscutella laevigata suitability to biological reclamation of areas contaminated with lead and cadmium.

PubMed

Muszyńska, Ewa; Hanus-Fajerska, Ewa; Piwowarczyk, Barbara; Augustynowicz, Joanna; Ciarkowska, Krystyna; Czech, Tomasz

2017-08-01

The aim of the work was to evaluate the usefulness of the in vitro multiplication of Biscutella laevigata calamine ecotype for in situ reclamation of post-flotation wastes polluted with Pb and Cd. The experiment was conducted on three steps: (i) plant shoots' production under in vitro condition, (ii) establishment of the material in greenhouse experiment, and finally (iii) field cultivation directly on the mining-waste heap of Olkusz Ore-Bearing Region, Poland. This region is known to be one of the most chemically-degraded area in central Europe. The laboratory-set in vitro analysis enabled to obtain the high-quality plant shoots, which multiply the most effectively (with growth tolerance index 130-150%) on medium containing 5.0μM CdCl 2 and 0.5mM Pb(NO 3 ) 2 . These plants were used for the next two ex vitro experiments. Several biometric and physiological analysis (i.e. of photosystem II activity F v /F m and PI, photosynthetic pigment contents) were done to indicate plant physiological status during these experiments. The main novelty of the work was to prove that in vitro-multiplied shoots of B. laevigata - the representative of native flora from Olkusz Ore-Bearing Region - can be successfully implemented in situ for the restoration of these degraded area. Moreover, the addition of sewage sludge as a source of organic compounds significantly improved plants' growth and development what is especially important due to the lack of other legal solutions for the management of the sewage sludge in some countries. Copyright © 2017 Elsevier Inc. All rights reserved.

Active locomotion of a paddling-based capsule endoscope in an in vitro and in vivo experiment (with videos).

PubMed

Kim, Hee Man; Yang, Sungwook; Kim, Jinseok; Park, Semi; Cho, Jae Hee; Park, Jeong Youp; Kim, Tae Song; Yoon, Eui-Sung; Song, Si Young; Bang, Seungmin

2010-08-01

Capsule endoscopy that could actively move and approach a specific site might be more valuable for the diagnosis or treatment of GI diseases. We tested the performance of active locomotion of a novel wired capsule endoscope with a paddling-based locomotion mechanism, using 3 models: a silicone tube, an extracted porcine colon, and a living pig. In vitro, ex vivo, and in vivo experiments in a pig model. Study in an animal laboratory. For the in vitro test, the locomotive capsule was controlled to actively move from one side of a silicone tube to the other by a controller-operated automatic traveling program. The velocity was calculated by following a video recording. We performed ex vivo tests by using an extracted porcine colon in the same manner we performed the in vitro test. In in vivo experiments, the capsule was inserted into the rectum of a living pig under anesthesia, and was controlled to move automatically forward. After 8 consecutive trials, the velocity was calculated. Elapsed time, velocity, and mucosal damage. The locomotive capsule showed stable and active movement inside the lumen both in vitro and ex vivo. The velocity was 60 cm/min in the silicone tube, and 36.8 and 37.5 cm/min in the extracted porcine colon. In the in vivo experiments, the capsule stably moved forward inside the colon of a living pig without any serious complications. The mean velocity was 17 cm/min over 40 cm length. We noted pinpoint erythematous mucosal injuries in the colon. Porcine model experiments, wired capsule endoscope. The novel paddling-based locomotive capsule endoscope performed fast and stable movement in a living pig colon with consistent velocity. Further investigation is necessary for practical use in humans. Copyright 2010 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.

Advances on the Transfer of Lipids by Lipid Transfer Proteins.

PubMed

Wong, Louise H; Čopič, Alenka; Levine, Tim P

2017-07-01

Transfer of lipid across the cytoplasm is an essential process for intracellular lipid traffic. Lipid transfer proteins (LTPs) are defined by highly controlled in vitro experiments. The functional relevance of these is supported by evidence for the same reactions inside cells. Major advances in the LTP field have come from structural bioinformatics identifying new LTPs, and from the development of countercurrent models for LTPs. However, the ultimate aim is to unite in vitro and in vivo data, and this is where much progress remains to be made. Even where in vitro and in vivo experiments align, rates of transfer tend not to match. Here we set out some of the advances that might test how LTPs work. Copyright © 2017. Published by Elsevier Ltd.

Immunomodulatory effect of morphine: therapeutic implications.

PubMed

Dinda, Amit; Gitman, Michael; Singhal, Pravin C

2005-07-01

The immunosuppressive as well as modulatory effects of morphine have been known in clinical medicine for > 100 years. Recent developments in molecular immunology, including experiments in mu (mu) opioid receptor knockout mice has led to a better understanding of central and peripheral mechanisms involved in this process. Though there is a large volume of literature documenting adverse effects of immunosupression following the use of morphine, several reports confirm its potential usefulness as an immunomodulator. In vitro and in vivo animal experiments have demonstrated wide-spectrum effects of morphine, including anti-inflammatory, antifibrotic, antitumour, cardioprotective and renoprotective. Immunomodulation is an important field in modern medicine with rapid advancement in recent years. Though a final statement regarding the clinical relevance of morphine-induced immunomodulation cannot be made at this juncture, nevertheless, it is worthwhile to review current developments. It may encourage further clinical studies to elucidate the influence of morphine treatment on immune regulation in different specialties of medicine.

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

PubMed

Martínez-Sierra, J Giner; Moreno Sanz, F; Herrero Espílez, P; Marchante Gayón, J M; Rodríguez Fernández, J; García Alonso, J I

2013-03-01

We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

Comparison of in vitro and in vivo acoustic response of a novel 50:50 PLGA contrast agent.

PubMed

Wheatley, Margaret A; Forsberg, Flemming; Oum, Kelleny; Ro, Raymond; El-Sherif, Dalia

2006-11-01

A comparison between in vitro and in vivo experiments conducted to investigate the acoustic properties of a novel, 1.2 microm diameter poly(lactic-co-glycolic acid) (50:50) (PLGA) ultrasound contrast agent, the development of which was described previously by us, is presented. A pulse-echo setup was used to determine enhancement in vitro. Additional in vitro studies further characterized the hollow microcapsules, including resonance frequency from attenuation measurements (from 2.25 to 15 MHz) and temperature effects (25 degrees C vs. 37 degrees C). In vivo, four rabbits received intravenous injections of the agent (dose range: 0.005-0.13 ml/kg). Quantitative in vivo dose-responses were calculated off-line using spectral power analysis of audio Doppler signals acquired from a custom-made 10 MHz cuff transducer placed around the surgically exposed distal aorta. This frequency was chosen since the very shallow scanning depths encountered in rabbits, in particular for the cuff transducer placed directly around the vessel, necessitates the use of high frequency imaging devices with sufficient spatial resolution to enable meaningful measurements. For qualitative assessments, two rabbits were imaged pre- and post-contrast administration (dose: 0.1 ml/kg) in power Doppler mode. Significant acoustic enhancements (up to 24 dB) were reported both in vitro and in vivo. Moreover, the rabbits did not show any adverse side effects from multiple injections (>20) of the agent. Measured in vitro resonance frequency between 3.09 and 3.49 MHz was lower than predicted for a similar sized free bubble, potentially due to capsule wall structure. Minimal loss of signal (approximately 4 dB) was observed at 25 degrees C over 20 min of insonation at 5 MHz but at 37 degrees C the signal dropped close to base line within the first 5 min. This temperature sensitivity could be due to loss of capsule integrity (and hence loss of gas). Potential causes include increased hydrolysis or polymer softening and increased water uptake by the shell at temperatures closer to the glass transition temperature (T(g)).

Yerba mate enhances probiotic bacteria growth in vitro but as a feed additive does not reduce Salmonella Enteritidis colonization in vivo.

PubMed

Gonzalez-Gil, Francisco; Diaz-Sanchez, Sandra; Pendleton, Sean; Andino, Ana; Zhang, Nan; Yard, Carrie; Crilly, Nate; Harte, Federico; Hanning, Irene

2014-02-01

Yerba mate (Ilex paraguariensis) is a tea known to have beneficial effects on human health and antimicrobial activity against some foodborne pathogens. Thus, the application of yerba mate as a feed additive for broiler chickens to reduce Salmonella colonization was evaluated. The first in vitro evaluation was conducted by suspending Salmonella Enteritidis and lactic acid bacteria (LAB) in yerba mate extract. The in vivo evaluations were conducted using preventative and horizontal transmission experiments. In all experiments, day-of-hatch chicks were treated with one of the following 1) no treatment (control); 2) ground yerba mate in feed; 3) probiotic treatment (Lactobacillus acidophilus and Pediococcus; 9:1 administered once on day of hatch by gavage); or 4) both yerba mate and probiotic treatments. At d 3, all chicks were challenged with Salmonella Enteritidis (preventative experiment) or 5 of 20 chicks (horizontal transmission experiment). At d 10, all birds were euthanized, weighed, and cecal contents enumerated for Salmonella. For the in vitro evaluation, antimicrobial activity was observed against Salmonella and the same treatment enhanced growth of LAB. For in vivo evaluations, none of the yerba mate treatments significantly reduced Salmonella Enteritidis colonization, whereas the probiotic treatment significantly reduced Salmonella colonization in the horizontal transmission experiment. Yerba mate decreased chicken BW and decreased the performance of the probiotic treatment when used in combination. In conclusion, yerba mate had antimicrobial activity against foodborne pathogens and enhanced the growth of LAB in vitro, but in vivo yerba mate did not decrease Salmonella Enteritidis colonization.

Mesenchymal Stem Cell Transplantation in Multiple Sclerosis

PubMed Central

Cohen, Jeffrey A.

2013-01-01

Mesenchymal stem cells (MSCs) are a pluripotent non-hematopoietic precursor cells that can be isolated from bone marrow and numerous other tissues, culture-expanded to purity, and induced to differentiate in vitro and in vivo into mesodermal derivatives. MSCs exhibit many phenotypic and functional similarities to pericytes. The immunomodulatory, tissue protective, and repair-promoting properties of MSCs demonstrated both in vitro and in animal models make them an attractive potential therapy for MS and other conditions characterized by inflammation and/or tissue injury. Other potential advantages of MSCs as a therapeutic include the relative ease of culture expansion, relative immunoprivilege allowing allogeneic transplantation, and their ability to traffic from blood to areas of tissue allowing intravascular administration. The overall published experience with MSC transplantation in MS is modest, but several small case series and preliminary studies yielded promising results. Several groups, including us, recently initiated formal studies of autologous, culture-expanded, bone-marrow-derived MSC transplantation in MS. Although there are several potential safety concerns, to date, the procedure has been well tolerated. Future studies that more definitively assess efficacy also will need to address several technical issues. PMID:23294498

Arsenic compounds as anticancer agents.

PubMed

Wang, Z Y

2001-08-01

In this paper the use of arsenic compounds as anticancer agents in clinical trials and in in vitro investigations is reviewed, including the experience at our institute. Treatment of newly diagnosed and relapsed patients with acute promyelocytic leukemia (APL) with arsenic trioxide (As2O3) has been found to result in complete remission (CR) rates of 85-93% when given by intravenous infusion for 2-3 h at a dose of 10 mg/day diluted in 5% glucose saline solution. Patients exhibit a response in 28-42 days. CR rates after administration of Composite Indigo Naturalis tablets containing arsenic sulfide and of pure tetraarsenic tetrasulfide reached 98% and 84.9%, respectively. At higher concentrations (1-2 microM), arsenic induced apoptosis, while at lower concentrations (0.1-0.5 microM), it triggered cell differentiation in vitro. As2O3-induced apoptosis has been observed in many cancer cell lines, including esophageal carcinoma, gastric cancer, neuroblastoma, lymphoid malignancies, and multiple myeloma. Its effectiveness was confirmed in the treatment of multiple myeloma. Arsenic compounds are effective agents in the treatment of APL and their activity against other types of cancer requires further investigation.

The influence of micronutrients in cell culture: a reflection on viability and genomic stability.

PubMed

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Machado, Miriana; Bordin, Diana Lilian; Bergter, Lothar; Prá, Daniel; Henriques, João Antonio Pêgas

2013-01-01

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

PubMed Central

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

2013-01-01

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Remineralization Potential of Three Tooth Pastes on Enamel Caries.

PubMed

Singhal, Rajnish K; Rai, Balwant

2017-08-15

Different formulations of dentifrices exist in the market. Usually, single toothpaste is used by all family members including children. There is a big concern of fluoride ingestion with the toothpaste containing high fluoride content in children. Recently, new toothpaste (including toothpaste) with remineralization potential without fluoride content has been formulated. There is an urgent need to compare remineralization potential of this new formulation with the exiting dentifrices. Therefore, the present study has been undertaken to assess and compare the remineralization potential of three dentifrices with different compositions on artificially induced carious lesions in vitro by using scanning electron microscopy and polarised light microscopy. The present in vitro study was conducted on 21 healthy extracted primary central incisor teeth surfaces, which were divided into three groups and were treated by three different dentifrices. Artificial demineralization was followed by remineralization using dentifrice slurry as per the group distribution. All the samples were studied for remineralization by using scanning electron microscopy and polarised light microscopy. Data were analysed using SPSS version 11 software. A significant difference was found between the remineralization potential of incudent toothpaste and other toothpaste groups based on the analysis of polarised light microscopy and stereomicroscope. The remineralizing ability of incudent toothpaste for artificial enamel lesions was found to be significantly higher than that of Colgate® and Crest toothpaste. The limitations of this study include, being a short term study, low sample size and in vitro experiment. incudent toothpaste has exhibited a higher remineralizing potential as compared to fluoride based toothpaste in our study.

Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

PubMed Central

2014-01-01

Background Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity. Results We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827. Conclusions We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents. PMID:24886633

PRK by Er:YAG laser: in-vitro studies and first in-vivo experiences

NASA Astrophysics Data System (ADS)

Steiner, Rudolf W.; Leiacker, Richard; Russ, Detlef; Seiler, Theo

1996-01-01

Photorefractive keratectomy (PRK) is usually performed by an excimer laser at 193 nm wavelength. Ablatio of corneal tissue is, however, not only possible in the UV region of the optical spectrum but also in the IR where water is an excellent absorber. Therefore, an Er:YAG laser was used at 2.94 micrometer wavelength as an alternative laser light source to perform in vitro studies of corneal ablation and also first clinical experiments to correct myopia of patients with blind eyes.

Skylab

NASA Image and Video Library

1973-01-01

This chart describes the Skylab student experiment In-Vitro Immunology, proposed by Todd A. Meister of Jackson Heights, New York. He suggested an in-vitro observation of the effects of zero-gravity on a presipitin-type antigen-antibody reaction, as compared with the same reaction carried out in an Earth-based laboratory. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

Arctigenin inhibits prostate tumor cell growth in vitro and in vivo

PubMed Central

Wang, Piwen; Solorzano, Walter; Diaz, Tanya; Magyar, Clara E.; Henning, Susanne M.; Vadgama, Jaydutt V.

2017-01-01

The low bioavailability of most phytochemicals limits their translation to humans. We investigated whether arctigenin, a novel anti-inflammatory lignan from the seeds of Arctium lappa, has favorable bioavailability/potency against prostate cancer. The anticarcinogenic activity of arctigenin was investigated both in vitro using the androgen-sensitive LNCaP and LAPC-4 human prostate cancer cells and pre-malignant WPE1-NA22 cells, and in vivo using xenograft mouse models. Arctigenin at lower doses (< 2μM) significantly inhibited the proliferation of LNCaP and LAPC-4 cells by 30-50% at 48h compared to control, and inhibited WPE1-NA22 cells by 75%, while did not affect normal prostate epithelial cells. Male severe combined immunodeficiency (SCID) mice were implanted subcutaneously with LAPC-4 cells for in vivo studies. In one experiment, the intervention started one week after tumor implantation. Mice received arctigenin at 50mg/kg (LD) or 100mg/kg (HD) b.w. daily or vehicle control by oral gavage. After 6 weeks, tumor growth was inhibited by 50% (LD) and 70% (HD) compared to control. A stronger tumor inhibitory effect was observed in a second experiment where arctigenin intervention started two weeks prior to tumor implantation. Arc was detectable in blood and tumors in Arc groups, with a mean value up to 2.0 μM in blood, and 8.3 nmol/g tissue in tumors. Tumor levels of proliferation marker Ki67, total and nuclear androgen receptor, and growth factors including VEGF, EGF, and FGF-β were significantly decreased by Arc, along with an increase in apoptosis marker of Bax/Bcl-2 ratio. Genes responsive to arctigenin were identified including TIMP3 and ZNF185, and microRNAs including miR-126-5p, and miR-21-5p. This study provides the first in vivo evidence of the strong anticancer activity of arctigenin in prostate cancer. The effective dose of arctigenin in vitro is physiologically achievable in vivo, which provides a high promise in its translation to human application. PMID:29062885

Arctigenin inhibits prostate tumor cell growth in vitro and in vivo.

PubMed

Wang, Piwen; Solorzano, Walter; Diaz, Tanya; Magyar, Clara E; Henning, Susanne M; Vadgama, Jaydutt V

2017-06-01

The low bioavailability of most phytochemicals limits their translation to humans. We investigated whether arctigenin, a novel anti-inflammatory lignan from the seeds of Arctium lappa , has favorable bioavailability/potency against prostate cancer. The anticarcinogenic activity of arctigenin was investigated both in vitro using the androgen-sensitive LNCaP and LAPC-4 human prostate cancer cells and pre-malignant WPE1-NA22 cells, and in vivo using xenograft mouse models. Arctigenin at lower doses (< 2μM) significantly inhibited the proliferation of LNCaP and LAPC-4 cells by 30-50% at 48h compared to control, and inhibited WPE1-NA22 cells by 75%, while did not affect normal prostate epithelial cells. Male severe combined immunodeficiency (SCID) mice were implanted subcutaneously with LAPC-4 cells for in vivo studies. In one experiment, the intervention started one week after tumor implantation. Mice received arctigenin at 50mg/kg (LD) or 100mg/kg (HD) b.w. daily or vehicle control by oral gavage. After 6 weeks, tumor growth was inhibited by 50% (LD) and 70% (HD) compared to control. A stronger tumor inhibitory effect was observed in a second experiment where arctigenin intervention started two weeks prior to tumor implantation. Arc was detectable in blood and tumors in Arc groups, with a mean value up to 2.0 μM in blood, and 8.3 nmol/g tissue in tumors. Tumor levels of proliferation marker Ki67, total and nuclear androgen receptor, and growth factors including VEGF, EGF, and FGF-β were significantly decreased by Arc, along with an increase in apoptosis marker of Bax/Bcl-2 ratio. Genes responsive to arctigenin were identified including TIMP3 and ZNF185, and microRNAs including miR-126-5p, and miR-21-5p. This study provides the first in vivo evidence of the strong anticancer activity of arctigenin in prostate cancer. The effective dose of arctigenin in vitro is physiologically achievable in vivo , which provides a high promise in its translation to human application.

IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA

EPA Science Inventory

A colorimetric microbicidal assay was adapted, optimized and applied in experiments to characterize the in vitro capacity of eastern oyster (Crassostrea virginica) hemocytes to kill cultured isolates of Perkinsus marinus, a protozoan parasite causing a highly destructive disease...

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis.

PubMed

Benelli, Carla; De Carlo, Anna; Engelmann, Florent

2013-01-01

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at -5 °C, and then cooled slowly to -30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants. Copyright © 2012 Elsevier Inc. All rights reserved.

Silver nanoparticles-quercetin conjugation to siRNA against drug-resistant Bacillus subtilis for effective gene silencing: in vitro and in vivo.

PubMed

Sun, Dongdong; Zhang, Weiwei; Li, Nuan; Zhao, Zhiwei; Mou, Zhipeng; Yang, Endong; Wang, Weiyun

2016-06-01

Quercetin (Qe) exhibited extremely low water solubility, and thus, it was modified using silver nanoparticles (AgNPs). We fabricated AgNPs combined with Qe (AgNPs-Qe). The modification suggested that the synergistic properties of Qe enhanced the antibacterial activity of AgNPs. However, AgNPs-Qe exerted no effect on many kinds of drug-resistant bacteria, including Pseudomonas aeruginosa and Bacillus subtilis. RNA interference has considerable therapeutic potential because of its high specificity and potential capability to evade drug resistance. Therefore, we stabilized AgNPs-Qe with a layer of molecules (siRNA). The newly fabricated nanoparticles exerted improved effect on many kinds of bacteria, including the most prominent drug-resistant species B. subtilis. Agarose gel electrophoresis showed that the highest critical nitrogen-to-phosphorus (N/P) ratio occurred at a vector/siRNA with a w/w ratio of 7:1. Characterization experiment indicated that the diameter of siRNA/AgNPs-Qe was approximately 40 nm (40 ± 10 nm). Moreover, AgNPs-Qe were stabilized with a layer of siRNA that was approximately 10nm thick. Results of the in vitro study suggested that siRNA/AgNPs-Qe could destroy the cell wall and inhibit bacterial propagation. Meanwhile, the in vivo experiment on the animal bacteremia model, as well as the optical imaging of nude mice and their isolated organs, demonstrated that bacteria accumulated in the blood, heart, liver, spleen, lungs, and kidneys after the intravenous injection of B. subtilis. The bacteria in the blood and organs, as well as the inflamed cells in the tissues, gradually decreased after the mice received intravenous tail injection of siRNA/AgNPs-Qe for treatment. Both the in vitro and the in vivo studies exhibit that siRNA/AgNPs-Qe can be a potential nanoscale drug delivery system for B. subtilis targeting bacterimia. Copyright © 2016 Elsevier B.V. All rights reserved.

Biochemical Characterization of Fungus Isolated during In vitro Propagation of Bambusa balcooa.

PubMed

Tyagi, Bhawna; Tewari, Salil; Dubey, Ashutosh

2018-01-01

Bambusa balcooa ( Poaceae: Bambusoideae ) is a multipurpose bamboo species, which is native of the Indian subcontinent. B. balcooa is regarded as one of the best species for scaffolding and building purposes because of its strong culm. Other uses include paper pulp, handicrafts, and products of the wood chip industry. Due to these various uses in industries, this species has been identified as one of the priority bamboos by the National Bamboo Mission. This study is designed to analyze the identification of fungus and develop the strategy to eliminate the contamination during in vitro establishment of B. balcooa through nodal part. Fungus contamination is a problem which is encountered during in vitro establishment of B. balcooa cultures. In the present study, fungus contamination from in vitro cultured plant has been isolated and subjected to partial sequence analysis of the 18S rRNA gene to identify the fungus strain. Experiments were designed to develop a strategy for removal of the fungus contamination with the help of antifungal compounds and commercial antimicrobial supplement supplied by HiMedia. Fusarium equiseti was identified as endophytic fungus. It was observed that antimicrobial supplement at concentration of 500 μl/l was more effective concentration to remove fungus contamination and not showed any detrimental effect on growth parameters of shoot. This experiment would help in identification and to get rid of fungal contamination and improve the in vitro establishment of B. balcooa cultures for large-scale propagation. Endogenous fungus was isolated from contaminated culture of B. balcooa , and it was identified as Fusarium equiseti and submitted to NCBI under accession no. KP274872. The endophytic fungus had shown substantial production of amylase, cellulase, and protease media. Gibberellic acid (GA 3 ) production by F. equiseti was maximum on the 7 th day on inoculation. Abbreviations used: B. balcooa : Bambusa balcooa , F. equiseti : Fusarium equiseti , PDA: Potato dextrose agar, PCR: Polymerase chain reaction, MS: Murashige and Skoog's, BAP: 6-Benzylaminopurine, ITS1/4: Internal transcribed spacer region 1/4, GA 3 : Gibberellic acid.

LOW PRESSURE ULTRAVEIOLET STUDIES FOR INACTIVATION OF GIARDIA MURIS CYSTS

EPA Science Inventory

Cysts of Giardia muris were inactivated using a low pressure ultravolet (UV) light source. Cyst viability was detemined by both in vitro excystation and animal infectivity. Cyst doeses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excy...

LOW PRESSURE ULTRAVIOLET STUDIES FOR INACTIVATION OF GIARDIA MURIS CYSTS

EPA Science Inventory

Cysts of Giardia muris were inactivated using a low pressure ultravolet (UV) light source. Cyst viability was detemined by both in vitro excystation and animal infectivity. Cyst doeses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excy...

Arsenic Metabolism by Human Gut Microbiota upon in Vitro Digestion of Contaminated Soils

EPA Science Inventory

Speciation analysis is essential when evaluating risks from, arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with animal microbiota. Howeve...

Experience of in vitro fertilization surrogacy in Finland.

PubMed

Söderström-Anttila, Viveca; Blomqvist, Tom; Foudila, Tuija; Hippeläinen, Maritta; Kurunmäki, Henri; Siegberg, Rita; Tulppala, Maija; Tuomi-Nikula, Merja; Vilska, Sirpa; Hovatta, Outi

2002-08-01

In vitro fertilization (IVF) surrogacy makes it possible for women who do not have a functional uterus to have their own genetic offspring. We describe here our experience of IVF surrogacy in Finland over a 10-year period. This retrospective study included 17 women who underwent ovarian stimulation in connection with surrogacy in 1991-2001 at four clinics. The surrogate mothers were unpaid volunteers: six sisters, three mothers, one husband's sister, one cousin, four friends and three other volunteers. Thorough counseling was given to the commissioning couples and to the surrogate mothers and their partners. The commissioning couples were prepared to adopt their biological children. Twenty-eight surrogate IVF cycles were started in 17 women. One couple received donated oocytes. Trans-vaginal oocyte retrieval was feasible in every case, including those five women with congenital absence of the vagina and uterus. An average of 1.8 embryos was transferred at a time, and 11 pregnancies were achieved [50% per fresh embryo transfer (ET) and 16% per frozen-thawed ET]. Nine healthy singletons and one set of twins were born. One pregnancy ended in miscarriage. The mean birth weight of singleton infants was 3498 g (2270-4650 g). The birth weights of the twins were 2900 and 2400 g. In all cases the genetic parents took care of the infant immediately after birth. Two surrogate mothers had postpartum depression. Altruistic IVF surrogacy works well, but careful counseling of all parties involved is essential.

Evolution of the Pseudomonas aeruginosa Aminoglycoside Mutational Resistome In Vitro and in the Cystic Fibrosis Setting.

PubMed

López-Causapé, Carla; Rubio, Rosa; Cabot, Gabriel; Oliver, Antonio

2018-04-01

Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments. Copyright © 2018 American Society for Microbiology.

Evidence for a dopamine intrinsic direct role in the regulation of the ovary reproductive function: in vitro study on rabbit corpora lutea.

PubMed

Parillo, Francesco; Maranesi, Margherita; Mignini, Fiorenzo; Marinelli, Lisa; Di Stefano, Antonio; Boiti, Cristiano; Zerani, Massimo

2014-01-01

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

Computational design and in vitro characterization of an integrated maglev pump-oxygenator.

PubMed

Zhang, Juntao; Taskin, M Ertan; Koert, Andrew; Zhang, Tao; Gellman, Barry; Dasse, Kurt A; Gilbert, Richard J; Griffith, Bartley P; Wu, Zhongjun J

2009-10-01

For the need for respiratory support for patients with acute or chronic lung diseases to be addressed, a novel integrated maglev pump-oxygenator (IMPO) is being developed as a respiratory assist device. IMPO was conceptualized to combine a magnetically levitated pump/rotor with uniquely configured hollow fiber membranes to create an assembly-free, ultracompact system. IMPO is a self-contained blood pump and oxygenator assembly to enable rapid deployment for patients requiring respiratory support or circulatory support. In this study, computational fluid dynamics (CFD) and computer-aided design were conducted to design and optimize the hemodynamics, gas transfer, and hemocompatibility performances of this novel device. In parallel, in vitro experiments including hydrodynamic, gas transfer, and hemolysis measurements were conducted to evaluate the performance of IMPO. Computational results from CFD analysis were compared with experimental data collected from in vitro evaluation of the IMPO. The CFD simulation demonstrated a well-behaved and streamlined flow field in the main components of this device. The results of hydrodynamic performance, oxygen transfer, and hemolysis predicted by computational simulation, along with the in vitro experimental data, indicate that this pump-lung device can provide the total respiratory need of an adult with lung failure, with a low hemolysis rate at the targeted operating condition. These detailed CFD designs and analyses can provide valuable guidance for further optimization of this IMPO for long-term use.

Impact of ketorolac administration around ovarian stimulation on in vivo and in vitro fertilization and subsequent embryo development.

PubMed

Jee, Byung Chul; Youm, Hye Won; Lee, Jae Ho; Kim, Jee Hyun; Suh, Chang Suk; Kim, Seok Hyun

2013-05-01

We performed this study to investigate the effect of ketorolac (a non-steroidal anti-inflammatory drug) administration around ovarian stimulation on in vivo and in vitro fertilization process. Sixty-four female mice (ICR) were injected with ketorolac (0, 7.5, 15 and 30 µg/d) for 3 d starting from the day of eCG treatment. In experiment 1, 41 mice were triggered by hCG and then mated; two-cell embryos were obtained and in vitro development up to blastocyst was observed. In experiment 2, 23 mice were triggered by hCG and mature oocytes were collected; in vitro fertilization rate and subsequent embryo development up to blastocyst was recorded. In experiment 1, the blastocyst-forming rates per in vivo fertilized two-cell embryo showed an inverse relationship with a dosage of ketorolac (97.6%, 64.2%, 35.4% and 25.9%). In experiment 2, degenerated oocytes were frequently observed in a dose-dependent manner (4.3%, 22.9%, 22.4% and 75.0%). Lower fertilization rates were noted in all the three ketorolac-treating groups; blastocyst-forming rate was significantly lower in 30-µg-treating group when compared with the control group. Administration of ketorolac around ovarian stimulation significantly affects the development of in vivo fertilized embryo in a dose-dependent manner. High-dose ketorolac could result in a poor oocyte quality and decreased embryo developmental competence.

Evaluation of sources of variation on in vitro fermentation kinetics of feedstuffs in a gas production system.

PubMed

Keim, Juan P; Alvarado-Gilis, Christian; Arias, Rodrigo A; Gandarillas, Mónica; Cabanilla, Jaime

2017-10-01

The aim of this study was to evaluate the effect of different sources of variation in gas production technique on the in vitro gas production kinetics of feedstuffs. Triplicates of commercial concentrate, grass silage, grass hay and grass pasture were incubated in three experiments: experiment 1 assessed two agitation methods; experiment 2 evaluated different rumen inocula (pooled or different donor cows for each incubation run); and experiment 3 used Goering-Van Soest or Mould buffers for media preparation. Gas production data were fitted into the Michaelis-Menten model and then subjected to analysis of variance. Gas production (GP) at 48 h and asymptote gas production (A) were lower when bottles were continuously under horizontal movement. Time to produce half and 75% of A, and A were affected by rumen inocula, while buffer type affected time to produce half and 25% of A and GP. No interactions between substrates and sources of variation were observed, suggesting that the effects of substrates on GP parameters were not modified. It is concluded that comparison of numerical data from in vitro experiments that follow different protocols must be done carefully. However, the ranking of different substrates is more robust and less affected by the sources of variation. © 2017 Japanese Society of Animal Science.

Comparison of two different in vivo models and an in vitro model for caloric determination of four novel fiber ingredients.

PubMed

Cervantes-Pahm, Sarah; Knapp, Brenda K; Kim, Beob G; Liu, Yanhong; Parsons, Carl M; Fahey, George C; Stein, Hans H

2013-12-18

The objective of this study was to compare two in vivo methods using pigs and roosters and an in vitro method for determining the caloric value of four fiber sources [i.e., two resistant starches (RS 60 and RS 75), soluble corn fiber (SCF 70), and pullulan]. Metabolizable energy (ME) in pigs and true metabolizable energy (TMEn) in roosters were determined by using 72 barrows and 24 roosters, respectively. A two-step in vitro procedure was used to quantify monosaccharides released. Results of the two in vivo experiments corresponded well with RS 75 having the least caloric value (7.55 MJ/kg in pigs; 6.19 MJ/kg in roosters) and pullulan having the greatest caloric value (12.21 MJ/kg in pigs; 13.94 MJ/kg in roosters). The caloric values for all the fiber ingredients were less (P < 0.05) than in MD both in pigs and in roosters. Despite some limitations, results of the in vitro procedure corresponded well with the in vivo experiments where the concentration of glucose hydrolyzed from RS 60, RS 75, and SCF 70, but not pullulan, was less (P < 0.05) than the concentration of glucose hydrolyzed from MD. However, the greatest accuracy was obtained in the in vivo experiments.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos.

PubMed

Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard; Callesen, Henrik

2015-06-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

Myocardial temperature distribution under cw Nd:YAG laser irradiation in in vitro and in vivo situations: theory and experiment

NASA Astrophysics Data System (ADS)

Splinter, Robert; Littmann, Laszlo; Tuntelder, Jan R.; Svenson, Robert H.; Chuang, Chi Hui; Tatsis, George P.; Semenov, Serguei Y.; Nanney, Glenn A.

1995-01-01

Tissue samples ranging from 2 to 16 mm in thickness were irradiated at 1064 nm with energies ranging from 40 to 2400 J. Coagulation lesions of in vitro and in vivo experiments were subjected to temperature profiling and submitted for histology. Irreversible damage was calculated with the damage integral formalism, following the bioheat equation solved with Monte Carlo computer light-distribution simula-tions. Numerical temperature rise and coagulation depth compared well with the in vitro results. The in vivo data required a change in the optical properties based on integrating sphere measurements for high irradiance to make the experimental and numerical data converge. The computer model has successfully solved several light-tissue interaction situations in which scattering dominates over absorption.

Activity of antibacterial, antiviral, anti-inflammatory in compounds andrographolide salt.

PubMed

Wen, Li; Xia, Nan; Chen, Xianghong; Li, Yingxiu; Hong, Yi; Liu, YaJie; Wang, ZiZhen; Liu, YaJie

2014-10-05

Andrographolide sulfonic acid sodium salt (ASS) was synthesized to increase the the solubility of Andrographolide in aqueous solution. We have studied its pharmacological effect of antibiosis, anti-inflammatory and immunoregulation. Cylinder-plate method was used to study ASS׳s in vitro antibacterial activity, and its protection for mice infected by Staphylococcus aureus and Shigella dysenteriae. Various inflammation models, including the auricular edema induced by xylene in mice, CMC-Na induced air pounch model and the paw edema induced by albumen in rats were used to explore the characteristic of ASS׳s anti-inflammation effect. We built up the immune model by injecting chicken red cells in enter celiac of mice and study the effect of ASS on immunoregulation, taking andrographolide as the positive control. bacteriostasis in vivo and in vitro experiments show that ASS has a weak antibacterial effect and no bactericidal effect, but can reduce the mice mortality of Staphylococcus aureus infected. Anti-inflammatory experiments show that ASS can reduce the mouse ear swelling induced by xylene and rat paw swelling induced by egg albumin, and lessen leukocytes in air bag caused by CMCNa, and lower IL1 not ably in rat serum. Immune tests indicate that ASS can get spleen and thymus gain weight and increase rate of abdominal macrophage phagocytosis of mice. The result of bacteriostasis shows that ASS has weak in vitro antibacterial effect. ASS shows significant effects of anti-inflammation and improving immunity, thus enables the mice against bacteria better. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation.

PubMed

Migheli, Rossana; Puggioni, Giulia; Dedola, Sonia; Rocchitta, Gaia; Calia, Giammario; Bazzu, Gianfranco; Esposito, Giovanni; Lowry, John P; O'Neill, Robert D; Desole, M S; Miele, Egidio; Serra, Pier A

2008-09-15

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous "treatment-control" experimental design was performed to detect K+-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC-EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment-control experiments performed on the same initial PC12 cell population.

Arsenic Metabolism by Human Gut Microbiota upon In Vitro Digestion of Contaminated Soils

EPA Science Inventory

Background: Speciation analysis is essential when evaluating risks from arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with ...

Silver-loaded nanotubular structures enhanced bactericidal efficiency of antibiotics with synergistic effect in vitro and in vivo.

PubMed

Xu, Na; Cheng, Hao; Xu, Jiangwen; Li, Feng; Gao, Biao; Li, Zi; Gao, Chenghao; Huo, Kaifu; Fu, Jijiang; Xiong, Wei

2017-01-01

Antibiotic-resistant bacteria have become a major issue due to the long-term use and abuse of antibiotics in treatments in clinics. The combination therapy of antibiotics and silver (Ag) nanoparticles is an effective way of both enhancing the antibacterial effect and decreasing the usage of antibiotics. Although the method has been proved to be effective in vitro, no in vivo tests have been carried out at present. Herein, we described a combination therapy of local delivery of Ag and systemic antibiotics treatment in vitro in an infection model of rat. Ag nanoparticle-loaded TiO 2 nanotube (NT) arrays (Ag-NTs) were fabricated on titanium implants for a customized release of Ag ion. The antibacterial properties of silver combined with antibiotics vancomycin, rifampin, gentamicin, and levofloxacin, respectively, were tested in vitro by minimum inhibitory concentration (MIC) assay, disk diffusion assay, and antibiofilm formation test. Enhanced antibacterial activity of combination therapy was observed for all the chosen bacterial strains, including gram-negative Escherichia coli (ATCC 25922), gram-positive Staphylococcus aureus (ATCC 25923), and methicillin-resistant Staphylococcus aureus (MRSA; ATCC 33591 and ATCC 43300). Moreover, after a relative short (3 weeks) combinational treatment, animal experiments in vivo further proved the synergistic antibacterial effect by X-ray and histological and immunohistochemical analyses. These results demonstrated that the combination of Ag nanoparticles and antibiotics significantly enhanced the antibacterial effect both in vitro and in vivo through the synergistic effect. The strategy is promising for clinical application to reduce the usage of antibiotics and shorten the administration time of implant-associated infection.

Modelling iron mismanagement in neurodegenerative disease in vitro: paradigms, pitfalls, possibilities & practical considerations.

PubMed

Healy, Sinead; McMahon, Jill M; FitzGerald, Una

2017-11-01

Although aberrant metabolism and deposition of iron has been associated with aging and neurodegeneration, the contribution of iron to neuropathology is unclear. Well-designed model systems that are suited to studying the putative pathological effect of iron are likely to be essential if such unresolved details are to be clarified. In this review, we have evaluated the utility and effectiveness of the reductionist in vitro platform to study the molecular mechanisms putatively underlying iron perturbations of neurodegenerative disease. The expression and function of iron metabolism proteins in glia and neurons and the extent to which this iron regulatory system is replicated in in vitro models has been comprehensively described, followed by an appraisal of the inherent suitability of different in vitro and ex vivo models that have been, or might be, used for iron loading. Next, we have identified and critiqued the relevant experimental parameters that have been used in in vitro iron loading experiments, including the choice of iron reagent, relevant iron loading concentrations and supplementation with serum or ascorbate, and propose optimal iron loading conditions. Finally, we have provided a synthesis of the differential iron accumulation and toxicity in glia and neurons from reported iron loading paradigms. In summary, this review has amalgamated the findings and paradigms of the published reports modelling iron loading in monocultures, discussed the limitations and discrepancies of such work to critically propose a robust, relevant and reliable model of iron loading to be used for future investigations. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro fermentation characteristics, in vivo ileal and total tract nutrient digestibilities, and fecal microbiota responses of dogs to α-cyclodextrin.

PubMed

Guevara, M A; Bauer, L L; Garleb, K A; Fahey, G C; de Godoy, M R C

2016-05-01

The objectives were to examine in vitro fermentation characteristics, in vivo nutrient digestibility, fecal microbiota, and serum lipid profiles as affected by α-cyclodextrin (ACD) supplementation. Short-chain fatty acid (SCFA) production was measured after in vitro fermentation for 3, 6, 9, and 12 h of ACD, β-cyclodextrin, and γ-cyclodextrin. Five mixed-breed hounds were used in a Latin square design. Each experimental period comprised 14 d, including 10 d for diet adaptation and 4 d for fecal collection. Dogs were fed, twice a day, an extruded diet made with poultry byproduct meal and brewer's rice as the main ingredients. Dogs were supplemented with 0, 1, 2, 3, or 4 g of ACD diluted in 15 mL of water twice daily for a total of 0, 2, 4, 6, and 8 g ACD/d. Maximal in vitro production of total SCFA was lowest for ACD. However, the greatest maximal production of propionate was noted for ACD treatment. Total tract nutrient digestibility and fecal DM concentration linearly decreased ( < 0.05) for treatment groups receiving ACD; no changes were observed for ileal digestibility. Serum cholesterol and triglyceride concentrations were within normal ranges for dogs and were not different among treatments. Similarly, no changes in fecal microbiota were observed. Overall, ACD supplementation appears to have no effect on nutrient absorption in the small intestine but may alter fermentation in the large bowel, which could lead to a higher proportion of propionate production as observed in the in vitro experiment.

Wireless energy transfer platform for medical sensors and implantable devices.

PubMed

Zhang, Fei; Hackworth, Steven A; Liu, Xiaoyu; Chen, Haiyan; Sclabassi, Robert J; Sun, Mingui

2009-01-01

Witricity is a newly developed technique for wireless energy transfer. This paper presents a frequency adjustable witricity system to power medical sensors and implantable devices. New witricity resonators are designed for both energy transmission and reception. A prototype platform is described, including an RF power source, two resonators with new structures, and inductively coupled input and output stages. In vitro experiments, both in open air and using a human head phantom consisting of simulated tissues, are employed to verify the feasibility of this platform. An animal model is utilized to evaluate in vivo energy transfer within the body of a laboratory pig. Our experiments indicate that witricity is an effective new tool for providing a variety of medical sensors and devices with power.

In vitro physical stimulation of tissue-engineered and native cartilage.

PubMed

Li, Kelvin W; Klein, Travis J; Chawla, Kanika; Nugent, Gayle E; Bae, Won C; Sah, Robert L

2004-01-01

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

In vitro biological outcome of laser application for modification or processing of titanium dental implants.

PubMed

Hindy, Ahmed; Farahmand, Farzam; Tabatabaei, Fahimeh Sadat

2017-07-01

There are numerous functions for laser in modern implant dentistry including surface treatment, surface coating, and implant manufacturing. As laser application may potentially improve osseointegration of dental implants, we systematically reviewed the literature for in vitro biological responses to laser-modified or processed titanium dental implants. The literature was searched in PubMed, ISI Web, and Scopus, using keywords "titanium dental implants," "laser," "biocompatibility," and their synonyms. After screening the 136 references obtained, 28 articles met the inclusion criteria. We found that Nd:YAG laser was the most commonly used lasers in the treatment or processing of titanium dental implants. Most of the experiments used cell attachment and cell proliferation to investigate bioresponses of the implants. The most commonly used cells in these assays were osteoblast-like cells. Only one study was conducted in stem cells. These in vitro studies reported higher biocompatibility in laser-modified titanium implants. It seems that laser radiation plays a vital role in cell response to dental implants; however, it is necessary to accomplish more studies using different laser types and parameters on various cells to offer a more conclusive result.

In vitro blood and fibroblast responses to BisGMA-TEGDMA/bioactive glass composite implants.

PubMed

Abdulmajeed, Aous A; Kokkari, Anne K; Käpylä, Jarmo; Massera, Jonathan; Hupa, Leena; Vallittu, Pekka K; Närhi, Timo O

2014-01-01

This in vitro study was designed to evaluate both blood and human gingival fibroblast responses to bisphenol A-glycidyl methacrylate-triethyleneglycol dimethacrylate (BisGMA-TEGDMA)/bioactive glass (BAG) composite, aimed to be used as composite implant abutment surface modifier. Three different types of substrates were investigated: (a) plain polymer (BisGMA 50 wt%-TEGDMA 50 wt%), (b) BAG-composite (50 wt% polymer + 50 wt% fraction of BAG-particles, <50 μm), and (c) plain BAG plates (100 wt% BAG). The blood response, including the blood-clotting ability and platelet adhesion morphology were evaluated. Human gingival fibroblasts were plated and cultured on the experimental substrates for up to 10 days, then the cell proliferation rate was assessed using AlamarBlue assay™. The BAG-composite and plain BAG substrates had a shorter clotting time than plain polymer substrates. Platelet activation and aggregation were most extensive, qualitatively, on BAG-composite. Analysis of the normalized cell proliferation rate on the different surfaces showed some variations throughout the experiment, however, by day 10 the BAG-composite substrate showed the highest (P < 0.001) cell proliferation rate. In conclusion, the presence of exposed BAG-particles enhances fibroblast and blood responses on composite surfaces in vitro.

Photodynamic Therapy Combined with Terbinafine Against Chromoblastomycosis and the Effect of PDT on Fonsecaea monophora In Vitro

PubMed Central

Hu, Yongxuan; Huang, Xiaowen; Lu, Sha; Hamblin, Michael R.; Mylonakis, Eleftherios; Zhang, Junmin

2014-01-01

Chromoblastomycosis, a chronic fungal infection of skin and subcutaneous tissue caused by dematiaceous fungi, is associated with low cure and high relapse rates. Among all factors affecting clinical outcome, etiological agents have an important position. In southern China, Fonsecaea pedrosoi and Fonsecaea monophora are main causative agents causing Chromoblastomycosis. We treated one case of chromoblastomycosis by photodynamic therapy (PDT) of 5-aminolevulinic acid (ALA) irradiation combined with terbinafine 250 mg a day. The lesions were improved after two sessions of ALA-PDT treatment, each including nine times, at an interval of 1 week, combined with terbinafine 250 mg/day oral, and clinical improvement could be observed. In the following study, based on the clinical treatment, the effect of PDT and antifungal drugs on this isolate was detected in vitro. It showed sensitivity to terbinafine, itraconazole or voriconazole, and PDT inhibited the growth. Both the clinic and experiments in vitro confirm the good outcome of ALA-PDT applied in the inhibition of F. monophora. It demonstrated that combination of antifungal drugs with ALA-PDT arises as a promising alternative method for the treatment of these refractory cases of chromoblastomycosis. PMID:25366276

Magnesium degradation influenced by buffering salts in concentrations typical of in vitro and in vivo models.

PubMed

Agha, Nezha Ahmad; Feyerabend, Frank; Mihailova, Boriana; Heidrich, Stefanie; Bismayer, Ulrich; Willumeit-Römer, Regine

2016-01-01

Magnesium and its alloys have considerable potential for orthopedic applications. During the degradation process the interface between material and tissue is continuously changing. Moreover, too fast or uncontrolled degradation is detrimental for the outcome in vivo. Therefore in vitro setups utilizing physiological conditions are promising for the material/degradation analysis prior to animal experiments. The aim of this study is to elucidate the influence of inorganic salts contributing to the blood buffering capacity on degradation. Extruded pure magnesium samples were immersed under cell culture conditions for 3 and 10 days. Hank's balanced salt solution without calcium and magnesium (HBSS) plus 10% of fetal bovine serum (FBS) was used as the basic immersion medium. Additionally, different inorganic salts were added with respect to concentration in Dulbecco's modified Eagle's medium (DMEM, in vitro model) and human plasma (in vivo model) to form 12 different immersion media. Influences on the surrounding environment were observed by measuring pH and osmolality. The degradation interface was analyzed by electron-induced X-ray emission (EIXE) spectroscopy, including chemical-element mappings and electron microprobe analysis, as well as Fourier transform infrared reflection micro-spectroscopy (FTIR). Copyright © 2015 Elsevier B.V. All rights reserved.

Ultrasound assisted extraction of polysaccharides from Lentinus edodes and its anti-hepatitis B activity in vitro.

PubMed

Zhao, Yong-Ming; Yang, Jian-Ming; Liu, Ying-Hui; Zhao, Ming; Wang, Jin

2018-02-01

The aim of this study was to optimize the extraction process of polysaccharides from the fruiting bodies of Lentinus edodes and investigate its anti-hepatitis B virus activity. The extracting parameters including ultrasonic power (240-320W), extraction temperature (40-60°C) and extraction time (15-25min) was optimized by using three-variable-three-level Box-Behnken design based on the single-factor experiments. Data analysis results showed that the optimal conditions for extracting LEPs were an extraction temperature of 45°C, extraction time of 21min and ultrasonic power of 290W. Under these optimal conditions, the experimental yield of LEPs was 9.75%, a 1.62-fold increase compared with conventional heat water extraction (HWE). In addition, crude polysaccharides were purified to obtain two fractions (LEP-1 and LEP-2). Chemical analysis showed that these components were rich in glucose, arabinose and mannose. Furthermore, HepG2.2.15 cells were used as in vitro models to evaluate their anti-hepatitis B virus (HBV) activity. The results suggest that LEPs possesses potent anti-HBV activity in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

A finite element method model to simulate laser interstitial thermo therapy in anatomical inhomogeneous regions

PubMed Central

Mohammed, Yassene; Verhey, Janko F

2005-01-01

Background Laser Interstitial ThermoTherapy (LITT) is a well established surgical method. The use of LITT is so far limited to homogeneous tissues, e.g. the liver. One of the reasons is the limited capability of existing treatment planning models to calculate accurately the damage zone. The treatment planning in inhomogeneous tissues, especially of regions near main vessels, poses still a challenge. In order to extend the application of LITT to a wider range of anatomical regions new simulation methods are needed. The model described with this article enables efficient simulation for predicting damaged tissue as a basis for a future laser-surgical planning system. Previously we described the dependency of the model on geometry. With the presented paper including two video files we focus on the methodological, physical and mathematical background of the model. Methods In contrast to previous simulation attempts, our model is based on finite element method (FEM). We propose the use of LITT, in sensitive areas such as the neck region to treat tumours in lymph node with dimensions of 0.5 cm – 2 cm in diameter near the carotid artery. Our model is based on calculations describing the light distribution using the diffusion approximation of the transport theory; the temperature rise using the bioheat equation, including the effect of microperfusion in tissue to determine the extent of thermal damage; and the dependency of thermal and optical properties on the temperature and the injury. Injury is estimated using a damage integral. To check our model we performed a first in vitro experiment on porcine muscle tissue. Results We performed the derivation of the geometry from 3D ultrasound data and show for this proposed geometry the energy distribution, the heat elevation, and the damage zone. Further on, we perform a comparison with the in-vitro experiment. The calculation shows an error of 5% in the x-axis parallel to the blood vessel. Conclusions The FEM technique proposed can overcome limitations of other methods and enables an efficient simulation for predicting the damage zone induced using LITT. Our calculations show clearly that major vessels would not be damaged. The area/volume of the damaged zone calculated from both simulation and in-vitro experiment fits well and the deviation is small. One of the main reasons for the deviation is the lack of accurate values of the tissue optical properties. In further experiments this needs to be validated. PMID:15631630

[Effect of vitamins B1, B2, B6, folic acid and vitamin C on the motor activity of chicken's intestines in chronic experiments and in vitro].

PubMed

Nagórna-Stasiak, B; Wawrzeńska, M

1987-01-01

The studies were carried out on 33 chickens of the broiler breed in chronic experiments and in vitro. In the chronic experiments the motility of the jejunum under the influence of vitamins of group B and vitamine C was recorded in 8 chickens. The vitamins were used at concentrations from 10 mg/l to 2.5 x 10(3) mg/l. In the experiments in vitro, the motility of the isolated segment of the jejunum was recorded by the method of Magnus. In this part of experiments the chickens were divided into 3 groups, of which group I (15 chickens) were fed with DKA finischer mixture, group II (5 hens) received, besides the mixture, per os 200 mg of vitamin C for 2 weeks, group III (5 hens) received the mixture and for 2 weeks intraperitoneally 200 mg of vitamin C. The effect of vitamins of group B in vitro was determined in chickens of group I, whereas that of vitamin C in chickens of group I, II and III. At the same time the level of vitamin C in the wall of the jejunum was determined by the method of Roe-Kuenther. It was shown that vitamin B2 and folic acid caused stimulation of intestine motility in the chickens, while vitamin B1, B6 and C decreased the motoric activity. Increased level of vitamin C in the intestinal wall resulted in increased intestine sensitivity. Chicken intestines sensitivity to vitamins was 10 times stronger to vitamins than that of the intestines of rabbits.

Assessment of early combination effects of colistin and meropenem against Pseudomonas aeruginosa and Acinetobacter baumannii in dynamic time-kill experiments.

PubMed

Tängdén, Thomas; Karvanen, Matti; Friberg, Lena E; Odenholt, Inga; Cars, Otto

2017-07-01

In view of the paucity of clinical evidence, in vitro studies are needed to find antibiotic combinations effective against multidrug-resistant Gram-negative bacteria. Interpretation of in vitro effects is usually based on bacterial growth after 24 h in time-kill and checkerboard experiments. However, the clinical relevance of the effects observed in vitro is not established. In this study we explored alternative output parameters to assess the activities of colistin and meropenem against Pseudomonas aeruginosa and Acinetobacter baumannii. Four strains each of P. aeruginosa and A. baumannii were exposed to colistin and meropenem, alone and in combination, in 8 h dynamic time-kill experiments. Initial (1 h), maximum and 8 h bacterial reductions and the area under the bacterial time-kill curve were evaluated. Checkerboards, interpreted based on fractional inhibitory concentration indices after 24 h, were performed for comparison. In the time-kill experiments, the combination resulted in enhanced 1 h, maximum and 8 h bacterial reductions against 2, 3 and 5 of 8 strains, respectively, as compared to the single drugs. A statistically significant reduction in the area under the time-kill curve was observed for three strains. In contrast, the checkerboards did not identify synergy for any of the strains. Combination effects were frequently found with colistin and meropenem against P. aeruginosa and A. baumannii in time-kill experiments but were not detected with the checkerboard method. We propose that the early dynamics of bacterial killing and growth, which may be of great clinical importance, should be considered in future in vitro combination studies.

Montreal electronic artificial urinary sphincters: Our futuristic alternatives to the AMS800™.

PubMed

Biardeau, Xavier; Hached, Sami; Loutochin, Oleg; Campeau, Lysanne; Sawan, Mohamad; Corcos, Jacques

2017-10-01

We aimed to present three novel remotely controlled hydromechanical artificial urinary sphincters (AUSs) and report their in-vitro and ex-vivo results. We successively developed three distinct hydromechanical AUSs on the basis of the existing AMS800 ™ device by incorporating an electronic pump. No changes were made to the cuff and balloon. The AUS#1 was designed as an electromagnetically controlled device. The AUS#2 and AUS#3 were conceived as Bluetooth 2.1 remotely controlled and Bluetooth 4.0 remotely-controlled, adaptive devices, respectively. In-vitro experiments profiled occlusive cuff pressure (OCP) during a complete device cycle, with different predetermined OCP. Ex-vivo experiments were performed on a fresh pig bladder with 4 cm cuff placed around the urethra. Leak point pressure with different predetermined OCP values was successively measured during cystometry via a catheter at the bladder dome. Our in-vitro and ex-vivo experiments demonstrated that these three novel AUSs provided stable and predetermined OCP - within the physiological range - and completely deflated the cuff, when required, in a limited time compatible with physiological voiding cycles. Our three novel, remotely controlled AUSs showed promising results that should be confirmed by in-vivo experiments focusing on efficacy and safety.

[In vitro and ex vivo EPR investigation of metabolic changes in blood under the action of radiotoxins obtained from irradiated potato tubers].

PubMed

Ibragimova, M I; Petukhov, V Iu; Zheglov, E P; Koniukhov, G V; Nizamov, R N

2004-01-01

The effect of radiotoxin (RT) obtained from y-irradiated potato tubes on blood of sheep and mice has been investigated by using in vitro and ex vivo EPR. In experiments in vitro, the action of different preparations (RT, extract from unirradiated potato tubers, 1%-HCl or 30%-hydrogen peroxide) on sheep blood has been compared. It has been established that RT is an effective oxidant (like 1%-HCl) of haem iron that leads to an increase of the methemoglobin concentration. The specific peculiarity of RT effect on blood in vitro is an appearance of two well-resolved lines from methemoglobin belonging, probably, to different paramagnetic centers. The signal from nonspecific complexes of Fe3+ has been also observed. Ex vivo EPR spectra markedly differ from these obtained in experiments in vitro. An additional line with g approximately 2.005 and width 6 G in 30 minutes after intraperitoneal RT injection in the lethal dose (0.2 ml of preparation containing of 2 mg RT) has been revealed. Subsequent intoxication of mice is accompanied by the appearance of the signal from nitrosyl complexes in EPR spectra. These differences in experimental results of in vitro and ex vivo EPR can be explained by launch of compensatory adaptive response of organism on the action of highly toxic preparation.

The Increased Endogenous Sulfur Dioxide Acts as a Compensatory Mechanism for the Downregulated Endogenous Hydrogen Sulfide Pathway in the Endothelial Cell Inflammation

PubMed Central

Zhang, Da; Wang, Xiuli; Tian, Xiaoyu; Zhang, Lulu; Yang, Guosheng; Tao, Yinghong; Liang, Chen; Li, Kun; Yu, Xiaoqi; Tang, Xinjing; Tang, Chaoshu; Zhou, Jing; Kong, Wei; Du, Junbao; Huang, Yaqian; Jin, Hongfang

2018-01-01

Endogenous hydrogen sulfide (H2S) and sulfur dioxide (SO2) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for in vivo experiments. We found that endogenous H2S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO2 level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO2 synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H2S donor could reverse the CSE knockdown-induced increase in the endogenous SO2 level and AAT activity. Moreover, H2S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H2S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartate-β-hydroxamate (HDX), which blocked the upregulation of endogenous SO2/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In in vivo experiment, H2S donor restored the deficiency of endogenous H2S production induced by MCT, and reversed the upregulation of endogenous SO2/AAT pathway via sulfhydrating AAT1 and AAT2. In accordance with the results of the in vitro experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H2S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H2S inhibited endogenous SO2 generation by inactivating AAT via the sulfhydration of AAT1/2; and the increased endogenous SO2 generation might play a compensatory role when H2S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses in vitro and in vivo. PMID:29760703

Antituberculous effect of silver nanoparticles

NASA Astrophysics Data System (ADS)

Kreytsberg, G. N.; Gracheva, I. E.; Kibrik, B. S.; Golikov, I. V.

2011-04-01

The in vitro experiment, involving 1164 strains of the tuberculosis mycobacteria, exhibited a potentiating effect of silver nanoparticles on known antituberculous preparations in respect of overcoming drug-resistance of the causative agent. The in vitro experiment, based on the model of resistant tuberculosis, was performed on 65 white mice. An evident antituberculous effect of the nanocomposite on the basis of silver nanoparticles and isoniazid was proved. Toxicological assessment of the of nanopreparations was carried out. The performed research scientifically establishes efficacy and safety of the nanocomposite application in combination therapy of patients suffering from drug-resistant tuberculosis.

Hydroxycinnamate conjugates as potential monolignol replacements: In vitro lignification and cell wall studies with rosmarinic acid

USDA-ARS?s Scientific Manuscript database

The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as rosmarinic acid (RA) and analogous catechol derivatives to create cell wall lignins that are less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that...

International standardization of cage designs and feeding regimes for honey bee in vitro experiments

USDA-ARS?s Scientific Manuscript database

The aim of this study was to improve and standardize cage systems for maintaining adult honey bee workers under in vitro laboratory conditions. To achieve this goal, we experimentally evaluated the impact of different cages, developed by scientists of the international research network COLOSS (Preve...

Pharmacokinetic-Pharmacodynamic Modeling of the In Vitro Activities of Oxazolidinone Antimicrobial Agents against Methicillin-Resistant Staphylococcus aureus▿

PubMed Central

Schmidt, Stephan; Sabarinath, Sreedharan Nair; Barbour, April; Abbanat, Darren; Manitpisitkul, Prasarn; Sha, Sue; Derendorf, Hartmut

2009-01-01

Linezolid is the first FDA-approved oxazolidinone with activity against clinically important gram-positive pathogens, including methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). RWJ-416457 is a new oxazolidinone with an antimicrobial spectrum similar to that of linezolid. The goal of the present study was to develop a general pharmacokinetic (PK)-pharmacodynamic (PD) model that allows the characterization and comparison of the in vitro activities of oxazolidinones, determined in time-kill curve experiments, against MRSA. The in vitro activities of RWJ-416457 and the first-in-class representative, linezolid, against MRSA OC2878 were determined in static and dynamic time-kill curve experiments over a wide range of concentrations: 0.125 to 8 μg/ml (MIC, 0.5 μg/ml) and 0.25 to 16 μg/ml (MIC, 1 μg/ml), respectively. After correction for drug degradation during the time-kill curve experiments, a two-subpopulation model was simultaneously fitted to all data in the NONMEM VI program. The robustness of the model and the precision of the parameter estimates were evaluated by internal model validation by nonparametric bootstrap analysis. A two-subpopulation model, consisting of a self-replicating, oxazolidinone-susceptible and a persistent, oxazolidinone-insusceptible pool of bacteria was appropriate for the characterization of the time-kill curve data. The PK-PD model identified was capable of accounting for saturation in growth, delays in the onsets of growth and drug-induced killing, as well as naturally occurring bacterial death. The simultaneous fit of the proposed indirect-response, maximum-effect model to the data resulted in concentrations that produced a half-maximum killing effect that were significantly (P < 0.05) lower for RWJ-416457 (0.41 μg/ml) than for linezolid (1.39 μg/ml). In combination with the appropriate PK data, the susceptibility-based two-subpopulation model identified may provide valuable guidance for the selection of oxazolidinone doses or dose regimens for use in clinical studies. PMID:19786607

Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

PubMed Central

Yoder-Himes, Deborah R.; Kroos, Lee

2006-01-01

The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Ω4400. To determine if FruA is a direct regulator of Ω4400 transcription, a combination of in vivo and in vitro experiments was performed. Ω4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter −35 region in vitro. Mutations between bp −86 and −77 greatly reduced binding. One of these mutations had been shown previously to reduce Ω4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Ω4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Ω4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp −80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp −86 and −77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain. PMID:16816188

Mesoporous Silica Nanoparticle Delivery of Chemically Modified siRNA Against TWIST1 Leads to Reduced Tumor Burden

PubMed Central

Finlay, James; Roberts, Cai M.; Dong, Juyao; Zink, Jeffrey I.; Tamanoi, Fuyuhiko; Glackin, Carlotta A.

2015-01-01

Growth and progression of solid tumors depends on the integration of multiple pro-growth and survival signals, including the induction of angiogenesis. TWIST1 is a transcription factor whose reactivation in tumors leads to epithelial to mesenchymal transition (EMT), including increased cancer cell stemness, survival, and invasiveness. Additionally, TWIST1 drives angiogenesis via activation of IL-8 and CCL2, independent of VEGF signaling. In this work, results suggest that chemically modified siRNA against TWIST1 reverses EMT both in vitro and in vivo. siRNA delivery with a polyethyleneimine-coated mesoporous silica nanoparticle (MSN) led to reduction of TWIST1 target genes and migratory potential in vitro. In mice bearing xenograft tumors, weekly intravenous injections of the siRNA-nanoparticle complexes resulted in decreased tumor burden together with a loss of CCL2 suggesting a possible anti-angiogenic response. Therapeutic use of TWIST1 siRNA delivered via MSNs has the potential to inhibit tumor growth and progression in many solid tumor types. Chemically modified siRNA against TWIST1 was complexed to cation-coated mesoporous silica nanoparticles and tested in vitro and in vivo. In cell culture experiments, siRNA reduced expression of TWIST1 and its target genes, and reduced cell migration. In mice, injections of the siRNA-nanoparticle complex led to reduced tumor weight. Data suggest that diminished tumor burden was the result of reduced CCL2 expression and angiogenesis following TWIST1 knockdown. PMID:26115637

Basic fibroblastic growth factor affects the osteogenic differentiation of dental pulp stem cells in a treatment-dependent manner.

PubMed

Qian, J; Jiayuan, W; Wenkai, J; Peina, W; Ansheng, Z; Shukai, S; Shafei, Z; Jun, L; Longxing, N

2015-07-01

To determine how basic fibroblastic growth factor (bFGF) affected the osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro and in vivo. Basic fibroblastic growth factor stimulation of DPSCs was divided into a pre-treatment period and an osteogenic differentiation period. Alizarin red quantification experiments and alkaline phosphatase activity quantification assay were performed to examine the osteogenic differentiation of DPSCs after different bFGF stimulation. Quantification reverse transcription polymerase chain reaction was used to analyze the osteogenic gene expression of DPSCs after different bFGF stimulation. In addition, DPSCs that received the 1 and 2 weeks bFGF pre-treatments as in the in vitro experiments were mineralized for 1 week and seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) pills and subcutaneously transplanted into naked mice for 2 or 3 months. The transplants were removed, sliced and stained using Modified Ponceau Trichrome Stain to observe the formation of mineralized tissue. Basic fibroblastic growth factor stimulation in the osteogenic differentiation period decreased the in vitro osteogenic differentiation ability of DPSCs. One week pre-treatment with bFGF increased the in vitro osteogenic differentiation ability of DPSCs, whereas 2 weeks pre-treatment with bFGF decreased the in vitro osteogenic differentiation ability of DPSCs. The pre-treatment period was vital for the osteogenic differentiation of DPSCs in vitro. The in vivo results were similar to the in vitro results. Basic fibroblastic growth factor affected the osteogenic differentiation of DPSCs in a treatment-dependent manner both in vitro and in vivo. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains.

PubMed

Falade, Titilayo D O; Syed Mohdhamdan, Sharifah H; Sultanbawa, Yasmina; Fletcher, Mary T; Harvey, Jagger J W; Chaliha, Mridusmita; Fox, Glen P

2016-07-01

In vitro experimental environments are used to study interactions between microorganisms, and to predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to match closely the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation were studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25 and 30°C). Competition experiments showed interaction between the main effects of aflatoxin accumulation and the environment at 25°C, but not so at 30°C. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-h incubation in different experimental environments. Whereas all fungi incubated within the soil environment survived, in the cotton wool environment none of the competitors of A. flavus survived at 30°C. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post-harvest.

Steroid Sulfates from Ophiuroids (Brittle Stars): Action on Some Factors of Innate and Adaptive Immunity.

PubMed

Gazha, Anna K; Ivanushko, Lyudmila A; Levina, Eleonora V; Fedorov, Sergey N; Zaporozets, Tatyana S; Stonik, Valentin A; Besednova, Nataliya N

2016-06-01

The action of seven polyhydroxylated sterol mono- and disulfates (1-7), isolated from ophiuroids, on innate and adaptive immunity was examined in in vitro and in vivo experiments. At least, three of them (1, 2 and 4) increased the functional activities of neutrophils, including levels of oxygen-dependent metabolism, adhesive and phagocytic properties, and induced the expression of pro-inflammatory cytokines TNF-α and IL-8. Compound 4 was the most active for enhancing the production of antibody forming cells in the mouse spleen.

Ginseng for Liver Injury: Friend or Foe?

PubMed Central

Kim, Tae-Woo

2016-01-01

Panax sp., including Panax ginseng Meyer, Panax quiquifolius L., or Panax notoginseng (Burk.) FH Chen, have been used as functional foods or for traditional Chinese medicine for diabetes, inflammation, stress, aging, hepatic injury, and cancer. In recent decades, a number of both in vitro and in vivo experiments as well as human studies have been conducted to investigate the efficacy and safety of various types of ginseng samples and their components. Of these, the hepatoprotective and hepatotoxic effects of ginseng and their ginsenosides and polysaccharides are reviewed and summarized. PMID:28930143

Cell therapy for spinal cord injury informed by electromagnetic waves.

PubMed

Finnegan, Jack; Ye, Hui

2016-10-01

Spinal cord injury devastates the CNS, besetting patients with symptoms including but not limited to: paralysis, autonomic nervous dysfunction, pain disorders and depression. Despite the identification of several molecular and genetic factors, a reliable regenerative therapy has yet to be produced for this terminal disease. Perhaps the missing piece of this puzzle will be discovered within endogenous electrotactic cellular behaviors. Neurons and stem cells both show mediated responses (growth rate, migration, differentiation) to electromagnetic waves, including direct current electric fields. This review analyzes the pathophysiology of spinal cord injury, the rationale for regenerative cell therapy and the evidence for directing cell therapy via electromagnetic waves shown by in vitro experiments.

Effects of replacing soybean meal with canola meal differing in rumen-undegradable protein content on ruminal fermentation and gas production kinetics using 2 in vitro systems.

PubMed

Paula, E M; Monteiro, H F; Silva, L G; Benedeti, P D B; Daniel, J L P; Shenkoru, T; Broderick, G A; Faciola, A P

2017-07-01

Previous research indicated that there were significant differences in rumen-undegradable protein (RUP) among canola meals (CM), which could influence the nutritional value of CM. The objectives of this study were to (1) evaluate the effects of feeding CM with different RUP contents on ruminal fermentation, nutrient digestion, and microbial growth using a dual-flow continuous culture system (experiment 1) and (2) evaluate ruminal gas production kinetics, in vitro organic matter (OM) digestibility, and methane (CH 4 ) production of soybean meal (SBM) and CM with low or high RUP in the diet or as a sole ingredient using a gas production system (experiments 2 and 3). In experiment 1, diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square. The only ingredient that differed among diets was the protein supplement. The treatments were (1) solvent-extracted SBM, (2) low-RUP solvent-extracted CM (38% RUP as a percentage of crude protein), and (3) high-RUP solvent-extracted CM (50% RUP). Diets were prepared as 3 concentrate mixtures that were combined with 25% orchardgrass hay and 15% wheat straw (dry matter basis). Experiments 2 and 3 had the same design with 24 bottles incubated 3 times for 48 h each. During the 48-h incubation, the cumulative pressure was recorded to determine gas production kinetics, in vitro OM digestibility, and CH 4 production. In experiment 1, N flow (g/d), efficiency of N use, efficiency of bacterial N synthesis, total volatile fatty acids (mM), and molar proportion of acetate, propionate, and isobutyrate were not affected by treatments. There were tendencies for a decrease in ruminal NH 3 -N and an increase in molar proportion of butyrate for the SBM diet compared with both CM diets. The molar proportion of valerate was greater in both CM diets, whereas the molar proportion of isovalerate and total branched-chain volatile fatty acids was lower for the CM diets compared with the SBM diet. In experiments 2 and 3, the SBM diet had a greater gas pool size than both CM diets. The SBM diet increased in vitro OM digestibility; however, it also tended to increase CH 4 production (mM and g/kg of DM) compared with both CM diets. Based on the results of this study, CM with RUP varying from 38 to 50% of crude protein does not affect ruminal fermentation, nutrient digestion, and microbial growth when CM is included at up to 34% of the diet. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

A model-based approach for automated in vitro cell tracking and chemotaxis analyses.

PubMed

Debeir, Olivier; Camby, Isabelle; Kiss, Robert; Van Ham, Philippe; Decaestecker, Christine

2004-07-01

Chemotaxis may be studied in two main ways: 1) counting cells passing through an insert (e.g., using Boyden chambers), and 2) directly observing cell cultures (e.g., using Dunn chambers), both in response to stationary concentration gradients. This article promotes the use of Dunn chambers and in vitro cell-tracking, achieved by video microscopy coupled with automatic image analysis software, in order to extract quantitative and qualitative measurements characterizing the response of cells to a diffusible chemical agent. Previously, we set up a videomicroscopy system coupled with image analysis software that was able to compute cell trajectories from in vitro cell cultures. In the present study, we are introducing a new software increasing the application field of this system to chemotaxis studies. This software is based on an adapted version of the active contour methodology, enabling each cell to be efficiently tracked for hours and resulting in detailed descriptions of individual cell trajectories. The major advantages of this method come from an improved robustness with respect to variability in cell morphologies between different cell lines and dynamical changes in cell shape during cell migration. Moreover, the software includes a very small number of parameters which do not require overly sensitive tuning. Finally, the running time of the software is very short, allowing improved possibilities in acquisition frequency and, consequently, improved descriptions of complex cell trajectories, i.e. trajectories including cell division and cell crossing. We validated this software on several artificial and real cell culture experiments in Dunn chambers also including comparisons with manual (human-controlled) analyses. We developed new software and data analysis tools for automated cell tracking which enable cell chemotaxis to be efficiently analyzed. Copyright 2004 Wiley-Liss, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, J.F.; Johnson, L.; Simpson, N.B.

In arctic tundra soil N is highly limiting, N mineralization is slow and organic N greatly exceeds inorganic N. We studied the effects of fungistatics (azoxystrobin [Quadris{reg_sign}] or propiconazole [Tilt{reg_sign}]) on the fungi isolated from ericaceous plant roots in vitro. In addition to testing the phytotoxicity of the two fungistatics we also tested their effects on growth and nitrogen uptake of an ericaceous plant (Vaccinium uliginosum) in a closed Petri plate system without root-associated fungi. Finally, to evaluate the fungistatic effects in an in vivo experiment we applied fungistatics and nitrogen isotopes to intact tundra soil cores from Toolik Lake,more » Alaska, and examined the ammonium-N and glycine-N use by Vaccinium vitis-idaea with and without fungistatics. The experiments on fungal pure cultures showed that Tilt{reg_sign} was more effective in reducing fungal colony growth in vitro than Quadris{reg_sign}, which was highly variable among the fungal strains. Laboratory experiments aiming to test the fungistatic effects on plant performance in vitro showed that neither Quadris{reg_sign} nor Tilt{reg_sign} affected V. uliginosum growth or N uptake. In this experiment V. uliginosum assimilated more than an order of magnitude more ammonium-N than glycine-N. The intact tundra core experiment provided contrasting results. After 10 wk of fungistatic application in the growth chamber V. vitis-idaea leaf %N was 10% lower and the amount of leaf {sup 15}N acquired was reduced from labeled ammonium (33%) and glycine (40%) during the 4 d isotope treatment. In contrast to the in vitro experiment leaf {sup 15}N assimilation from glycine was three times higher than from {sup 15}NH{sub 4} in the treatments that received no-fungistatics. We conclude that the function of the fungal communities is essential to the acquisition of N from organic sources and speculate that N acquisition from inorganic sources is mainly inhibited by competition with complex soil microbial communities.« less

Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro.

PubMed

Roasa, L M; Choi, Y H; Love, C C; Romo, S; Varner, D D; Hinrichs, K

2007-09-01

In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates were influenced by factors associated with the freezing process (Experiment 2), a single ejaculate from a second stallion was frozen using eight variations in timing of steps in the freezing protocol. There were no differences among treatments in fertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; P<0.01). We inferred that rates of equine IVF with frozen-thawed sperm were influenced by ejaculate, the composition and age of the media used, and freezing extender. To our knowledge, this is the first report of ejaculate or extender differences affecting in vitro fertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.

In vitro-in vivo study on the effects of plant compounds on rumen fermentation, microbial abundances and methane emissions in goats.

PubMed

Martínez-Fernández, G; Abecia, L; Martín-García, A I; Ramos-Morales, E; Hervás, G; Molina-Alcaide, E; Yáñez-Ruiz, D R

2013-12-01

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P≥ 0.366). Total volatile fatty-acid (VFA) concentration decreased (P≤ 0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P≤ 0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P≤ 0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P≤ 0.004) by CAR and PTS. The addition of all compounds linearly decreased (P≤ 0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet-dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P≤ 0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.

In vitro study on effect of germinated wheat on human breast cancer cells

USDA-ARS?s Scientific Manuscript database

This research investigated the possible anti-cancer effects of germinated wheat flours (GWF) on cell growth and apoptosis of human breast cancer cells. In a series of in vitro experiments, estrogen receptor-positive (MCF-7) and negative (MDA-MB-231) cells were cultured and treated with GWF that wer...

Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

COMPARISON OF IN VIVO DERIVED AND SCALED IN VITRO METABOLIC RATE CONSTANTS FOR SOME VOLATILE ORGANIC COMPOUNDS (VOCS)

EPA Science Inventory

The reliability of physiologically based pharmacokinetic (PBPK) models is directly related to the accuracy of the metabolic rate parameters used as model inputs. When metabolic rate parameters derived from in vivo experiments are unavailable, they can be estimated from in vitro d...

Formulation of diazepam containing rectal suppositories and experiences of their biopharmaceutical study.

PubMed

Regdon, G; Bácskay, I; Kata, M; Selmeczi, B; Szikszay, M; Sánta, A; Bálint, G S

1994-05-01

Methodology and the results of the in vitro membrane diffusion and in vivo bioavailability studies are presented. The results confirm a correlation between in vitro and in vivo findings. Hydrophilic macrogol-mixture with great molecular mass can be recommended as the optimal vehicle for formulation of diazepam suppositories.

Enhancing osseointegration of orthopedic implants with titania nanotube surfaces

NASA Astrophysics Data System (ADS)

Baker, Erin A.

Introduction: As joint arthroplasty surgical procedures increase annually, the development of new strategies, including novel materials and surface modifications, to attain solid bone-implant fixation are needed to increase implant terms of service. In this study, we evaluate two morphologies of titania nanotubes in both in vitro and in vivo experiments to quantify osseointegrative potential and material-level biocompatibility. Materials and Methods: Samples were prepared via an electrochemical etching process. Two different titania nanotube (TiNT) morphologies were produced, Aligned and Trabecular. For the in vitro experiment, Sprague Dawley (SD) rat marrow-derived bone marrow cells (BMC) were seeded on samples. Alkaline phosphatase (ALP) activity, osteocalcin (OC) expression, expression of relevant genes as well as cell attachment and morphology were assessed. In the first in vivo experiment, Kirschner wires were implanted unilaterally into SD rat femora with a TiNT-etched or unmodified (Control) implant. General health assessments and weekly body weights were recorded. At a 12-week endpoint, hematologic, systemic metal ion, and histologic analyses were performed. For the second in vivo experiment, Kirschner wires were implanted bilaterally into SD rat femora, with a TiNT-etched implant in one femora and unmodified (Control) implant as an internal control. At 4- and 12-week endpoints, femora were assessed via biomechanics, undecalcified histology, micro-computed tomography (muCT), and backscattered electron imaging (BEI) to characterize de novo bone formation. Results: In vitro experiments demonstrated BMC attachment and differentiation into osteoblasts as well as greater ALP activity, OC expression, total cell counts, and gene expression (of Col1a1, IGF-1, and osteonectin) on TiNT surfaces versus Controls. Cells on TiNT-etched substrates were smaller in diameter and more eccentric than Controls. In the first in vivo experiment, there were significant differences in body weight between groups at Weeks 9 and 11. There were no significant differences in red or white blood cell function between TiNT groups and Control. Aluminum levels in the lungs were significantly greater in the Trabecular TiNT group compared to Control. Histologic analysis showed significantly fewer granulocytes and neutrophils in the distal region of Trabecular TiNT-implanted femora as well as significantly fewer foreign body giant/multinucleated cells and neutrophils in the midshaft region of Aligned TiNT-implanted femora versus Controls. In the second in vivo experiment, at 12 weeks, microCT analysis showed TiNT implants generated greater bone formation than Controls. Histologic analysis demonstrated 1.5 times greater bone-implant contact in TiNT groups than Controls at 12 weeks. TiNT groups exhibited 1.3 to 3.7 times greater strength of fixation than Controls during pull-out testing. Discussion and Conclusions: In vitro data confirmed BMC attachment and differentiation into osteoblasts as well as osteoblastic phenotypic behavior. A clinically-relevant in vivo model of femoral intramedullary fixation, showed increased bone formation and quality in femora implanted with TiNT-etched implants versus Controls. A second in vivo study showed that TiNT surfaces do not generate systemic effects and may beneficially modulate the periprosthetic inflammatory environment.

Toxic effects of ethylene oxide residues on bovine embryos in vitro.

PubMed

Holyoak, G R; Wang, S; Liu, Y; Bunch, T D

1996-04-15

The potential of ethylene oxide (EtO) residues in exposed plastic tissue culture dishes to adversely affect bovine oocyte maturation, fertilization and subsequent embryonic development was monitored. In experiment 1, the effects of aeration time and aeration combined with washing of EtO-gassed culture dishes on the extent of residual toxicity were investigated. There was no cleavage in any treatment in which oocytes were matured and fertilized in dishes exposed to EtO. EtO residues caused functional degeneration of oocytes even when culture dishes were aerated for more than 12 days post EtO-exposure and repeatedly washed. In experiment 2, the residual toxicity of EtO gas on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were evaluated. Cleavage rate significantly decreased and post-cleavage development was retarded in ova maintained in dishes treated with EtO either during IVM or IVF. EtO residues may be more detrimental to spermatozoa than to oocytes which may have been the primary cause of fertilization failure during IVF.

Meta-analysis of the relationship between dietary tannin level and methane formation in ruminants from in vivo and in vitro experiments.

PubMed

Jayanegara, A; Leiber, F; Kreuzer, M

2012-06-01

A meta-analysis was conducted to evaluate the extent to which dietary tannin level is related to methane emissions from ruminants. Data from a total of 30 experiments comprising 171 treatments were entered in a database. In vitro batch culture and in vivo measurements were distinguished as experimental approaches. With any approach, methane declined when dietary tannins increased. The in vitro approach predicted the in vivo response quite accurately. However, in vitro, the response followed a quadratic response pattern (R(2) = 0.66; lower response with increasing tannin level), whereas in vivo, this decline was linear (R(2) = 0.29). This indicates that the in vitro batch culture is of limited accuracy for estimating effects at levels >100 g tannin/kg dry matter. The large variation in methane/digestible organic matter (OM) found at low tannin levels may explain contrasting literature reports. Methane reduction with tannins was associated with a reduced apparent digestion of OM, and especially fibre, but methane/apparently digestible OM declined also. The present findings are helpful as they identified an underlying general antimethanogenic effect of tannins across tannin sources and experimental conditions, thus allowing concentrating the search on sources with satisfactory palatability and low adverse effects on animal performance. © 2011 Blackwell Verlag GmbH.

Design of experiments to optimize an in vitro cast to predict human nasal drug deposition.

PubMed

Shah, Samir A; Dickens, Colin J; Ward, David J; Banaszek, Anna A; George, Chris; Horodnik, Walter

2014-02-01

Previous studies showed nasal spray in vitro tests cannot predict in vivo deposition, pharmacokinetics, or pharmacodynamics. This challenge makes it difficult to assess deposition achieved with new technologies delivering to the therapeutically beneficial posterior nasal cavity. In this study, we determined best parameters for using a regionally divided nasal cast to predict deposition. Our study used a model suspension and a design of experiments to produce repeatable deposition results that mimic nasal deposition patterns of nasal suspensions from the literature. The seven-section (the nozzle locator, nasal vestibule, front turbinate, rear turbinate, olfactory region, nasopharynx, and throat filter) nylon nasal cast was based on computed tomography images of healthy humans. It was coated with a glycerol/Brij-35 solution to mimic mucus. After assembling and orienting, airflow was applied and nasal spray containing a model suspension was sprayed. After disassembling the cast, drug depositing in each section was assayed by HPLC. The success criteria for optimal settings were based on nine in vivo studies in the literature. The design of experiments included exploratory and half factorial screening experiments to identify variables affecting deposition (angles, airflow, and airflow time), optimization experiments, and then repeatability and reproducibility experiments. We found tilt angle and airflow time after actuation affected deposition the most. The optimized settings were flow rate of 16 L/min, postactuation flow time of 12 sec, a tilt angle of 23°, nozzle angles of 0°, and actuation speed of 5 cm/sec. Neither cast nor operator caused significant variation of results. We determined cast parameters to produce results resembling suspension nasal sprays in the literature. The results were repeatable and unaffected by operator or cast. These nasal spray parameters could be used to assess deposition from new devices or formulations. For human deposition studies using radiolabeled formulations, this cast could show that radiolabel deposition represents drug deposition. Our methods could also be used to optimize settings for other casts.

Inactivation of foot-and-mouth disease virus in various bovine tissues used for the production of natural sausage casings.

PubMed

Wijnker, Joris J; Haas, Bernd; Berends, Boyd R

2012-02-01

Bovine intestines, bladders and oesophagus are used for the production of natural casings ("beef casings") as edible sausage containers. Derived from cattle experimentally infected with FMDV (initial dosage 10(4) TCID(50)/mL, strain A Iran 97), these beef casings were treated with sodium chloride (NaCl) or phosphate supplemented salt (P-salt). In addition, different in-vitro experiments using beef casings were done on a small scale with other FMDV strains (A Turkey 06, C-Oberbayern and O(1) Manisa) as "proof of principle". Based on the combined results of the in-vivo and in-vitro experiments, it can be concluded that the storage period of 30 days at 20 °C in NaCl is sufficiently effective to inactivate a possible contamination with FMDV in beef casings and that the usage of P-salt does not clearly enhance the inactivation of FMDV infectivity. Storage of salted beef casings at about 20 °C for 30 days is already part of the Standard Operating Procedures (included in HACCP) of the international casing industry and can therefore be considered as a protective measure for the international trade in natural casings. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of ultra-high pressure homogenization on the interaction between bovine casein micelles and ritonavir.

PubMed

Corzo-Martínez, M; Mohan, M; Dunlap, J; Harte, F

2015-03-01

The aim of this work was to develop a milk-based powder formulation appropriate for pediatric delivery of ritonavir (RIT). Ultra-high pressure homogenization (UHPH) at 0.1, 300 and 500 MPa was used to process a dispersion of pasteurized skim milk (SM) and ritonavir. Loading efficiency was determined by RP-HPLC-UV; characterization of RIT:SM systems was carried out by apparent average hydrodynamic diameter and rheological measurements as well as different analytical techniques including Trp fluorescence, UV spectroscopy, DSC, FTIR and SEM; and delivery capacity of casein micelles was determined by in vitro experiments promoting ritonavir release. Ritonavir interacted efficiently with milk proteins, especially, casein micelles, regardless of the processing pressure; however, results suggest that, at 0.1 MPa, ritonavir interacts with caseins at the micellar surface, whilst, at 300 and 500 MPa, ritonavir is integrated to the protein matrix during UHPH treatment. Likewise, in vitro experiments showed that ritonavir release from micellar casein systems is pH dependent; with a high retention of ritonavir during simulated gastric digestion and a rapid delivery under conditions simulating the small intestine environment. Skim milk powder, especially, casein micelles are potentially suitable and efficient carrier systems to develop novel milk-based and low-ethanol powder formulations of ritonavir appropriate for pediatric applications.

Fission neutron source in Rome

NASA Astrophysics Data System (ADS)

Coppola, Mario; Di Majo, V.; Ingrao, G.; Rebessi, S.; Testa, A.

1997-02-01

A fission neutron source is operating in Rome at the ENEA Casaccia Research Center since 1971, consisting of a low power fast reactor named RSV-Tapiro. it is employed for a variety of experiments, including dosimetry, material testing, radiation protection and biology. In particular, application to experimental radiobiology includes studies of the biological action of neutrons in the whole-body irradiated animal, or in specialized systems in vivo or in vitro. For his purpose a vertical irradiation facility was originally constructed. Recently, a new horizontal irradiation facility has been designed to allow the exposure of larger samples or larger sample batches at one time. Dosimetry at the sample irradiation positions is routinely carried out by the conventional method of using two ion chambers. This physical dosimetry has recently been compared with the results of biological dosimetry based on the detection of chromosomal aberrations in peripheral blood human lymphocytes irradiated in vitro. A characterization of the radiation quality in the two configurations has been carried out by tissue equivalent proportional counter microdosimetry measurements. Information about the main characteristics of the reactor and the two irradiation facilities is provided and relevant results of the various measurements are summarized. Radiobiological results obtained using this neutron source are also briefly outlined.

How to assess the mutagenic potential of cosmetic products without animal tests?

PubMed

Speit, Günter

2009-08-01

Animal experiments (in vivo tests) currently play a key role in genotoxicity testing. Results from in vivo tests are, in many cases, decisive for the assessment of a mutagenic potential of a test compound. The Seventh Amendment to the European Cosmetics Directive will, however, ban the European marketing of cosmetic/personal care products that contain ingredients that have been tested in animal experiments. If genotoxicity testing is solely based on the currently established in vitro tests, the attrition rate for chemicals used in cosmetic products will greatly increase due to irrelevant positive in vitro test results. There is urgent need for new and/or improved in vitro genotoxicity tests and for modified test strategies. Test strategies should consider all available information on chemistry of the test substance/the chemical class (e.g. SAR, metabolic activation and dermal adsorption). Test protocols for in vitro genotoxicity tests should be sensitive and robust enough to ensure that negative results can be accepted with confidence. It should be excluded that positive in vitro test results are due to high cytotoxicity or secondary genotoxic effects which may be thresholded and/or only occur under in vitro test conditions. Consequently, further research is needed to establish the nature of thresholds in in vitro assays and to determine the potential for incorporation of mode of action data into future risk assessments. New/improved tests have to be established and validated, considering the use of (metabolically competent) primary (skin) cells, 3D skin models and cells with defined capacity for metabolic activation (e.g. genetically engineered cell lines). The sensitivity and specificity of new and improved genotoxicity tests has to be determined by testing a battery of genotoxic and non-genotoxic chemicals. New or adapted international guidelines will be needed for these tests. The establishment of such a new genotoxicity testing strategy will take time and the new in vitro genotoxicity testing will become much more complex and will require greater mechanistic understanding to build a weight of evidence decision, which will be demanding and time-consuming. At present, no validated alternative methods for the follow-up of positive results from the standard genotoxicity battery are available and an appropriate evaluation of the mutagenic potential of cosmetic ingredients without animal experiments is therefore not possible in many cases.

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition.

PubMed

Sørli, Jorid B; Huang, Yishi; Da Silva, Emilie; Hansen, Jitka S; Zuo, Yi Y; Frederiksen, Marie; Nørgaard, Asger W; Ebbehøj, Niels E; Larsen, Søren T; Hougaard, Karin S

2018-01-01

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

Mathematical modeling of HIV-like particle assembly in vitro.

PubMed

Liu, Yuewu; Zou, Xiufen

2017-06-01

In vitro, the recombinant HIV-1 Gag protein can generate spherical particles with a diameter of 25-30 nm in a fully defined system. It has approximately 80 building blocks, and its intermediates for assembly are abundant in geometry. Accordingly, there are a large number of nonlinear equations in the classical model. Therefore, it is difficult to compute values of geometry parameters for intermediates and make the mathematical analysis using the model. In this work, we develop a new model of HIV-like particle assembly in vitro by using six-fold symmetry of HIV-like particle assembly to decrease the number of geometry parameters. This method will greatly reduce computational costs and facilitate the application of the model. Then, we prove the existence and uniqueness of the positive equilibrium solution for this model with 79 nonlinear equations. Based on this model, we derive the interesting result that concentrations of all intermediates at equilibrium are independent of three important parameters, including two microscopic on-rate constants and the size of nucleating structure. Before equilibrium, these three parameters influence the concentration variation rates of all intermediates. We also analyze the relationship between the initial concentration of building blocks and concentrations of all intermediates. Furthermore, the bounds of concentrations of free building blocks and HIV-like particles are estimated. These results will be helpful to guide HIV-like particle assembly experiments and improve our understanding of the assembly dynamics of HIV-like particles in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

Testing strategies for embryo-fetal toxicity of human pharmaceuticals. Animal models vs. in vitro approaches: a workshop report.

PubMed

van der Laan, Jan Willem; Chapin, Robert E; Haenen, Bert; Jacobs, Abigail C; Piersma, Aldert

2012-06-01

Reproductive toxicity testing is characterized by high animal use. For registration of pharmaceutical compounds, developmental toxicity studies are usually conducted in both rat and rabbits. Efforts have been underway for a long time to design alternatives to animal use. Implementation has lagged, partly because of uncertainties about the applicability domain of the alternatives. The reproductive cycle is complex and not all mechanisms of development can be mimicked in vitro. Therefore, efforts are underway to characterize the available alternative tests with regard to the mechanism of action they include. One alternative test is the mouse embryonic stem cell test (EST), which has been studied since the late 1990s. It is a genuine 3R "alternative" assay as it is essentially animal-free. A meeting was held to review the state-of-the-art of various in vitro models for prediction of developmental toxicity. Although the predictivity of individual assays is improving, a battery of several assays is likely to have even higher predictivity, which is necessary for regulatory acceptance. The workshop concluded that an important first step is a thorough survey of the existing rat and rabbit studies, to fully characterize the frequency of responses and the types of effects seen. At the same time, it is important to continue the optimization of in vitro assays. As more experience accumulates, the optimal conditions, assay structure, and applicability of the alternative assays are expected to emerge. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

PubMed

Fisher, T; Golan, H; Schiby, G; PriChen, S; Smoum, R; Moshe, I; Peshes-Yaloz, N; Castiel, A; Waldman, D; Gallily, R; Mechoulam, R; Toren, A

2016-03-01

Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma

PubMed Central

Fisher, T.; Golan, H.; Schiby, G.; PriChen, S.; Smoum, R.; Moshe, I.; Peshes-Yaloz, N.; Castiel, A.; Waldman, D.; Gallily, R.; Mechoulam, R.; Toren, A.

2016-01-01

Background Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. Methods We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ9-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Results Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Conclusions Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl. PMID:27022310

A combined theoretical and in vitro modeling approach for predicting the magnetic capture and retention of magnetic nanoparticles in vivo

PubMed Central

David, Allan E.; Cole, Adam J.; Chertok, Beata; Park, Yoon Shin; Yang, Victor C.

2011-01-01

Magnetic nanoparticles (MNP) continue to draw considerable attention as potential diagnostic and therapeutic tools in the fight against cancer. Although many interacting forces present themselves during magnetic targeting of MNP to tumors, most theoretical considerations of this process ignore all except for the magnetic and drag forces. Our validation of a simple in vitro model against in vivo data, and subsequent reproduction of the in vitro results with a theoretical model indicated that these two forces do indeed dominate the magnetic capture of MNP. However, because nanoparticles can be subject to aggregation, and large MNP experience an increased magnetic force, the effects of surface forces on MNP stability cannot be ignored. We accounted for the aggregating surface forces simply by measuring the size of MNP retained from flow by magnetic fields, and utilized this size in the mathematical model. This presumably accounted for all particle-particle interactions, including those between magnetic dipoles. Thus, our “corrected” mathematical model provided a reasonable estimate of not only fractional MNP retention, but also predicted the regions of accumulation in a simulated capillary. Furthermore, the model was also utilized to calculate the effects of MNP size and spatial location, relative to the magnet, on targeting of MNPs to tumors. This combination of an in vitro model with a theoretical model could potentially assist with parametric evaluations of magnetic targeting, and enable rapid enhancement and optimization of magnetic targeting methodologies. PMID:21295085

Energy metabolism in isolated chick (Gallus domesticus) gastrocnemius and tilapia (Tilapia mossambica) epaxial muscle at various temperatures in vitro.

PubMed

Suzuki, E Y; Early, R J; Patterson, P H

1994-09-01

Muscle respiration experiments on inhibitor dosage (experiment 1), muscle preparation (tendons removed vs. unstretched vs. stretched muscles; chick muscle only; experiment 2) and media temperature (26.5, 32, 37, 42 degrees C; experiment 3) were conducted on chick (Gallus domesticus) gastrocnemius and tilapia (Tilapia mossambica) epaxial muscle in vitro. Experiment 1: The dosage of cycloheximide and ouabain required for maximum inhibition of protein synthesis and Na+,K+ ATPase, respectively, in chick and tilapia muscle was approximately 6 x 10(-5) M. Experiment 2: Removing the tendons of chick muscle decreased (% inhibition, P = 0.05) cycloheximide-sensitive respiration compared to stretched and unstretched muscles (tendons intact). However, muscle preparation had little influence on ouabain-sensitive respiration. Experiment 3: Cycloheximide-sensitive respiration tended to increase (microliter O2/mg DNA.hr, P = 0.054) with media temperature in tilapia muscle. Chick muscle was less responsive in this respect. Ouabain-sensitive respiration increased at lower temperature in chick muscle (% inhibition, cubic relationship, P = 0.001) and at higher temperature in tilapia muscle (% inhibition, quadratic relationship, P = 0.0002).

Are there optical communication channels in the brain?

PubMed

Zarkeshian, Parisa; Kumar, Sourabh; Tuszynski, Jack; Barclay, Paul; Simon, Christoph

2018-03-01

Despite great progress in neuroscience, there are still fundamental unanswered questions about the brain, including the origin of subjective experience and consciousness. Some answers might rely on new physical mechanisms. Given that biophotons have been discovered in the brain, it is interesting to explore if neurons use photonic communication in addition to the well-studied electro-chemical signals. Such photonic communication in the brain would require waveguides. Here we review recent work (S. Kumar, K. Boone, J. Tuszynski, P. Barclay, and C. Simon, Scientific Reports 6, 36508 (2016)) suggesting that myelinated axons could serve as photonic waveguides. The light transmission in the myelinated axon was modeled, taking into account its realistic imperfections, and experiments were proposed both in vivo and in vitro to test this hypothesis. Potential implications for quantum biology are discussed.

[The effect of acupuncture and endogenous c-Fos, c-Jun on regeneration of neuronal dendrite of spared DRG in vitro following partial ganglionectomy].

PubMed

Wang, Te-wei; Wang, Ting-hua; Zhou, Xue; Zhang, Lian-shuang; Xu, Xin-yun

2007-09-01

To explore the effect of acupuncture, endogenous c-Fos and c-Jun on the regeneration of neuronal dendrite of spared dorsal root ganglion (DRG) in vitro following partial ganglionectomy. Five adult male cats were used in this experiment. Their bilateral L1-L5, L7-S2 DRG were removed, and L6 DRG were spared. Then unilaterally, two sets of acupoints (Zusanli(St. 36) and Xuanzhong(G. B. 39); Futu (St. 32) and Sanyinjiao (Sp. 6) located in the distribution area of spinal nerve L6) were electro-stimulated alternately 30 min everyday by electro-needling. Seven days after operation, bilateral L6 DRGs were taken out and were cultured respectively in vitro. Some cultured mediums of the acupuncture lateral wells were totally replaced by each corresponding antibody-cultured medium including respectively 100 ng/mL anti-c-Fos and anti-c-Jun antibody at the 24th hour and terminated after 7 days. The length of the neurite was measured by upside-down light microscopy. Then, cultured cells were stained by the immunohistochemistry ABC method. Data were analyzed by One-way ANOVA and q test. Immunocytochemical staining revealed that over 95% cells were NSE positive cells which were the typical neuron of DRG in vitro. On the 7th day, the average neurite length of the spared DRG group, the anti-c-Fos antibody and the anti-c-Jun antibody group were shorter than that of the acupuncture group (P < 0.05); the average neurite length of the two antibody groups were longer than that of the spared DRG group (P < 0.05). These results indicate that acupuncture, endogenous c-Fos and c-Jun probably promote regeneration of neuronal dendrite of spared DRG in vitro.

In vitro fabrication of a tissue engineered human cardiovascular patch for future use in cardiovascular surgery.

PubMed

Yang, Chao; Sodian, Ralf; Fu, Ping; Lüders, Cora; Lemke, Thees; Du, Jing; Hübler, Michael; Weng, Yuguo; Meyer, Rudolf; Hetzer, Roland

2006-01-01

One approach to tissue engineering has been the development of in vitro conditions for the fabrication of functional cardiovascular structures intended for implantation. In this experiment, we developed a pulsatile flow system that provides biochemical and biomechanical signals in order to regulate autologous, human patch-tissue development in vitro. We constructed a biodegradable patch scaffold from porous poly-4-hydroxy-butyrate (P4HB; pore size 80 to 150 microm). The scaffold was seeded with pediatric aortic cells. The cell-seeded patch constructs were placed in a self-developed bioreactor for 7 days to observe potential tissue formation under dynamic cell culture conditions. As a control, cell-seeded scaffolds were not conditioned in the bioreactor system. After maturation in vitro, the analysis of the tissue engineered constructs included biochemical, biomechanical, morphologic, and immunohistochemical examination. Macroscopically, all tissue engineered constructs were covered by cells. After conditioning in the bioreactor, the cells were mostly viable, had grown into the pores, and had formed tissue on the patch construct. Electron microscopy showed confluent smooth surfaces. Additionally, we demonstrated the capacity to generate collagen and elastin under in vitro pulsatile flow conditions in biochemical examination. Biomechanical testing showed mechanical properties of the tissue engineered human patch tissue without any statistical differences in strength or resistance to stretch between the static controls and the conditioned patches. Immunohistochemical examination stained positive for alpha smooth muscle actin, collagen type I, and fibronectin. There was minor tissue formation in the nonconditioned control samples. Porous P4HB may be used to fabricate a biodegradable patch scaffold. Human vascular cells attached themselves to the polymeric scaffold, and extracellular matrix formation was induced under controlled biomechanical and biodynamic stimuli in a self-developed pulsatile bioreactor system.

The context of transcription start site regions is crucial for transcription of a plant tRNA(Lys)(UUU) gene group both in vitro and in vivo.

PubMed

Yukawa, Yasushi; Akama, Kazuhito; Noguchi, Kanta; Komiya, Masaaki; Sugiura, Masahiro

2013-01-10

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5' and 3' flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5' flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a β-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes. Copyright © 2012 Elsevier B.V. All rights reserved.

Chrondrogenesis in micromass cultures of embryonic mouse limb mesenchymal cells exposed to microgravity (7-IML-1)

NASA Technical Reports Server (NTRS)

Duke, Jackie

1992-01-01

A basic question of space biology is whether changes in gravity are perceived at the cellular level. Previous studies with a variety of cells have shown that this is the case, but to date the response of skeletal cells has not been examined, even though the skeleton is sensitive to gravitational changes. The objective of the CELLS Experiment is to examine the effect of microgravity in vitro on a skeletal cell known to be sensitive to gravitational changes both in vivo and in vitro - the mammalian chondrocyte. Various aspects of the experiment are discussed.

Fretting corrosion behavior of nitinol spinal rods in conjunction with titanium pedicle screws.

PubMed

Lukina, Elena; Kollerov, Mikhail; Meswania, Jay; Khon, Alla; Panin, Pavel; Blunn, Gordon W

2017-03-01

Untypical corrosion damage including erosions combined with the build-up of titanium oxide as a corrosion product on the surface of explanted Nitinol spinal rods in the areas where it was in contact with titanium pedicle screw head is reported. It was suggested that Nitinol rods might have inferior fretting corrosion resistance compared with that made of titanium or CoCr. Fretting corrosion of Nitinol spinal rods with titanium (Ti6Al4V) pedicle screws were tested in-vitro by conducting a series of potentiostatic measurements of the peak-to-peak values of fretting corrosion current under bending in a 10% solution of calf serum in PBS. The test included Nitinol rods locked in titanium pedicle screws of different designs. Performance of commercially available titanium (Ti6Al4V) and CoCr spinal rods was also investigated for a comparison. Corrosion damage observed after the in-vitro tests was studied using SEM and EDAX analysis and was compared with patterns on Nitinol rods retrieved 12months after initial surgery. Metal ions level was measured in the test media after in-vitro experiments and in the blood and tissues of the patients who had the rods explanted. The results of this study revealed that Nitinol spinal rods locked in Ti pedicle screws are susceptible to fretting corrosion demonstrating higher fretting corrosion current compared with commercially used Ti6Al4V and CoCr rods. On the surface of Nitinol rods after in-vitro tests and on those retrieved from the patients similar corrosion patterns were observed. Improved resistance to fretting corrosion was observed with Nitinol rods in the in-vitro tests where pedicle screws were used with a stiffer locking mechanism. Since the development of the localized corrosion damage might increase the risk of premature fatigue failure of the rods and result in leaching of Ni ions, it is concluded that Nitinol rods should not be used in conjunction with Ti pedicle screws without special protection especially where the design provides a high degree of mobility to the rods. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations.

PubMed

Choi, Young-Ho; Ross, Pablo; Velez, Isabel C; Macías-García, B; Riera, Fernando L; Hinrichs, Katrin

2015-07-01

Equine embryos develop in vitro in the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31-46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively for POU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did. GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation in in vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse. © 2015 Society for Reproduction and Fertility.

MicroRNA-301a-3p promotes pancreatic cancer progression via negative regulation of SMAD4

PubMed Central

Zhang, Kundong; Cen, Gang; Jiang, Tao; Cao, Jun; Huang, Kejian; Zhao, Qian; Qiu, Zhengjun

2015-01-01

Background Aim to determine the clinicopathological and prognostic role of miR-301a-3p in pancreatic ductal adenocarcinoma(PDAC), to investigate the biological mechanism of miR-301a-3p in vitro and in vivo. Methods By tissue microarray analysis, we studied miR-301a-3p expression in PDAC patients and its clinicopathological correlations as well as prognostic significance. qRT-PCR was used to test miR-301a-3p expression in PDAC tissues and cell lines. Functional experiments including in vitro and in vivo were performed. Results Significantly higher expression of miR-301a-3p were found in PDAC patients with lymph node metastasis and advanced pathological stages and identified as an independent prognostic factor for worse survival. In PDAC samples and cell lines, miR-301a-3p was significantly up-regulated compared with matched non-tumor tissues and normal pancreatic ductal cells, respectively. Overexpression of miR-301a-3p enhanced PDAC cells colony, invasion and migration abilities in vitro as well as tumorigenicity in vivo. Furthermore, SMAD4 was identified as a target gene of miR-301a-3p by cell as well as mice xenograft experiments. In PDAC tissue microarray, a significantly inverse correlation between miR-301a-3p ISH scores and SMAD4 IHC scores were observed in both tumor and corresponding non-tumor tissues. Conclusion MiR-301a-3p functions as a novel oncogene in PDAC and the oncogenic activity may involve its inhibition of the target gene SMAD4. PMID:26019136

Presence of nano-sized silica during in vitro digestion of foods containing silica as a food additive.

PubMed

Peters, Ruud; Kramer, Evelien; Oomen, Agnes G; Rivera, Zahira E Herrera; Oegema, Gerlof; Tromp, Peter C; Fokkink, Remco; Rietveld, Anton; Marvin, Hans J P; Weigel, Stefan; Peijnenburg, Ad A C M; Bouwmeester, Hans

2012-03-27

The presence, dissolution, agglomeration state, and release of materials in the nano-size range from food containing engineered nanoparticles during human digestion is a key question for the safety assessment of these materials. We used an in vitro model to mimic the human digestion. Food products subjected to in vitro digestion included (i) hot water, (ii) coffee with powdered creamer, (iii) instant soup, and (iv) pancake which either contained silica as the food additive E551, or to which a form of synthetic amorphous silica or 32 nm SiO(2) particles were added. The results showed that, in the mouth stage of the digestion, nano-sized silica particles with a size range of 5-50 and 50-500 nm were present in food products containing E551 or added synthetic amorphous silica. However, during the successive gastric digestion stage, this nano-sized silica was no longer present for the food matrices coffee and instant soup, while low amounts were found for pancakes. Additional experiments showed that the absence of nano-sized silica in the gastric stage can be contributed to an effect of low pH combined with high electrolyte concentrations in the gastric digestion stage. Large silica agglomerates are formed under these conditions as determined by DLS and SEM experiments and explained theoretically by the extended DLVO theory. Importantly, in the subsequent intestinal digestion stage, the nano-sized silica particles reappeared again, even in amounts higher than in the saliva (mouth) digestion stage. These findings suggest that, upon consumption of foods containing E551, the gut epithelium is most likely exposed to nano-sized silica. © 2012 American Chemical Society

In vitro permeation of levothyroxine across the skin.

PubMed

Padula, Cristina; Pappani, Alice; Santi, Patrizia

2008-02-12

The aim of this work was to investigate the in vitro transdermal permeation characteristics of sodium levothyroxine, in view of its topical application. Permeation experiments were performed in vitro, using rabbit ear skin as barrier. At the end of the experiments levothyroxine retained in the skin was extracted and quantified by HPLC. The formulations tested were solutions and a commercial cream. The use of dimethyl beta-cyclodextrin as solubilizing agent increased to a significant extent levothyroxine solubility, but reduced its skin accumulation. Skin stripping before drug application produced a considerable increase in the amount retained and levothyroxine was found also in the receptor compartment. The application of the commercial cream in occlusive conditions increased to a significant extent drug retention in the skin. In conclusion, levothyroxine skin administration is promising in view of a localized effect, because it was retained in the skin. On the contrary, transdermal administration in view of systemic effect does not represent a concrete possibility.

Calcification of in vitro developed hypertrophic cartilage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tacchetti, C.; Quarto, R.; Campanile, G.

1989-04-01

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix. In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level ofmore » alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and /sup 45/Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.« less

Novel monitoring of corneal surface hydration during photorefractive keratectomy using pulsed photothermal radiometry: in-vitro study

NASA Astrophysics Data System (ADS)

Kawauchi, Satoko; Matsuyama, Hiroko; Obara, Minoru; Ishihara, Miya; Arai, Tsunenori; Kikuchi, Makoto; Katoh, Masayoshi

1997-05-01

We developed novel monitoring methodology for corneal surface hydration during photorefractive keratectomy (PRK) in order to solve undercorrection issue at the central part of cornea (Central island). We employed pulsed photothermal radiometry to monitor corneal surface hydration. We performed two experiments; gelatin gel experiments and porcine cornea experiments in vitro. In the case of the gelatin gel experiments, the e-folding decay time of transient infrared radiation waveform from the ArF laser irradiated surface was prolonged from 420 microsecond(s) to 30 ms with decreasing gelatin density from 15% to 0.15%. These measured e-folding decay times were good agreements with theoretical calculations. Using porcine cornea, we observed the e-folding decay time increase during the series of ArF excimer laser irradiations. Our method may be available to know ablation efficiency change to improve the controllability of refractive correction on the PRK.

Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation

USDA-ARS?s Scientific Manuscript database

In this study, plants (14) and essential oils (EO; 88) from plants that are naturalized to, or can be successfully grown in North America were evaluated in a batch culture in vitro screening experiments with ruminal fluid as potential anti-methanogenic additives for ruminant diets. Essential oils we...

In vitro/in vivo characterization of nanoemulsion formulation of metronidazole with improved skin targeting and anti-rosacea properties.

PubMed

Yu, Meng; Ma, Huixian; Lei, Mingzhu; Li, Nan; Tan, Fengping

2014-09-01

Topical skin treatment was limited due to the lack of suitable delivery system with significant cutaneous localization and systemic safety. The aim of this study was to develop and optimize a nanoemulsion (NE) to enhance targeting localization of metronidazole (MTZ) in skin layers. In vitro studies were used to optimize NE formulations, and a series of experiments were carried in vitro and in vivo to validate the therapeutic efficacy of MTZ-loaded optimal NE. NE type selection and D-optimal design study were applied to optimize NE formulation with maximum skin retention and minimum skin penetration. Three formulation variables: Oil X1 (Labrafil), Smix X2 (a mixture of Cremophor EL/Tetraethylene glycol, 2:1 w/w) and water X3 were included in D-design. The system was assessed for skin retention Y1, cumulative MTZ amount after 24 h Y2 and droplet size Y3. Following optimization, the values of formulation components (X1, X2 and X3) were 4.13%, 16.42% and 79.45%, respectively. The optimized NE was assessed for viscosity, droplet size, morphological study and in vitro permeation in pig skin. Distributions of MTZ were validated by confocal laser scanning microscopy (CLSM). Active agent of NE transferred into deeper skin and localized in epidermal/dermal layers after 24 h, which showed significant advantages of the optimal NE over Gel. The skin targeting localization and minimal systemic escape of optimal NE was further proved by in vivo study on rat skin. Current in vitro-in vivo correlation (IVIVC) enabled the prediction of pharmacokinetic profile of MTZ from in vitro permeation results. Further, the in vivo anti-rosacea efficacy of optimal formulation was investigated by pharmacodynamics study on mice ear. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro-in vivo extrapolation of zolpidem as a perpetrator of metabolic interactions involving CYP3A.

PubMed

Polasek, Thomas M; Sadagopal, Janani S; Elliot, David J; Miners, John O

2010-03-01

To evaluate zolpidem as a mechanism-based inactivator of human CYP3A in vitro, and to assess its metabolic interaction potential with CYP3A drugs (in vitro-in vivo extrapolation; IV-IVE). A co- vs. pre-incubation strategy was used to quantify time-dependent inhibition of human liver microsomal (HLM) and recombinant CYP3A4 (rCYP3A4) by zolpidem. Experiments involving a 10-fold dilution step were employed to determine the kinetic constants of inactivation (K (I) and k (inact)) and to assess the in vitro mechanism-based inactivation (MBI) criteria. Inactivation data were entered into the Simcyp population-based ADME simulator to predict the increase in the area under the plasma concentration-time curve (AUC) for orally administered midazolam. Consistent with MBI, the inhibitory potency of zolpidem toward CYP3A was increased following pre-incubation. In HLMs, the concentration required for half maximal inactivation (K (I)) was 122 microM and the maximal rate of inactivation (k (inact)) was 0.094 min(-1). In comparison, K (I) and k (inact) values with rCYP3A4 were 50 microM and 0.229 min(-1), respectively. Zolpidem fulfilled all other in vitro MBI criteria, including irreversible inhibition. The mean oral AUC for midazolam in healthy volunteers was predicted to increase 1.1- to 1.7-fold due to the inhibition of metabolic clearance by zolpidem. Elderly subjects were more sensitive to the interaction, with mean increases in midazolam AUC of 1.2- and 2.2-fold for HLM IV-IVE and rCYP3A4 IV-IVE, respectively. Zolpidem is a relatively weak mechanism-based inactivator of human CYP3A in vitro. Zolpidem is unlikely to act as a significant perpetrator of metabolic interactions involving CYP3A.

Fertility preservation 2

PubMed Central

De Vos, Michel; Smitz, Johan; Woodruff, Teresa K

2014-01-01

Enhanced long-term survival rates of young women with cancer and advances in reproductive medicine and cryobiology have culminated in an increased interest in fertility preservation methods in girls and young women with cancer. Present data suggest that young patients with cancer should be referred for fertility preservation counselling quickly to help with their coping process. Although the clinical application of novel developments, including oocyte vitrification and oocyte maturation in vitro, has resulted in reasonable success rates in assisted reproduction programmes, experience with these techniques in the setting of fertility preservation is in its infancy. It is hoped that these and other approaches, some of which are still regarded as experimental (eg, ovarian tissue cryopreservation, pharmacological protection against gonadotoxic agents, in-vitro follicle growth, and follicle transplantation) will be optimised and become established within the next decade. Unravelling the complex mechanisms of activation and suppression of follicle growth will not only expand the care of thousands of women diagnosed with cancer, but also inform the care of millions of women confronted with reduced reproductive fitness because of ageing. PMID:25283571

Application of hydroxyapatite nanoparticles in development of an enhanced formulation for delivering sustained release of triamcinolone acetonide

PubMed Central

Koocheki, Saeid; Madaeni, Sayed Siavash; Niroomandi, Parisa

2011-01-01

We report an analysis of in vitro and in vivo drug release from an in situ formulation consisting of triamcinolone acetonide (TR) and poly(d,l-lactide-co-glycolide) (PLGA) and the additives glycofurol (GL) and hydroxyapatite nanoparticles (HA). We found that these additives enhanced drug release rate. We used the Taguchi method to predict optimum formulation variables to minimize the initial burst. This method decreased the burst rate from 8% to 1.3%. PLGA-HA acted as a strong buffer, thereby preventing tissue inflammation at the injection site caused by the acidic degradation products of PLGA. Characterization of the optimized formulation by a variety of techniques, including scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, and Fourier transform near infrared spectroscopy, revealed that the crystalline structure of TR was converted to an amorphous form. Therefore, this hydrophobic agent can serve as an additive to modify drug release rates. Data generated by in vitro and in vivo experiments were in good agreement. PMID:21589650

Promotion of the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles combined with polypropylene electret.

PubMed

Tu, Ye; Wang, Xinxia; Lu, Ying; Zhang, He; Yu, Yuan; Chen, Yan; Liu, Junjie; Sun, Zhiguo; Cui, Lili; Gao, Jing; Zhong, Yanqiang

We recently reported that electret, which was prepared by a corona charging system with polypropylene film, could enhance the transdermal delivery of several drugs of low molecular weight. The aim of this study was to investigate whether electret could enhance the transdermal delivery of protein drugs by N -trimethyl chitosan nanoparticles (TMC NPs) prepared by an ionic gelation method. A series of experiments were performed, including in vitro skin permeation assays and anti-inflammatory effects, to evaluate the transdermal delivery of protein drugs by TMC NPs in the presence of electret. The results showed that in the presence of electret, the transdermal delivery of protein drugs in TMC NPs was significantly enhanced, as demonstrated by in vitro permeation studies and confocal laser scanning microscopy. Notably, superoxide dismutase-loaded TMC NPs combined with electret exhibited the best inhibitory effect on the edema of the mouse ear. TMC NPs combined with electret represent a novel platform for the transdermal delivery of protein drugs.

Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration.

PubMed

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.

Promotion of the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles combined with polypropylene electret

PubMed Central

Tu, Ye; Wang, Xinxia; Lu, Ying; Zhang, He; Yu, Yuan; Chen, Yan; Liu, Junjie; Sun, Zhiguo; Cui, Lili; Gao, Jing; Zhong, Yanqiang

2016-01-01

We recently reported that electret, which was prepared by a corona charging system with polypropylene film, could enhance the transdermal delivery of several drugs of low molecular weight. The aim of this study was to investigate whether electret could enhance the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles (TMC NPs) prepared by an ionic gelation method. A series of experiments were performed, including in vitro skin permeation assays and anti-inflammatory effects, to evaluate the transdermal delivery of protein drugs by TMC NPs in the presence of electret. The results showed that in the presence of electret, the transdermal delivery of protein drugs in TMC NPs was significantly enhanced, as demonstrated by in vitro permeation studies and confocal laser scanning microscopy. Notably, superoxide dismutase-loaded TMC NPs combined with electret exhibited the best inhibitory effect on the edema of the mouse ear. TMC NPs combined with electret represent a novel platform for the transdermal delivery of protein drugs. PMID:27822034

Environmental estrogenic compounds and the induction of hepatic vitellogenin synthesis using cultured goldfish hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Z.; Kraak, G.J. Van Der; Squires, E.J.

1995-12-31

A series of experiments were conducted to evaluate the estrogenic activity of some environmental contaminants including the {beta}-sitosterol, nonylphenol (major components of pulp mill effluent) and 3-trifluoromethyl-4-nitrophenol (TFM, a lampricide widely used in the Great Lakes), using the goldfish (Carassius auratus L.) as a model species. The in vivo exposure studies have demonstrated that all three compounds tested possess various degrees of estrogenic activity as measured by increased plasma vitellogenin (VTG) production in both the male and female fish. To understand how these compounds induce hepatic VTG synthesis and determine their potency of VTG induction, an in vitro hepatocyte culturemore » system of goldfish was established and the induction of VTG synthesis by these compounds in the cultured hepatocytes was studied. The concentration of VTG in the plasma and in the cell culture medium was determined with a enzyme-linked immunosorbent assay. Both in vivo and in vitro studies suggest that {beta}-sitosterol has the highest estrogenic activity of VTG induction.« less

Effects of the flavonoid drug quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo.

PubMed

Asea, A; Ara, G; Teicher, B A; Stevenson, M A; Calderwood, S K

2001-01-01

Tumour hyperthermia, although potentially a powerful therapeutic agent and radiation sensitizer, is hindered by a number of considerations including inhomogeneous heating of deep seated tumours due to energy deposition and perfusion issues. One solution is to design hyperthermia sensitizers to amplify the effects of hyperthermia, particularly at cold spots within the tumour undergoing treatment. This study examined the use of Quercetin, a flavonoid drug shown previously to antagonize the expression of HSP72 and induce apoptosis as a sensitizer of prostate cancer growth in vivo. Quercetin dose-dependently suppressed PC-3 tumour growth in vitro and in vivo. When combined in a treatment protocol with hyperthermia, quercetin drastically inhibited tumour growth and potently amplified the effects of hyperthermia on two prostate tumour types, PC-3 and DU-145 in vivo. These experiments, thus, suggest the use of Quercetin as a hyperthermia sensitizer in the treatment of prostate carcinoma.

Molecular and Clinical Aspects of Protein Aggregation Assays in Neurodegenerative Diseases.

PubMed

Villar-Piqué, Anna; Schmitz, Matthias; Candelise, Niccolò; Ventura, Salvador; Llorens, Franc; Zerr, Inga

2018-02-10

The presence of protein deposits is a common pathological hallmark in patients suffering from neurodegenerative conditions and other proteinopathies. Deciphering the molecular basis of protein misfolding and aggregation is a crucial step towards the full comprehension of the factors that trigger the onset of these diseases and for the development of efficient therapeutical strategies. In this regard, in vitro aggregation assays for misfolded proteins offer an excellent tool to study pathological processes of protein deposition under controlled conditions, where confounders can be easily discriminated. These methods are generally cost-effective and have been proved useful in many fields, including drug discovery and clinical diagnostics. Here, we review the bases of in vitro aggregation and seeding assays, recapitulate their main applications and offer a critical evaluation of their limitations. Comprehending the molecular mechanisms behind these assays and combining them with in vivo or cell-based experiments will maximize their potential and allow the necessary improvement to overcome some of the current drawbacks.

Estimation of intra-operator variability in perfusion parameter measurements using DCE-US

PubMed Central

Gauthier, Marianne; Leguerney, Ingrid; Thalmensi, Jessie; Chebil, Mohamed; Parisot, Sarah; Peronneau, Pierre; Roche, Alain; Lassau, Nathalie

2011-01-01

AIM: To investigate intra-operator variability of semi-quantitative perfusion parameters using dynamic contrast-enhanced ultrasonography (DCE-US), following bolus injections of SonoVue®. METHODS: The in vitro experiments were conducted using three in-house sets up based on pumping a fluid through a phantom placed in a water tank. In the in vivo experiments, B16F10 melanoma cells were xenografted to five nude mice. Both in vitro and in vivo, images were acquired following bolus injections of the ultrasound contrast agent SonoVue® (Bracco, Milan, Italy) and using a Toshiba Aplio® ultrasound scanner connected to a 2.9-5.8 MHz linear transducer (PZT, PLT 604AT probe) (Toshiba, Japan) allowing harmonic imaging (“Vascular Recognition Imaging”) involving linear raw data. A mathematical model based on the dye-dilution theory was developed by the Gustave Roussy Institute, Villejuif, France and used to evaluate seven perfusion parameters from time-intensity curves. Intra-operator variability analyses were based on determining perfusion parameter coefficients of variation (CV). RESULTS: In vitro, different volumes of SonoVue® were tested with the three phantoms: intra-operator variability was found to range from 2.33% to 23.72%. In vivo, experiments were performed on tumor tissues and perfusion parameters exhibited values ranging from 1.48% to 29.97%. In addition, the area under the curve (AUC) and the area under the wash-out (AUWO) were two of the parameters of great interest since throughout in vitro and in vivo experiments their variability was lower than 15.79%. CONCLUSION: AUC and AUWO appear to be the most reliable parameters for assessing tumor perfusion using DCE-US as they exhibited the lowest CV values. PMID:21512654

Estimation of intra-operator variability in perfusion parameter measurements using DCE-US.

PubMed

Gauthier, Marianne; Leguerney, Ingrid; Thalmensi, Jessie; Chebil, Mohamed; Parisot, Sarah; Peronneau, Pierre; Roche, Alain; Lassau, Nathalie

2011-03-28

To investigate intra-operator variability of semi-quantitative perfusion parameters using dynamic contrast-enhanced ultrasonography (DCE-US), following bolus injections of SonoVue(®). The in vitro experiments were conducted using three in-house sets up based on pumping a fluid through a phantom placed in a water tank. In the in vivo experiments, B16F10 melanoma cells were xenografted to five nude mice. Both in vitro and in vivo, images were acquired following bolus injections of the ultrasound contrast agent SonoVue(®) (Bracco, Milan, Italy) and using a Toshiba Aplio(®) ultrasound scanner connected to a 2.9-5.8 MHz linear transducer (PZT, PLT 604AT probe) (Toshiba, Japan) allowing harmonic imaging ("Vascular Recognition Imaging") involving linear raw data. A mathematical model based on the dye-dilution theory was developed by the Gustave Roussy Institute, Villejuif, France and used to evaluate seven perfusion parameters from time-intensity curves. Intra-operator variability analyses were based on determining perfusion parameter coefficients of variation (CV). In vitro, different volumes of SonoVue(®) were tested with the three phantoms: intra-operator variability was found to range from 2.33% to 23.72%. In vivo, experiments were performed on tumor tissues and perfusion parameters exhibited values ranging from 1.48% to 29.97%. In addition, the area under the curve (AUC) and the area under the wash-out (AUWO) were two of the parameters of great interest since throughout in vitro and in vivo experiments their variability was lower than 15.79%. AUC and AUWO appear to be the most reliable parameters for assessing tumor perfusion using DCE-US as they exhibited the lowest CV values.

The in vivo antitumor effects of type I-interferon against hepatocellular carcinoma: the suppression of tumor cell growth and angiogenesis.

PubMed

Enomoto, Hirayuki; Tao, Lihua; Eguchi, Ryoji; Sato, Ayuko; Honda, Masao; Kaneko, Shuichi; Iwata, Yoshinori; Nishikawa, Hiroki; Imanishi, Hiroyasu; Iijima, Hiroko; Tsujimura, Tohru; Nishiguchi, Shuhei

2017-09-22

Type I-interferon (IFN) is considered to exert antitumor effects through the inhibition of cancer cell proliferation and angiogenesis. Based on the species-specific biological activity of IFN, we evaluated each antitumor mechanism separately. We further examined the antitumor effects of type I-IFN combined with sorafenib. Human IFN (hIFN) significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) Hep3B cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Although mouse IFN (mIFN) did not inhibit the proliferation of Hep3B cells in vitro, mIFN, as well as hIFN, showed significant antitumor effects in mouse Hep3B cell-xenograft model. Furthermore, mIFN treatment amplified the antitumor effects of sorafenib in vivo with the suppression of angiogenesis. The DNA chip analysis showed that the mIFN treatment promoted the antitumor signal pathways of sorafenib, including anti-angiogenic effects. Unlike the effects observed in in vitro experiments, mIFN showed an antitumor effect in the mouse Hep3B cell-xenograft model, suggesting a role of the anti-angiogenic activity in the in vivo tumoricidal effects of type I-IFN. In addition, our findings suggested the clinical utility of combination therapy with type І-IFN and sorafenib for HCC.

A milk-based wolfberry preparation prevents prenatal stress-induced cognitive impairment of offspring rats, and inhibits oxidative damage and mitochondrial dysfunction in vitro.

PubMed

Feng, Zhihui; Jia, Haiqun; Li, Xuesen; Bai, Zhuanli; Liu, Zhongbo; Sun, Lijuan; Zhu, Zhongliang; Bucheli, Peter; Ballèvre, Olivier; Wang, Junkuan; Liu, Jiankang

2010-05-01

Lycium barbarum (Fructus Lycii, Wolfberry, or Gouqi) belongs to the Solanaceae. The red-colored fruits of L. barbarum have been used for a long time as an ingredient in Chinese cuisine and brewing, and also in traditional Chinese herbal medicine for improving health. However, its effects on cognitive function have not been well studied. In the present study, prevention of a milk-based wolfberry preparation (WP) on cognitive dysfunction was tested in a prenatal stress model with rats and the antioxidant mechanism was tested by in vitro experiments. We found that prenatal stress caused a significant decrease in cognitive function (Morris water maze test) in female offspring. Pretreatment of the mother rats with WP significantly prevented the prenatal stress-induced cognitive dysfunction. In vitro studies showed that WP dose-dependently scavenged hydroxyl and superoxide radicals (determined by an electron spin resonance spectrometric assay), and inhibited FeCl(2)/ascorbic acid-induced dysfunction in brain tissue and tissue mitochondria, including increases in reactive oxygen species and lipid peroxidation and decreases in the activities of complex I, complex II, and glutamate cysteine ligase. These results suggest that dietary supplementation with WP may be an effective strategy for preventing the brain oxidative mitochondrial damage and cognitive dysfunction associated with prenatal stress.

Ilexsaponin A attenuates ischemia-reperfusion-induced myocardial injury through anti-apoptotic pathway.

PubMed

Zhang, Shuang-Wei; Liu, Yu; Wang, Fang; Qiang, Jiao; Liu, Pan; Zhang, Jun; Xu, Jin-Wen

2017-01-01

The protective effects of ilexsaponin A on ischemia-reperfusion-induced myocardial injury were investigated. Myocardial ischemia/reperfusion model was established in male Sprague-Dawley rats. Myocardial injury was evaluated by TTC staining and myocardial marker enzyme leakage. The in vitro protective potential of Ilexsaponin A was assessed on hypoxia/reoxygenation cellular model in neonatal rat cardiomyocytes. Cellular viability and apoptosis were evaluated by MTT and TUNEL assay. Caspase-3, cleaved caspase-3, bax, bcl-2, p-Akt and Akt protein expression levels were detected by western-blot. Ilexsaponin A treatment was able to attenuate the myocardial injury in ischemia/reperfusion model by reducing myocardial infarct size and lower the serum levels of LDH, AST and CK-MB. The in vitro study also showed that ilexsaponin A treatment could increase cellular viability and inhibit apoptosis in hypoxia/reoxygenation cardiomyocytes. Proapoptotic proteins including caspase-3, cleaved caspase-3 and bax were significantly reduced and anti-apoptotic protein bcl-2 was significantly increased by ilexsaponin A treatment in hypoxia/reoxygenation cardiomyocytes. Moreover, Ilexsaponin A treatment was able to increase the expression levels of p-Akt in hypoxia/reoxygenation cellular model and myocardial ischemia/reperfusion animal model. Coupled results from both in vivo and in vitro experiments indicate that Ilexsaponin A attenuates ischemia-reperfusion-induced myocardial injury through anti-apoptotic pathway.

The S-acyl-2-thioethyl pronucleotide approach applied to acyclovir: part I. Synthesis and in vitro anti-hepatitis B virus activity of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir.

PubMed

Périgaud, C; Gosselin, G; Girardet, J L; Korba, B E; Imbach, J L

1999-01-01

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

In Vitro and In Vivo Effects of Norathyriol and Mangiferin on α-Glucosidase.

PubMed

Shi, Zhi-Long; Liu, Yi-Dan; Yuan, Yun-Yun; Song, Da; Qi, Mei-Feng; Yang, Xu-Juan; Wang, Ping; Li, Xiao-Ying; Shang, Jian-Hua; Yang, Zhao-Xiang

2017-01-01

Norathyriol is a metabolite of mangiferin. Mangiferin has been reported to inhibit α -glucosidase. To the best of our knowledge, no study has been conducted to determine or compare those two compounds on inhibiting α -glucosidase in vitro and in vivo by far. In this study, we determined the inhibitory activity of norathyriol and mangiferin on α -glucosidase in vitro and evaluated their antidiabetic effect in diabetic mice. The results showed that norathyriol inhibited α -glucosidase in a noncompetitive manner with an IC 50 value of 3.12  μ M, which is more potent than mangiferin (IC 50 = 358.54  μ M) and positive drug acarbose (IC 50 = 479.2  μ M) in the zymological experiment. Both of norathyriol and mangiferin caused significant ( p < 0.05) reduction in fasting blood glucose and the blood glucose levels at two hours after carbohydrate loading and it was interesting that mangiferin and norathyriol can make the decline of the blood glucose earlier than other groups ever including normal group in the starch tolerance test. However, norathyriol and mangiferin did not significantly influence carbohydrate absorption in the glucose tolerance test. Therefore, the antidiabetic effects of norathyriol and mangiferin might be associated with α -glucosidase, and norathyriol was more potent than mangiferin.

Cancer gene therapy with targeted adenoviruses.

PubMed

Bachtarzi, Houria; Stevenson, Mark; Fisher, Kerry

2008-11-01

Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.

Berry Phenolic Antioxidants – Implications for Human Health?

PubMed Central

Olas, Beata

2018-01-01

Antioxidants present in the diet may have a significant effect on the prophylaxis and progression of various diseases associated with oxidative stress. Berries contain a range of chemical compounds with antioxidant properties, including phenolic compounds. The aim of this review article is to provide an overview of the current knowledge of such phenolic antioxidants, and to discuss whether these compounds may always be natural gifts for human health, based on both in vitro and in vivo studies. It describes the antioxidant properties of fresh berries (including aronia berries, grapes, blueberries, sea buckthorn berries, strawberries and other berries) and their various products, especially juices and wines. Some papers report that these phenolic compounds may sometimes behave like prooxidants, and sometimes demonstrate both antioxidant and prooxidant activity, while others note they do not behave the same way in vitro and in vivo. However, no unwanted or toxic effects (i.e., chemical, hematological or urinary effect) have been associated with the consumption of berries or berry juices or other extracts, especially aronia berries and aronia products in vivo, and in vitro, which may suggest that the phenolic antioxidants found in berries are natural gifts for human health. However, the phenolic compound content of berries and berry products is not always well described, and further studies are required to determine the therapeutic doses of different berry products for use in future clinical studies. Moreover, further experiments are needed to understand the beneficial effects reported so far from the mechanistic point of view. Therefore, greater attention should be paid to the development of well-controlled and high-quality clinical studies in this area. PMID:29662448

Pacific oyster (Crassostrea gigas) hemocyte are not affected by a mixture of pesticides in short-term in vitro assays.

PubMed

Moreau, Pierrick; Burgeot, Thierry; Renault, Tristan

2014-04-01

Pesticides are frequently detected in estuaries among the pollutants found in estuarine and coastal areas and may have major ecological consequences. They could endanger organism growth, reproduction, or survival. In the context of high-mortality outbreaks affecting Pacific oysters, Crassostrea gigas, in France since 2008, it appears of importance to determine the putative effects of pesticides on oyster susceptibility to infectious agents. Massive mortality outbreaks reported in this species, mainly in spring and summer, may suggest an important role played by the seasonal use of pesticides and freshwater input in estuarine areas where oyster farms are frequently located. To understand the impact of some pesticides detected in French waters, their effects on Pacific oyster hemocytes were studied through short-term in vitro experiments. Bivalve immunity is mainly supported by hemocytes eliminating pathogens by phagocytosis and producing compounds including lysosomal enzymes and antimicrobial molecules. In this study, oyster hemocytes were incubated with a mixture of 14 pesticides and metaldehyde alone, a molecule used to eliminate land mollusks. Hemocyte parameters including dead/alive cells, nonspecific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. No significant effect of pesticides tested at different concentrations was reported on oyster hemocytes maintained in vitro for short-term periods in the present study. It could be assumed that these oyster cells were resistant to pesticide exposure in tested conditions and developing in vivo assays appears as necessary to better understand the effects of pollutants on immune system in mollusks.

Carbon Nanotubes in Biology and Medicine: In vitro and in vivo Detection, Imaging and Drug Delivery

PubMed Central

Liu, Zhuang; Tabakman, Scott; Welsher, Kevin; Dai, Hongjie

2010-01-01

Carbon nanotubes exhibit many unique intrinsic physical and chemical properties and have been intensively explored for biological and biomedical applications in the past few years. In this comprehensive review, we summarize the main results from our and other groups in this field and clarify that surface functionalization is critical to the behavior of carbon nanotubes in biological systems. Ultrasensitive detection of biological species with carbon nanotubes can be realized after surface passivation to inhibit the non-specific binding of biomolecules on the hydrophobic nanotube surface. Electrical nanosensors based on nanotubes provide a label-free approach to biological detection. Surface-enhanced Raman spectroscopy of carbon nanotubes opens up a method of protein microarray with detection sensitivity down to 1 fmol/L. In vitro and in vivo toxicity studies reveal that highly water soluble and serum stable nanotubes are biocompatible, nontoxic, and potentially useful for biomedical applications. In vivo biodistributions vary with the functionalization and possibly also size of nanotubes, with a tendency to accumulate in the reticuloendothelial system (RES), including the liver and spleen, after intravenous administration. If well functionalized, nanotubes may be excreted mainly through the biliary pathway in feces. Carbon nanotube-based drug delivery has shown promise in various In vitro and in vivo experiments including delivery of small interfering RNA (siRNA), paclitaxel and doxorubicin. Moreover, single-walled carbon nanotubes with various interesting intrinsic optical properties have been used as novel photoluminescence, Raman, and photoacoustic contrast agents for imaging of cells and animals. Further multidisciplinary explorations in this field may bring new opportunities in the realm of biomedicine. PMID:20174481

Gallic Acid Content in Taiwanese Teas at Different Degrees of Fermentation and Its Antioxidant Activity by Inhibiting PKCδ Activation: In Vitro and in Silico Studies.

PubMed

Kongpichitchoke, Teeradate; Chiu, Ming-Tzu; Huang, Tzou-Chi; Hsu, Jue-Liang

2016-10-12

Teas can be classified according to their degree of fermentation, which has been reported to affect both the bioactive components in the teas and their antioxidative activity. In this study, four kinds of commercial Taiwanese tea at different degrees of fermentation, which include green (non-fermented), oolong (semi-fermented), black (fully fermented), and Pu-erh (post-fermented) tea, were profiled for catechin levels by using high performance liquid chromatography (HPLC). The result indicated that the gallic acid content in tea was directly proportional to the degree of fermentation in which the lowest and highest gallic acid content were 1.67 and 21.98 mg/g from green and Pu-erh tea, respectively. The antioxidative mechanism of the gallic acid was further determined by in vitro and in silico analyses. In vitro assays included the use of phorbol ester-induced macrophage RAW264.7 cell model for determining the inhibition of reactive oxygen species (ROS) production, and PKCδ and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit (p47) activations. The results showed that only at a concentration of 5.00 μM could gallic acid significantly ( p < 0.05) reduce ROS levels in phorbol ester-activated macrophages. Moreover, protein immunoblotting expressed similar results in which activations of PKCδ and p47 were only significantly ( p < 0.05) attenuated by 5.00 μM treatment. Lastly, in silico experiments further revealed that gallic acid could block PKCδ activation by occupying the phorbol ester binding sites of the protein.

From in vivo to in vitro: How the Guatemala STD Experiments Transformed Bodies Into Biospecimens.

PubMed

Spector-Bagdady, Kayte; Lombardo, Paul A

2018-06-01

Policy Points: While most scholarship regarding the US Public Health Service's STD experiments in Guatemala during the 1940s has focused on the intentional exposure experiments, secondary research was also conducted on biospecimens collected from these subjects. These biospecimen experiments continued after the Guatemala grant ended, and the specimens were used in conjunction with those from the Tuskegee syphilis experiments for ongoing research. We argue there should be a public accounting of whether there are still biospecimens from the Guatemala and Tuskegee experiments held in US government biorepositories today. If such specimens exist, they should be retired from US government research archives because they were collected unethically as understood at the time. The US Public Health Service's Guatemala STD experiments (1946-1948) included intentional exposure to pathogens and testing of postexposure prophylaxis methods for syphilis, gonorrhea, and chancroid in over 1,300 soldiers, commercial sex workers, prison inmates, and psychiatric patients. Though the experiments had officially ended, the biospecimens collected from these subjects continued to be used for research at least into the 1950s. We analyzed historical documents-including clinical and laboratory records, correspondence, final reports, and medical records-for information relevant to these biospecimen experiments from the US National Archives. In addition, we researched material from past governmental investigations into the Guatemala STD experiments, including those of the US Presidential Commission for the Study of Bioethical Issues and the Guatemalan Comisión Presidencial para el Esclarecimiento de los Experimentos Practicados con Humanos en Guatemala. Identified spinal fluid, blood specimens, and tissue collected during the Guatemala diagnostic methodology and intentional exposure experiments were subsequently distributed to laboratories throughout the United States for use in ongoing research until at least 1957. Five psychiatric patient subjects involved in these biospecimen experiments died soon after experimental exposure to STDs. The same US government researchers working with the Guatemala biospecimens after the exposure experiments ended were also working with specimens taken from the Tuskegee syphilis study. There should be a complete public accounting of whether biospecimens from the Guatemala and Tuskegee experiments are held in US government biorepositories today. If they still exist, these specimens should be retired from such biorepositories and their future disposition determined by stakeholders, including representatives from the communities from which they were derived. © 2018 Milbank Memorial Fund.

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts.

PubMed

Kubisch, H M; Larson, M A; Ealy, A D; Murphy, C N; Roberts, R M

2001-04-30

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.

Evaluation of permeability alteration and epithelial-mesenchymal transition induced by transforming growth factor-β1 in A549, NCI-H441, and Calu-3 cells: Development of an in vitro model of respiratory epithelial cells in idiopathic pulmonary fibrosis.

PubMed

Togami, Kohei; Yamaguchi, Kotaro; Chono, Sumio; Tada, Hitoshi

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease, which is accompanied by changes in lung structure. With regard to treatment, aerosolized drugs administered intrapulmonarily are rapidly distributed into the plasma and do not remain in the lungs due to damage to the alveolar epithelium that occurs from pulmonary fibrosis. In this study, we sought to develop an in vitro model of respiratory epithelial cells in IPF for the evaluation of the intrapulmonary distribution of aerosolized drugs. We investigated transforming growth factor (TGF)-β 1 -induced epithelial-mesenchymal transition (EMT) and permeability alteration in A549, NCI-H441, and Calu-3 cell monolayers. After TGF-β 1 treatment of A549, NCI-H441, and Calu-3 cells, EMT markers including E-cadherin and vimentin and tight junction proteins including claudins-1, -3, and -5 were stained using immunofluorescence methods and detected using immunoblotting methods. Transport experiments were performed using TGF-β 1 -treated cell monolayers and fluorescein isothiocyanate dextrans (FD; 4.4, 10, and 70kDa). In addition, TGF-β 1 -induced apoptosis and necrosis were evaluated by flow cytometry using Annexin V and ethidium homodimer III, respectively. In NCI-H441 cells, incomplete EMT, destruction of claudins-1 and -3, and enhancement of FD permeability were caused by TGF-β 1 treatment. In A549 cells, complete EMT occurred but was not adequate for transport experiments because of low transepithelial electrical resistance. Whereas in Calu-3 cells, no changes were observed. TGF-β 1 -induced apoptosis and necrosis were not observed in any of the cell lines. Incomplete EMT and permeability enhancement were observed in the alveolar epithelium of IPF. Therefore, our results indicate that TGF-β 1 -treated NCI-H441 cell monolayers may serve as a useful in vitro model of respiratory epithelial cells for IPF. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure transition in lipids and nucleic acids of tumor cells under anticancer drugs applications

NASA Astrophysics Data System (ADS)

Dovbeshko, G. I.; Repnytska, O. P.; Tryndiak, V. P.; Todor, I. N.

2003-12-01

Interaction of DNA and phospholipids from Carcinoma Guerina resistant and sensitive cells of Wistar line rats with anti-cancer drugs - cis-platin and doxorubicin (DOX) have been studied in vivo and in vitro experiments. Surface enhanced infrared absorption (SEIRA) spectroscopy was applied for registration of conformational changes in DNA and lipids induced by anti-cancer drugs. It has been shown in vivo experiment that doxorubicin influences less structural disordering of the membrane than cis-platin. Cis-platin creates irreversible complex with memebrane phospholipids, strongly interacting with phosophates and carbohydrate chains. Doxorubicin influences the ordering of carbohydrate chains and does not strongly influence phosphate heads. This change seems to be partially reversible. In contrast, in vivo experiment the doxorubicin strongly influences the DNA structure, leading to DNA stabilization and formation of new H-bonds in DNA-doxorubicin complex. We have not registered the interaction of DNA with cis-platin in vivo experiment. Experiment in vitro for cis-platin incubation with phospholipids from cancer cells during 0.5 hour at 37°C has not shown those drastic structural peculiarities that it was observed in vivo experiments.

Rules of good practice in the care of laboratory animals used in biomedical research.

PubMed

Valanzano, Angelina

2004-01-01

In recent years, the use of laboratory animals has decreased as a result of the adoption of alternative methods such as in vitro experiments and simulation studies. Nonetheless, animal models continue to be necessary in many fields of biomedical research, giving rise to ethical issues regarding the treatment of these animals. In the present work, a general overview of the rules of good practise in caring for laboratory animals is provided, focussing on housing conditions and the proper means of handling animals, including the importance of the relationship or "bond" between the researcher and the animal.

Octenidine dihydrochloride, a modern antiseptic for skin, mucous membranes and wounds.

PubMed

Hübner, N-O; Siebert, J; Kramer, A

2010-01-01

Octenidine dihydrochloride (octenidine) was introduced for skin, mucous membrane and wound antisepsis more than 20 years ago. Until now, a wealth of knowledge has been gained, including in vitro and animal studies on efficacy, tolerance, safety and clinical experience both from case reports and prospective controlled trials. Nowadays, octenidine is an established antiseptic in a large field of applications and represents an alternative to older substances such as chlorhexidine, polyvidone-iodine or triclosan. The review is based on the current literature and unpublished original data as well. Copyright 2010 S. Karger AG, Basel.

Site and extent of starch degradation in the dairy cow - a comparison between in vivo, in situ and in vitro measurements.

PubMed

Hindle, V A; Vuuren van, A M; Klop, A; Mathijssen-Kamman, A A; van Gelder, A H; Cone, J W

2005-01-01

Prediction of the supply of glycogenic precursors to dairy cows and the site of degradation of wheat, maize and potato starch (PS) were determined in an in vivo experiment and the results were compared with data obtained from experiments involving in situ nylon bag and in vitro gas production techniques. In a Latin square design experiment four lactating dairy cows fitted with a rumen cannula and T-piece cannulae in the duodenum and terminal ileum, received either a low-starch control diet or diets in which sugar beet pulp in the concentrate mixture had been replaced by wheat, maize or PS. Starch from the different sources was almost completely degraded in the total gastrointestinal tract. For all starches, the rumen was the main site of degradation in vivo. No digestion of PS in the small intestine was observed. In situ results suggested that 14% of wheat starch (WS), 47% of maize starch and 34% of PS escaped rumen fermentation. According to the gas production technique WS ferments quickest and potato slowest. PS had a low degradability during the first 8 (gas production) to 11 (in situ) h. However, according to both in vitro and in vivo measurements rumen degradability of PS was high. The results suggest that in situ and in vitro techniques should be performed in animals that have adapted to starch source to provide a more accurate simulation of the in vivo situation.

A gene expression biomarker accurately predicts estrogen ...

EPA Pesticide Factsheets

The EPA’s vision for the Endocrine Disruptor Screening Program (EDSP) in the 21st Century (EDSP21) includes utilization of high-throughput screening (HTS) assays coupled with computational modeling to prioritize chemicals with the goal of eventually replacing current Tier 1 screening tests. The ToxCast program currently includes 18 HTS in vitro assays that evaluate the ability of chemicals to modulate estrogen receptor α (ERα), an important endocrine target. We propose microarray-based gene expression profiling as a complementary approach to predict ERα modulation and have developed computational methods to identify ERα modulators in an existing database of whole-genome microarray data. The ERα biomarker consisted of 46 ERα-regulated genes with consistent expression patterns across 7 known ER agonists and 3 known ER antagonists. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments in MCF-7 cells. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% or 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) OECD ER reference chemicals including “very weak” agonists and replicated predictions based on 18 in vitro ER-associated HTS assays. For 114 chemicals present in both the HTS data and the MCF-7 c

Oncostatic action of melatonin: facts and question marks.

PubMed

Pawlikowski, Marek; Winczyk, Katarzyna; Karasek, Michal

2002-04-01

The paper presents the data concerning the in vivo effects of melatonin on experimentally-induced tumors in animals and the in vitro effects on animal and human tumor cells. The majority of experimental tumors responded to the melatonin treatment with growth inhibition. However, some negative or opposite results (i.e. stimulation of tumor instead of inhibition) were also reported. Some of the negative results can be attributed to the improper timing of melatonin administration. Melatonin was also shown to inhibit the growth of several animal and human tumor cell lines in vitro. On the basis of these experiments, a hypothesis of the oncostatic action of melatonin was put forward. The mechanism of the postulated action is complex and probably includes: 1) modulation of the endocrine system; 2) modulation of the immune system; 3) the direct oncostatic action of melatonin on tumor cells. The latter includes the recently discovered anti-oxidative action which probably plays an important role in the countering the DNA damage during the radiation challenge or the exposure to chemical carcinogens. It also includes the antiproliferative and pro-apoptotic effects exerted via melatonin receptors expressed by tumor cells. The involvement of the membrane melatonin receptors is mainly assumed. However, the recent data from our and other laboratories suggest also the involvement of RZR/ROR receptors (the putative melatonin nuclear receptors) in both melatonin-induced proliferation inhibition and apoptosis.

A short term quality control tool for biodegradable microspheres.

PubMed

D'Souza, Susan; Faraj, Jabar A; Dorati, Rossella; DeLuca, Patrick P

2014-06-01

Accelerated in vitro release testing methodology has been developed as an indicator of product performance to be used as a discriminatory quality control (QC) technique for the release of clinical and commercial batches of biodegradable microspheres. While product performance of biodegradable microspheres can be verified by in vivo and/or in vitro experiments, such evaluation can be particularly challenging because of slow polymer degradation, resulting in extended study times, labor, and expense. Three batches of Leuprolide poly(lactic-co-glycolic acid) (PLGA) microspheres having varying morphology (process variants having different particle size and specific surface area) were manufactured by the solvent extraction/evaporation technique. Tests involving in vitro release, polymer degradation and hydration of the microspheres were performed on the three batches at 55°C. In vitro peptide release at 55°C was analyzed using a previously derived modification of the Weibull function termed the modified Weibull equation (MWE). Experimental observations and data analysis confirm excellent reproducibility studies within and between batches of the microsphere formulations demonstrating the predictability of the accelerated experiments at 55°C. The accelerated test method was also successfully able to distinguish the in vitro product performance between the three batches having varying morphology (process variants), indicating that it is a suitable QC tool to discriminate product or process variants in clinical or commercial batches of microspheres. Additionally, data analysis utilized the MWE to further quantify the differences obtained from the accelerated in vitro product performance test between process variants, thereby enhancing the discriminatory power of the accelerated methodology at 55°C.

In vitro shoot growth of Brugmansia x candida Pers.

USDA-ARS?s Scientific Manuscript database

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida 'Creamsickle'. Several mineral nutrient experiments were conducted to determine the effect of NH4+, NO3-, K+, FeSO4/EDTA, ZnSO4, MnSO4, and CuSO4 on quality, leaf width and length, size and weigh...

A Classroom Activity Debating the Ethical Issues of Preimplantation Genetic Diagnosis: A Role-Playing Exercise

ERIC Educational Resources Information Center

Staddon, William

2008-01-01

Almost three decades ago, the birth of Louise Brown in England marked the beginning of the "in vitro" fertilization (IVF) era. IVF is done to overcome infertility problems that some couples experience. Sperm and eggs are collected and fertilized in the lab. ("In vitro" means "in glass" and is used to refer to…

Cross-study projections of genomic biomarkers: an evaluation in cancer genomics.

PubMed

Lucas, Joseph E; Carvalho, Carlos M; Chen, Julia Ling-Yu; Chi, Jen-Tsan; West, Mike

2009-01-01

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a "common currency" that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies--in cancer and other diseases--have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.

Duration of senescent cell survival in vitro as a characteristic of organism longevity, an additional to the proliferative potential of fibroblasts.

PubMed

Yegorov, Yegor E; Zelenin, Alexander V

2003-04-24

More than 40 years have passed since the original publication by Hayflick and Moorhead led to the concept of the 'Hayflick limit' of the maximum number of divisions which somatic cells undergo in vitro. This concept is still regarded as a fundamental characteristic of species longevity. Here we want to emphasize another characteristic of somatic cells, namely, the duration of their survival in vitro in the non-dividing state after cessation of proliferation. This is suggested on the basis of results of recent experiments with so-called Japanese accelerated senescent mice. Results of these experiments reveal a good correlation between the longevity of the mice, the number of duplications of their fibroblasts in vitro, and the survival time of these cells in the non-dividing state. In routine culture conditions, cell survival time may be very long, as much as a few years. However, when the cells are grown under conditions of oxidative stress, cellular longevity is markedly shortened. This new test may serve as an additional marker of organismic longevity. The comparative value of both tests, the classical 'Hayflick limit' and the new test, is discussed.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations.

PubMed

Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Bakala-N'Goma, Jean-Claude; Calderone, Marilyn; Jannin, Vincent; Igonin, Annabel; Partheil, Anette; Marchaud, Delphine; Jule, Eduardo; Vertommen, Jan; Maio, Mario; Blundell, Ross; Benameur, Hassan; Carrière, Frédéric; Müllertz, Anette; Porter, Christopher J H; Pouton, Colin W

2012-09-01

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation. Copyright © 2012 Wiley Periodicals, Inc.

In vitro and in vivo Efficacy of New Blue Light Emitting Diode Phototherapy Compared to Conventional Halogen Quartz Phototherapy for Neonatal Jaundice

PubMed Central

Chang, Yun Sil; Hwang, Jong Hee; Kwon, Hyuk Nam; Choi, Chang Won; Ko, Sun Young; Park, Won Soon; Shin, Son Moon

2005-01-01

High intensity light emitting diodes (LEDs) are being studied as possible light sources for the phototherapy of neonatal jaundice, as they can emit high intensity light of narrow wavelength band in the blue region of the visible light spectrum corresponding to the spectrum of maximal bilirubin absorption. We developed a prototype blue gallium nitride LED phototherapy unit with high intensity, and compared its efficacy to commercially used halogen quartz phototherapy device by measuring both in vitro and in vivo bilirubin photodegradation. The prototype device with two focused arrays, each with 500 blue LEDs, generated greater irradiance than the conventional device tested. The LED device showed a significantly higher efficacy of bilirubin photodegradation than the conventional phototherapy in both in vitro experiment using microhematocrit tubes (44±7% vs. 35±2%) and in vivo experiment using Gunn rats (30±9% vs. 16±8%). We conclude that high intensity blue LED device was much more effective than conventional phototherapy of both in vitro and in vivo bilirubin photodegradation. Further studies will be necessary to prove its clinical efficacy. PMID:15716604

Radiolabeled cholesteryl ethers: A need to analyze for biological stability before use.

PubMed

Manual Kollareth, Denny Joseph; Chang, Chuchun L; Hansen, Inge H; Deckelbaum, Richard J

2018-03-01

Radiolabeled cholesteryl ethers are widely used as non-metabolizable tracers for lipoproteins and lipid emulsions in a variety of in vitro and in vivo experiments. Since cholesteryl ethers do not leave cells after uptake and are not hydrolyzed by mammalian cellular enzymes, these compounds can act as markers for cumulative cell uptakes of labeled particles. We have employed [ 3 H]cholesteryl oleoyl ether to study the uptake and distribution of triglyceride-rich emulsion particles on animal models. However, questionable unexpected results compelled us to analyze the stability of these ethers. We tested the stability of two commercially available radiolabeled cholesteryl ethers - [ 3 H]cholesteryl oleoyl ether and [ 3 H]cholesteryl hexadecyl ether from different suppliers, employing in vitro , in vivo and chemical model systems. Our results show that, among the two cholesteryl ethers tested, one ether was hydrolyzed to free cholesterol in vitro , in vivo and chemically under alkaline hydrolyzing agent. Free cholesterol, unlike cholesteryl ether, can then re-enter the circulation leading to confounding results. The other ether was not hydrolyzed to free cholesterol and remained as a stable ether. Hence, radiolabeled cholesteryl ethers should be analyzed for biological stability before utilizing them for in vitro or in vivo experiments.

Methods to study legionella transcriptome in vitro and in vivo.

PubMed

Faucher, Sebastien P; Shuman, Howard A

2013-01-01

The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of virulence factors. Microarrays have been previously used to study gene regulation in Legionella pneumophila. In the past few years a sharp reduction of the costs associated with microarray experiments together with the availability of relatively inexpensive custom-designed commercial microarrays has made microarray technology an accessible tool for the majority of researchers. Here we describe the methodologies to conduct microarray experiments from in vitro and in vivo samples.

A new low-profile visualized intraluminal support (LVIS) device, LVIS Blue: laboratory comparison between old and new LVIS.

PubMed

Chung, Joonho; Matsuda, Yoshikazu; Nelson, Jessica; Keigher, Kiffon; Lopes, Demetrius K

2018-01-01

Objectives The Low-profile Visualized Intraluminal Support (LVIS) device is a flexible intracranial stent. The first generation of this system had significant challenges in consistently providing good wall apposition and aneurysm neck coverage. A new modified LVIS, LVIS Blue (Blue), has been developed to address these issues. The purpose of this study is to report a laboratory comparison of wall apposition and aneurysm neck coverage between the original LVIS and Blue. Methods In bench-top experiments, we noted the visual appearance of the devices and evaluated changes in stent cell angles, neck coverage surface area (%), and stent cell crossing profile for microcatheters using a fusiform aneurysm model. Our in vitro experiments included evaluation of wall apposition and aneurysm neck coverage of the devices under direct radiographic visualization. Results Blue showed three definite different zones (a mid-zone, a high-density zone, and a transitional zone) and higher metal coverage in the straight fusiform aneurysm model compared to LVIS. Two commercially available microcatheters easily crossed the stent cell at the greater curvature for both devices. In in vitro experiments, Blue showed better wall apposition in tortuous arteries and achieved higher neck coverage in the bifurcation aneurysm compared to LVIS. Discussion Blue achieved better wall apposition in tortuous arteries and higher aneurysm neck coverage (higher metal-to-artery ratio) in bifurcation aneurysms than LVIS. Our results may provide informative physical properties of LVIS and Blue to be expected when those are used for stent-assisted coil embolization of a large-giant fusiform aneurysm or a bifurcation saccular aneurysm clinically.

Utilization of third-party in vitro fertilization in the United States.

PubMed

Kushnir, Vitaly A; Darmon, Sarah K; Shapiro, Alice J; Albertini, David F; Barad, David H; Gleicher, Norbert

2017-03-01

The use of in vitro fertilization that includes third-party in vitro fertilization is increasing. However, the relative contribution of third-party in vitro fertilization that includes the use of donor oocytes, sperm, or embryo and a gestational carrier to the birth cohort after in vitro fertilization is unknown. The purpose of this study was to examine the contribution of third-party in vitro fertilization to the in vitro fertilization birth cohort over the past decade. This retrospective analysis investigated 1,349,874 in vitro fertilization cycles that resulted in 421,525 live births and 549,367 liveborn infants in the United States from 2004-2013. Cycles were self-reported by fertility centers to a national registry: Society for Assisted Reproductive Technologies Clinic Outcome Reporting System. Third-party in vitro fertilization accounted for 217,030 (16.1%) of all in vitro fertilization cycles, 86,063 (20.4%) of all live births, and 115,024 (20.9%) of all liveborn infants. Overall, 39.7% of third-party in vitro fertilization cycles resulted in a live birth, compared with 29.6% of autologous in vitro fertilization cycles. Use of third-party in vitro fertilization increased with maternal age and accounted for 42.2% of all in vitro fertilization cycles and 75.3% of all liveborn infants among women >40 years old. Oocyte donation was the most common third-party in vitro fertilization technique, followed by sperm donation. Over the study period, annual cycle volume and live birth rates gradually increased for both autologous in vitro fertilization and third-party in vitro fertilization (P<.0001 for all). Live birth rates were the highest when multiple third-party in vitro fertilization modalities were used, followed by oocyte donation. Third-party in vitro fertilization use and efficacy have increased over the past decade, now comprising >20% of the total in vitro fertilization birth cohort. In women who are >40 years old, third-party in vitro fertilization has become the dominant treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro activity of the polyether ionophorous antibiotic monensin against the cyst form of Toxoplasma gondii.

PubMed

Couzinet, S; Dubremetz, J F; Buzoni-Gatel, D; Jeminet, G; Prensier, G

2000-10-01

Toxoplasma gondii. The experiments were conducted in vitro using 2 methods; cysts produced either in mice or in cell culture were exposed to monensin in vitro, and the infectivity of the parasites was then assessed in vivo or in vitro. The data obtained from these 2 systems of evaluation showed that monensin inhibits the infectivity and the viability of the bradyzoites. Its activity was time and concentration dependent. The first effects were observed at very low drug concentrations (i.e. 0.0001 microg/ml). Immunofluorescence and electron microscopy analysis showed significant cytological alterations of the monensin-treated bradyzoites: they were swollen, had a large number of vacuoles in their cytoplasm and were found lysed at higher concentrations in ionophore.

Fibril growth kinetics link buffer conditions and topology of 3D collagen I networks.

PubMed

Kalbitzer, Liv; Pompe, Tilo

2018-02-01

Three-dimensional fibrillar networks reconstituted from collagen I are widely used as biomimetic scaffolds for in vitro and in vivo cell studies. Various physicochemical parameters of buffer conditions for in vitro fibril formation are well known, including pH-value, ion concentrations and temperature. However, there is a lack of a detailed understanding of reconstituting well-defined 3D network topologies, which is required to mimic specific properties of the native extracellular matrix. We screened a wide range of relevant physicochemical buffer conditions and characterized the topology of the reconstituted 3D networks in terms of mean pore size and fibril diameter. A congruent analysis of fibril formation kinetics by turbidimetry revealed the adjustment of the lateral growth phase of fibrils by buffer conditions to be key in the determination of pore size and fibril diameter of the networks. Although the kinetics of nucleation and linear growth phase were affected by buffer conditions as well, network topology was independent of those two growth phases. Overall, the results of our study provide necessary insights into how to engineer 3D collagen matrices with an independent control over topology parameters, in order to mimic in vivo tissues in in vitro experiments and tissue engineering applications. The study reports a comprehensive analysis of physicochemical conditions of buffer solutions to reconstitute defined 3D collagen I matrices. By a combined analysis of network topology, i.e., pore size and fibril diameter, and the kinetics of fibril formation we can reveal the dependence of 3D network topology on buffer conditions, such as pH-value, phosphate concentration and sodium chloride content. With those results we are now able to provide engineering strategies to independently tune the topology parameters of widely used 3D collagen scaffolds based on the buffer conditions. By that, we enable the straightforward mimicking of extracellular matrices of in vivo tissues for in vitro cell culture experiments and tissue engineering applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Development of a statistical model for cervical cancer cell death with irreversible electroporation in vitro.

PubMed

Yang, Yongji; Moser, Michael A J; Zhang, Edwin; Zhang, Wenjun; Zhang, Bing

2018-01-01

The aim of this study was to develop a statistical model for cell death by irreversible electroporation (IRE) and to show that the statistic model is more accurate than the electric field threshold model in the literature using cervical cancer cells in vitro. HeLa cell line was cultured and treated with different IRE protocols in order to obtain data for modeling the statistical relationship between the cell death and pulse-setting parameters. In total, 340 in vitro experiments were performed with a commercial IRE pulse system, including a pulse generator and an electric cuvette. Trypan blue staining technique was used to evaluate cell death after 4 hours of incubation following IRE treatment. Peleg-Fermi model was used in the study to build the statistical relationship using the cell viability data obtained from the in vitro experiments. A finite element model of IRE for the electric field distribution was also built. Comparison of ablation zones between the statistical model and electric threshold model (drawn from the finite element model) was used to show the accuracy of the proposed statistical model in the description of the ablation zone and its applicability in different pulse-setting parameters. The statistical models describing the relationships between HeLa cell death and pulse length and the number of pulses, respectively, were built. The values of the curve fitting parameters were obtained using the Peleg-Fermi model for the treatment of cervical cancer with IRE. The difference in the ablation zone between the statistical model and the electric threshold model was also illustrated to show the accuracy of the proposed statistical model in the representation of ablation zone in IRE. This study concluded that: (1) the proposed statistical model accurately described the ablation zone of IRE with cervical cancer cells, and was more accurate compared with the electric field model; (2) the proposed statistical model was able to estimate the value of electric field threshold for the computer simulation of IRE in the treatment of cervical cancer; and (3) the proposed statistical model was able to express the change in ablation zone with the change in pulse-setting parameters.

Supplementing Rhodobacter sphaeroides in the diet of lactating Holstein cows may naturally produce coenzyme Q10-enriched milk

PubMed Central

Bae, Gui-Seck; Choi, Ahreum; Yeo, Joon Mo; Kim, Jong Nam; Song, Jaeyong; Kim, Eun Joong; Chang, Moon Baek

2018-01-01

Objective To examine the effects of Rhodobacter sphaeroides (R. sphaeroides) supplementation as a direct-fed microbial (DFM) on rumen fermentation in dairy cows and on coenzyme Q10 (CoQ10) transition into milk, an in vitro rumen simulation batch culture and an in vivo dairy cow experiment were conducted. Methods The characteristics of in vitro ruminal fermentation were investigated using rumen fluids from six cannulated Holstein dairy cows at 2 h post-afternoon feeding. A control treatment was included in the experiments based on a typified total mixed ration (TMR) for lactating dairy cows, which was identical to the one used in the in vivo study, plus R. sphaeroides at 0.1%, 0.3%, and 0.5% TMR dry matter. The in vivo study employed six ruminally cannulated lactating Holstein cows randomly allotted to either the control TMR (C-TMR) treatment or to a diet supplemented with a 0.5% R. sphaeroides culture (S-TMR, dry matter basis) ad libitum. The presence of R. sphaeroides was verified using denaturing gradient gel electrophoresis (DGGE) applied to the bacterial samples obtained from the in vivo study. The concentration of CoQ10 in milk and in the supernatant from the in vitro study was determined using high performance liquid chromatography. Results The results of the in vitro batch culture and DGGE showed that the concentration of CoQ10 significantly increased after 2 h of R. sphaeroides supplementation above 0.1%. When supplemented to the diet of lactating cows at the level of 0.5%, R. sphaeroides did not present any adverse effect on dry matter intake and milk yield. However, the concentration of CoQ10 in milk dramatically increased, with treated cows producing 70.9% more CoQ10 than control cows. Conclusion The CoQ10 concentration in milk increased via the use of a novel DFM, and R. sphaeroides might be used for producing value-added milk and dairy products in the future. PMID:28427254

A new in vitro lipid digestion - in vivo absorption model to evaluate the mechanisms of drug absorption from lipid-based formulations.

PubMed

Crum, Matthew F; Trevaskis, Natalie L; Williams, Hywel D; Pouton, Colin W; Porter, Christopher J H

2016-04-01

In vitro lipid digestion models are commonly used to screen lipid-based formulations (LBF), but in vitro-in vivo correlations are in some cases unsuccessful. Here we enhance the scope of the lipid digestion test by incorporating an absorption 'sink' into the experimental model. An in vitro model of lipid digestion was coupled directly to a single pass in situ intestinal perfusion experiment in an anaesthetised rat. The model allowed simultaneous real-time analysis of the digestion and absorption of LBFs of fenofibrate and was employed to evaluate the influence of formulation digestion, supersaturation and precipitation on drug absorption. Formulations containing higher quantities of co-solvent and surfactant resulted in higher supersaturation and more rapid drug precipitation in vitro when compared to those containing higher quantities of lipid. In contrast, when the same formulations were examined using the coupled in vitro lipid digestion - in vivo absorption model, drug flux into the mesenteric vein was similar regardless of in vitro formulation performance. For some drugs, simple in vitro lipid digestion models may underestimate the potential for absorption from LBFs. Consistent with recent in vivo studies, drug absorption for rapidly absorbed drugs such as fenofibrate may occur even when drug precipitation is apparent during in vitro digestion.

In vitro development of Haemoproteus parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages.

PubMed

Valkiūnas, Gediminas; Palinauskas, Vaidas; Ilgūnas, Mikas; Bernotienė, Rasa; Iezhova, Tatjana A

2014-04-01

Recent in vitro experimental studies reported the complex patterns of haemosporidian (Haemosporida) between-lineage interactions, which prevent mixing of lineages during simultaneous sexual process. Numerous anomalous ookinetes have been observed; these are not involved in sporogony. Massive development of such ookinetes might influence parasite transmission but is insufficiently investigated. The simultaneous sexual process of several lineages is a common phenomenon in vectors due to high prevalence of haemosporidian co-infections in wildlife. It remains unclear if the number of anomalous ookinetes changes during dual-infection sporogony in comparison with the single-infection process. We calculated proportions of the anomalous and normal ookinetes, which developed during single-infection (control) and dual-infection experiments in vitro conditions. Three mitochondrial cytochrome b lineages belonging to three Haemoproteus spp. (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. Sexual process and ookinete development were initiated in vitro by mixing blood containing mature gametocytes of two different parasites; the following experiments were performed: (1) Haemoproteus tartakovskyi (lineage hSISKIN1) × Haemoproteus lanii (lineage hRBS4) and (2) Haemoproteus belopolskyi (hHIICT3) × H. lanii (hRBS4). Genetic difference between lineages was 5.0-5.9%. Normal and anomalous ookinetes developed in all control and dual-infection experiments. The number of anomalous ookinetes markedly decreased, and normal ookinetes increased in all dual-infection experiments in comparison with those in controls, except for H. belopolskyi, in which proportion of the anomalous and normal ookinetes did not change. This study shows that simultaneous sexual process of two genetically distant lineages of haemosporidian parasites might increase the efficiency of reproductive cells, resulting in the development of a greater number of normal ookinetes. The marked increase of the number of normal ookinetes, which is involved in sporogony, indicates the success of sporogony in dual infections. Some haemosporidian lineages might benefit from simultaneous sporogony. Widespread avian Haemoproteus spp. are convenient and laboratory-friendly organisms for in vitro experimental research addressing between-lineage interaction in parasites during the sexual process.

NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience.

PubMed

Singh-Taylor, A; Molet, J; Jiang, S; Korosi, A; Bolton, J L; Noam, Y; Simeone, K; Cope, J; Chen, Y; Mortazavi, A; Baram, T Z

2018-03-01

Resilience to stress-related emotional disorders is governed in part by early-life experiences. Here we demonstrate experience-dependent re-programming of stress-sensitive hypothalamic neurons, which takes place through modification of neuronal gene expression via epigenetic mechanisms. Specifically, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.

78 FR 65338 - Agency Information Collection Activities; Submission for Office of Management and Budget Review...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-31

..., other clinical studies, or in vitro testing. Reports from animal testing are not included. \\3\\ Includes... serious risks from clinical trials within 15 calendar days for findings from in vitro testing that suggest...

76 FR 4113 - Independent Scientific Peer Review Panel Meeting on an In Vitro

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-24

... Vitro Estrogen Receptor Transcriptional Activation Test Method for Endocrine Disruptor Chemical... Vitro Estrogen Receptor Transcriptional Activation Test Method for Endocrine Disruptor Chemical... the information included in the BRD supports ICCVAM's draft test method recommendations. NICEATM...

Investigation of mRNA quadruplex formation in Escherichia coli.

PubMed

Wieland, Markus; Hartig, Jörg S

2009-01-01

The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

Cultivation of pathogenic Treponema pallidum in vitro.

PubMed

Horváth, I; Duncan, W P; Bullard, J C

1981-01-01

Treponema pallidum was discovered relatively late and was not cultured in vitro. Both the delineation of T. pallidum biology and the eradication of syphilis suggest the necessity of cultivation in vitro. An attempt has been made with an improved medium to cultivate pathogenic T. pallidum Budapest strain in vitro. Only in the first passage, evidence of in vitro multiplication of T. pallidum has been established by (i) macroscopic observation, (ii) darkfield examination, (iii) electron microscopic examination, (iv) optical densities, (v) tritium labelled thymidine incorporation, and (vi) the pathogenicity off the cultured organisms was evidenced by rabbit challenge. Explanation of the oxygen utilization of T. pallidum suspension is discussed. Unidentified formations were observed on electron micrographs from the 96 h cultures. They may belong to the multiplication forms of treponemes. Further experiments are needed for their identification and for expansion of the multiplication of T. pallidum beyond the first passage.

In Vivo and In Vitro Antimalarial Properties of Azithromycin-Chloroquine Combinations That Include the Resistance Reversal Agent Amlodipine ▿ †

PubMed Central

Pereira, Marcus R.; Henrich, Philipp P.; Sidhu, Amar bir Singh; Johnson, David; Hardink, Joel; Van Deusen, Jeffrey; Lin, Jian; Gore, Katrina; O'Brien, Connor; Wele, Mamadou; Djimde, Abdoulaye; Chandra, Richa; Fidock, David A.

2011-01-01

Evidence of emerging Plasmodium falciparum resistance to artemisinin-based combination therapies, documented in western Cambodia, underscores the continuing need to identify new antimalarial combinations. Given recent reports of the resurgence of chloroquine-sensitive P. falciparum parasites in Malawi, after the enforced and prolonged withdrawal of this drug, and indications of a possible synergistic interaction with the macrolide azithromycin, we sought to further characterize chloroquine-azithromycin combinations for their in vitro and in vivo antimalarial properties. In vitro 96-h susceptibility testing of chloroquine-azithromycin combinations showed mostly additive interactions against freshly cultured P. falciparum field isolates obtained from Mali. Some evidence of synergy, however, was apparent at the fractional 90% inhibitory concentration level. Additional in vitro testing highlighted the resistance reversal properties of amlodipine for both chloroquine and quinine. In vivo experiments, using the Peters 4-day suppressive test in a P. yoelii mouse model, revealed up to 99.9% suppression of parasitemia following treatment with chloroquine-azithromycin plus the R enantiomer of amlodipine. This enantiomer was chosen because it does not manifest the cardiac toxicities observed with the racemic mixture. Pharmacokinetic/pharmacodynamic analyses in this rodent model and subsequent extrapolation to a 65-kg adult led to the estimation that 1.8 g daily of R-amlodipine would be required to achieve similar efficacy in humans, for whom this is likely an unsafe dose. While these data discount amlodipine as an additional partner for chloroquine-based combination therapy, our studies continue to support azithromycin as a safe and effective addition to antimalarial combination therapies. PMID:21464242

Evaluating In Vitro-In Vivo Extrapolation of Toxicokinetics

PubMed Central

MacMillan, Denise K; Ford, Jermaine; Fennell, Timothy R; Black, Sherry R; Snyder, Rodney W; Sipes, Nisha S; Westerhout, Joost; Setzer, R Woodrow; Pearce, Robert G; Simmons, Jane Ellen; Thomas, Russell S

2018-01-01

Abstract Prioritizing the risk posed by thousands of chemicals potentially present in the environment requires exposure, toxicity, and toxicokinetic (TK) data, which are often unavailable. Relatively high throughput, in vitro TK (HTTK) assays and in vitro-to-in vivo extrapolation (IVIVE) methods have been developed to predict TK, but most of the in vivo TK data available to benchmark these methods are from pharmaceuticals. Here we report on new, in vivo rat TK experiments for 26 non-pharmaceutical chemicals with environmental relevance. Both intravenous and oral dosing were used to calculate bioavailability. These chemicals, and an additional 19 chemicals (including some pharmaceuticals) from previously published in vivo rat studies, were systematically analyzed to estimate in vivo TK parameters (e.g., volume of distribution [Vd], elimination rate). For each of the chemicals, rat-specific HTTK data were available and key TK predictions were examined: oral bioavailability, clearance, Vd, and uncertainty. For the non-pharmaceutical chemicals, predictions for bioavailability were not effective. While no pharmaceutical was absorbed at less than 10%, the fraction bioavailable for non-pharmaceutical chemicals was as low as 0.3%. Total clearance was generally more under-estimated for nonpharmaceuticals and Vd methods calibrated to pharmaceuticals may not be appropriate for other chemicals. However, the steady-state, peak, and time-integrated plasma concentrations of nonpharmaceuticals were predicted with reasonable accuracy. The plasma concentration predictions improved when experimental measurements of bioavailability were incorporated. In summary, HTTK and IVIVE methods are adequately robust to be applied to high throughput in vitro toxicity screening data of environmentally relevant chemicals for prioritizing based on human health risks. PMID:29385628

Graphene Functionalized with Arginine Decreases the Development of Glioblastoma Multiforme Tumor in a Gene-Dependent Manner

PubMed Central

Sawosz, Ewa; Jaworski, Sławomir; Kutwin, Marta; Vadalasetty, Krishna Prasad; Grodzik, Marta; Wierzbicki, Mateusz; Kurantowicz, Natalia; Strojny, Barbara; Hotowy, Anna; Lipińska, Ludwika; Jagiełło, Joanna; Chwalibog, André

2015-01-01

Our previous studies revealed that graphene had anticancer properties in experiments in vitro with glioblastoma multiforme (GBM) cells and in tumors cultured in vivo. We hypothesized that the addition of arginine or proline to graphene solutions might counteract graphene agglomeration and increase the activity of graphene. Experiments were performed in vitro with GBM U87 cells and in vivo with GBM tumors cultured on chicken embryo chorioallantoic membranes. The measurements included cell morphology, mortality, viability, tumor morphology, histology, and gene expression. The cells and tumors were treated with reduced graphene oxide (rGO) and rGO functionalized with arginine (rGO + Arg) or proline (rGO + Pro). The results confirmed the anticancer effect of graphene on GBM cells and tumor tissue. After functionalization with amino acids, nanoparticles were distributed more specifically, and the flakes of graphene were less agglomerated. The molecule of rGO + Arg did not increase the expression of TP53 in comparison to rGO, but did not increase the expression of MDM2 or the MDM2/TP53 ratio in the tumor, suggesting that arginine may block MDM2 expression. The expression of NQO1, known to be a strong protector of p53 protein in tumor tissue, was greatly increased. The results indicate that the complex of rGO + Arg has potential in GBM therapy. PMID:26512645

EFFECT OF ULTRA-HIGH PRESSURE HOMOGENIZATION ON THE INTERACTION BETWEEN BOVINE CASEIN MICELLES AND RITONAVIR

PubMed Central

Corzo-Martínez, M.; Mohan, M.; Dunlap, J.; Harte, F.

2014-01-01

Purpose The aim of this work was to develop a milk-based powder formulation appropriate for pediatric delivery of ritonavir (RIT). Methods Ultra-high pressure homogenization (UHPH) at 0.1, 300 and 500 MPa was used to process a dispersion of pasteurized skim milk (SM) and ritonavir. Loading efficiency was determined by RP-HPLC-UV; characterization of RIT:SM systems was carried out by apparent average hydrodynamic diameter and rheological measurements as well as different analytical techniques including Trp fluorescence, UV spectroscopy, DSC, FTIR and SEM; and delivery capacity of casein micelles was determined by in vitro experiments promoting ritonavir release. Results Ritonavir interacted efficiently with milk proteins, especially, casein micelles, regardless of the processing pressure; however, results suggest that, at 0.1 MPa, ritonavir interacts with caseins at the micellar surface, whilst, at 300 and 500 MPa, ritonavir is integrated to the protein matrix during UHPH treatment. Likewise, in vitro experiments showed that ritonavir release from micellar casein systems is pH dependent; with a high retention of ritonavir during simulated gastric digestion and a rapid delivery under conditions simulating the small intestine environment. Conclusions Skim milk powder, especially, casein micelles are potentially suitable and efficient carrier systems to develop novel milk-based and low-ethanol powder formulations of ritonavir appropriate for pediatric applications. PMID:25270571

Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

PubMed Central

Bao, Jialing; Yura, Renee E.; Matters, Gail L.; Bradley, S. Gaylen; Shi, Pan; Tian, Fang

2013-01-01

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation. PMID:23804454

Evaluation of automated time-lapse microscopy for assessment of in vitro activity of antibiotics.

PubMed

Ungphakorn, Wanchana; Malmberg, Christer; Lagerbäck, Pernilla; Cars, Otto; Nielsen, Elisabet I; Tängdén, Thomas

2017-01-01

This study aimed to evaluate the potential of a new time-lapse microscopy based method (oCelloScope) to efficiently assess the in vitro antibacterial effects of antibiotics. Two E. coli and one P. aeruginosa strain were exposed to ciprofloxacin, colistin, ertapenem and meropenem in 24-h experiments. Background corrected absorption (BCA) derived from the oCelloScope was used to detect bacterial growth. The data obtained with the oCelloScope were compared with those of the automated Bioscreen C method and standard time-kill experiments and a good agreement in results was observed during 6-24h of experiments. Viable counts obtained at 1, 4, 6 and 24h during oCelloScope and Bioscreen C experiments were well correlated with the corresponding BCA and optical density (OD) data. Initial antibacterial effects during the first 6h of experiments were difficult to detect with the automated methods due to their high detection limits (approximately 10 5 CFU/mL for oCelloScope and 10 7 CFU/mL for Bioscreen C), the inability to distinguish between live and dead bacteria and early morphological changes of bacteria during exposure to ciprofloxacin, ertapenem and meropenem. Regrowth was more frequently detected in time-kill experiments, possibly related to the larger working volume with an increased risk of pre-existing or emerging resistance. In comparison with Bioscreen C, the oCelloScope provided additional information on bacterial growth dynamics in the range of 10 5 to 10 7 CFU/mL and morphological features. In conclusion, the oCelloScope would be suitable for detection of in vitro effects of antibiotics, especially when a large number of regimens need to be tested. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of calcium oxide level and time of exposure to sugarcane on in vitro and in situ digestion kinetics

USDA-ARS?s Scientific Manuscript database

Experiments were carried out to evaluate, using in vitro and in situ techniques, the effects of three inclusion levels of calcium oxide (0, 5, and 10 g/kg of sugarcane fresh matter) and four exposure times (0, 24, 48, and 72 h) of sugarcane to calcium oxide on the chemical composition and digestive ...

The ovine uterus as a host for in vitro-produced bovine embryos.

PubMed

Rexroad, C E; Powell, A M

1999-07-15

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.

Circular RNA hsa_circ_0016788 regulates hepatocellular carcinoma tumorigenesis through miR-486/CDK4 pathway.

PubMed

Guan, Zheng; Tan, Jing; Gao, Wei; Li, Xin; Yang, Yuandong; Li, Xiaogang; Li, Yingchao; Wang, Qiang

2018-06-19

Recent studies have revealed that circular RNAs (circRNAs) play important roles in the tumorigenesis of human cancer, including hepatocellular carcinoma (HCC). In present study, we screen the circular RNA expression profiles in HCC tissue and investigate the molecular roles on HCC tumorigenesis. Human circRNA microarray analysis showed there were total 1,245 differently expressed circular RNAs, including 756 up-regulated circRNAs and 489 down-regulated circRNAs, in three pairs of HCC tissue and adjacent normal tissue. Hsa_circ_0016788 was identified to be up-regulated in both HCC tissue and cell lines. Loss-of-functional experiments in vivo and vitro revealed that hsa_circ_0016788 silencing inhibited the proliferation, invasion and promoted the apoptosis in vitro, and inhibited the tumor growth in vivo. Bioinformatics tools and luciferase reporter assay validated that miR-486 targeted hsa_circ_0016788 and CDK4 accompanying with negatively correlated expression, suggesting the hsa_circ_0016788/miR-486/CDK4 pathway. Receiver operating characteristic (ROC) curve showed that hsa_circ_0016788 had high diagnostic value (AUC = 0.851). In summary, results reveal the role of hsa_circ_0016788/miR-486/CDK4 in HCC tumorigenesis, providing a novel therapeutic target for HCC. © 2018 Wiley Periodicals, Inc.

Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures

NASA Technical Reports Server (NTRS)

Klaus, David M.; Benoit, Michael R.; Nelson, Emily S.; Hammond, Timmothy G.

2004-01-01

Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

Framing the ethical and legal issues of human artificial gametes in research, therapy, and assisted reproduction: A German perspective.

PubMed

Advena-Regnery, Barbara; Dederer, Hans-Georg; Enghofer, Franziska; Cantz, Tobias; Heinemann, Thomas

2018-06-01

Recent results from studies on animals suggest that functional germ cells may be generated from human pluripotent stem cells, giving rise to three possibilities: research with these so-called artificial gametes, including fertilization experiments in vitro; their use in vivo for therapy for the treatment of human infertility; and their use in assisted reproductive technologies in vitro. While the legal, philosophical, and ethical questions associated with these possibilities have been already discussed intensively in other countries, the debate in Germany is still at its beginning. A systematic and detailed analysis of the legal framework in Germany is provided with regard to the three possibilities, including the applicable statutory laws as well as the constitutional law. The question emerges as to whether the statutory laws as well as the constitution justify a distinction to be made between embryos of artificial and natural origin. This question is subject to philosophical analysis, discussing the distinction between person and thing, dignity and price, personality and property, and nature and technique. As a result, the criterion of naturalness alone may not be sufficient to differentiate between embryos of natural and artificial origin, and other criteria need to be identified. © 2018 The Authors. Bioethics Published by John Wiley & Sons Ltd.

Current limitations and recommendations to improve testing ...

EPA Pesticide Factsheets

In this paper existing regulatory frameworks and test systems for assessing potential endocrine-active chemicals are described, and associated challenges discussed, along with proposed approaches to address these challenges. Regulatory frameworks vary somewhat across organizations, but all basically evaluate whether a chemical possesses endocrine activity and whether this activity can result in adverse outcomes either to humans or the environment. Current test systems include in silico, in vitro and in vivo techniques focused on detecting potential endocrine activity, and in vivo tests that collect apical data to detect possible adverse effects. These test systems are currently designed to robustly assess endocrine activity and/or adverse effects in the estrogen, androgen, and thyroid hormonal pathways; however, there are some limitations of current test systems for evaluating endocrine hazard and risk. These limitations include a lack of certainty regarding: 1)adequately sensitive species and life-stages, 2) mechanistic endpoints that are diagnostic for endocrine pathways of concern, and 3) the linkage between mechanistic responses and apical, adverse outcomes. Furthermore, some existing test methods are resource intensive in regard to time, cost, and use of animals. However, based on recent experiences, there are opportunities to improve approaches to, and guidance for existing test methods, and to reduce uncertainty. For example, in vitro high throughput

The interplay of α-amylase and amyloglucosidase activities on the digestion of starch in in vitro enzymic systems.

PubMed

Warren, Frederick J; Zhang, Bin; Waltzer, Gina; Gidley, Michael J; Dhital, Sushil

2015-03-06

In vitro hydrolysis assays are a key tool in understanding differences in rate and extent of digestion of starchy foods. They offer a greater degree of simplicity and flexibility than dynamic in vitro models or in vivo experiments for quantifiable, mechanistic exploration of starch digestion. In the present work the influence of α-amylase and amyloglucosidase activities on the digestion of maize and potato starch granules was measured using both glucose and reducing sugar assays. Data were analysed through initial rates of digestion, and by 1st order kinetics, utilising logarithm of slope (LOS) plots. The rate and extent of starch digestion was dependent on the activities of both enzymes and the type of starch used. Potato required more enzyme than maize to achieve logarithmic reaction curves, and complete digestion. The results allow targeted design of starch digestion experiments through a thorough understanding of the contributions of α-amylase and amyloglucosidase to digestion rates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Polyketide synthases from poison hemlock (Conium maculatum L.).

PubMed

Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

2015-11-01

Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. © 2015 FEBS.

Rational Design of a New Class of Toll-Like Receptor 4 (TLR4) Tryptamine Related Agonists by Means of the Structure- and Ligand-Based Virtual Screening for Vaccine Adjuvant Discovery.

PubMed

Honegr, Jan; Dolezal, Rafael; Malinak, David; Benkova, Marketa; Soukup, Ondrej; Almeida, Joyce S F D de; Franca, Tanos C C; Kuca, Kamil; Prymula, Roman

2018-01-04

In order to identify novel lead structures for human toll-like receptor 4 ( h TLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the h TLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate h TLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

The first dedicated life sciences mission - Spacelab 4

NASA Technical Reports Server (NTRS)

Cramer, D. R.; Reid, D. H.; Klein, H. P.

1983-01-01

The details of the payload and the experiments in Spacelab 4, the first Spacelab mission dedicated entirely to the life sciences, are discussed. The payload of Spacelab 4, carried in the bay of the Shuttle Orbiter, consists of 25 tentatively selected investigations combined into a comprehensive integrated exploration of the effects of acute weightlessness on living systems. The payload contains complementary designs in the human and animal investigations in order to validate animal models of human physiology in weightlessness. Animals used as experimental subjects will include squirrel monkeys, laboratory rats, several species of plants, and frog eggs. The main scientific objectives of the investigations include the study of the acute cephalic fluid shift, cardiovascular adaptation to weightlessness, including postflight reductions in orthostatic tolerance and exercise capacity, and changes in vestibular function, including space motion sickness, associated with weightlessness. Other scientific objective include the study of red cell mass reduction, negative nitrogen balance, altered calcium metabolism, suppressed in vitro lymphocyte reactivity, gravitropism and photropism in plants, and fertilization and early development in frog eggs.

Animal experimental studies using small intestine endoscope

PubMed Central

Liu, Jin-Hua; Liu, Dan-Yang; Wang, Li; Han, Li-Ping; Qi, Zhe-Yu; Ren, Hai-Jun; Feng, Yan; Luan, Feng-Ming; Mi, Liang-Tian; Shan, Shu-Mei

2017-01-01

AIM To assess the feasibility and safety of a novel enteroscope, negative-pressure suction endoscope in examining the small intestine of a porcine model. METHODS In vitro experiments in small intestinal loops from 20 pigs and in vivo experiments in 20 living pigs were conducted. RESULTS In in vitro experiments, a negative pressure of > 0.06 MPa was necessary for optimal visualization of the intestine, and this pressure did not cause gross or histological damage to the mucosa. For satisfactory examination of the small intestine in vivo, higher negative pressure (> 1.00 MPa) was required. Despite this higher pressure, the small intestine did not show any gross or microscopic damage in the suctioned areas. The average time of examination in the living animals was 60 ± 7.67 min. The animals did not experience any apparent ill effects from the procedure. CONCLUSION Small intestine endoscope was safely performed within a reasonable time period and enabled complete visualization of the intestine in most cases. PMID:28611521

Effect of the dose of exogenous fibrolytic enzyme preparations on preingestive fiber hydrolysis, ruminal fermentation, and in vitro digestibility of bermudagrass haylage.

PubMed

Romero, J J; Zarate, M A; Adesogan, A T

2015-01-01

Our objectives were to evaluate the effects of the dose rates of 5 Trichoderma reesei and Aspergillus oryzae exogenous fibrolytic enzymes (EFE; 1A, 2A, 11C, 13D, and 15D) on in vitro digestibility, fermentation characteristics, and preingestive hydrolysis of bermudagrass haylage and to identify the optimal dose of each EFE for subsequent in vitro and in vivo studies. In experiment 1, EFE were diluted in citrate-phosphate buffer (pH 6) and applied in quadruplicate in each of 2 runs at 0× (control), 0.5×, 1×, 2×, and 3×; where 1× was the respective manufacturer-recommended dose (2.25, 2.25, 10, 15, and 15g of EFE/kg of dry matter). The suspension was incubated for 24h at 25°C and for a further 24h at 39°C after the addition of ruminal fluid. In experiment 2, a similar approach to that in experiment 1 was used to evaluate simulated preingestive effects, except that sodium azide (0.02% wt/vol) was added to the EFE solution. The suspension was incubated for 24h at 25°C and then 15mL of water was added before filtration to extract water-soluble compounds. For both experiments, data for each enzyme were analyzed separately as a completely randomized block design with a model that included effects of EFE dose, run, and their interaction. In experiment 1, increasing the EFE dose rate nonlinearly increased the DM digestibility of 1A, 2A, 11C, and 13D and the neutral detergent fiber digestibility (NDFD) of 1A, 2A, 11C, and 13D. Optimal doses of 1A, 2A, 11C, 13D, and 15D, as indicated by the greatest increases in NDFD at the lowest dose tested, were 2×, 2×, 1×, 0.5×, and 0.5×, respectively. Increasing the dose rate of 2A, 11C, and 13D nonlinearly increased concentrations of total volatile fatty acids and propionate (mM), decreased their acetate-to-propionate ratios and linearly decreased those of samples treated with 1A and 15D. In experiment 2, increasing the dose rate of each EFE nonlinearly decreased concentrations of netural detergent fiber; also, increasing the dose rate of 1A, 2A, 11C, and 1D nonlinearly increased concentrations of water-soluble carbohydrates and free ferulic acid (μg/g). Application of increasing doses of the EFE increased NDF hydrolysis, NDFD, and ruminal fluid fermentation of bermudagrass haylage, but the optimal dose varied with the EFE. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects.

PubMed

Pauloin, Thierry; Dutot, Mélody; Liang, Hong; Chavinier, Emilie; Warnet, Jean-Michel; Rat, Patrice

2009-10-01

The aim of this study was to investigate high-molecular-weight hyaluronan (HA-HMW) corneal protection against sodium lauryl sulfate (SLS)-induced toxic effects with in vitro and in vivo experimental approaches. In vitro experiments consisted of a human corneal epithelial cell line incubated with HA-HMW, rinsed, and incubated with SLS. Cell viability, oxidative stress, chromatin condensation, caspase-3, -8, -9, and P2X7 cell death receptor activation, interleukin-6, and interleukin-8 production were investigated. In vivo experiments consisted of 36 New Zealand white rabbits treated for 3 days, 3 times per day, with HA-HMW or phosphate-buffered salt solution. At day 4, eyes were treated with SLS. Clinical observation and in vivo confocal microscopy using the Rostock Cornea Module of the Heidelberg Retina Tomograph-II were performed to evaluate and to compare SLS-induced toxicity between eyes treated with HA-HMW and eyes treated with phosphate-buffered salt solution. In vitro data indicate that exposure of human corneal epithelial cells to HA-HMW significantly decreased SLS-induced oxidative stress, apoptosis, and inflammation cytokine production. In vivo data indicate that SLS cornea injuries, characterized by damaged corneal epithelium, damaged anterior stroma, and inflammatory infiltrations, were attenuated with HA-HMW treatment. A good correlation was seen between in vitro and in vivo findings showing that HA-HMW decreases SLS-induced toxic effects and protects cornea.

CALCIFICATION IN ECHINODERMS: EFFECTS OF TEMPERATURE AND DIAMOX ON INCORPORATION OF CALCIUM-45 IN VITRO BY REGENERATING SPINES OF STRONGYLOCENTROTUS PURPURATUS.

PubMed

Heatfield, Barry M

1970-08-01

1. Calcification during regeneration of experimentally fractured spines of the sea urchin, Strongylocentrotus purpuratus (Stimpson), was studied quantitatively under different conditions with calcium-45 as a tracer. 2. Fractured spines rapidly incorporated 45 Ca in vivo or in vitro after a lag period of about two days. The lag period is attributed to wound healing and reorganization of tissue at the site of fracture. 3. Additional experiments were conducted while calcification was in progress by allowing fractured spines to regenerate for four days in vivo followed by incubation in 45 Ca in vivo or in vitro up to 24 hours. In these experiments incorporation of the label was nearly linear with time and no significant difference was observed in the rate of uptake of 45 Ca between regenerating spines incubated in vivo and those from the same urchin incubated simultaneously in vitro. 4. Incorporation of 45 Ca in vitro was directly proportional to temperature between 4.7° and 20° C, at which a maximum occurred. A temperature of 26° C appeared to be lethal and little incorporation of 45 Ca took place. Values of Q 10 and the energy of activation varied inversely with temperature, with overall means of 2.72 and 15,504 calories per mole, respectively, between 4.7° and 20° C. 5. Diamox (acetazolamide) at concentrations from 10 -3 to 10 -6 M, inhibited incorporation of 45 Ca in vitro by 50% to 61%. It is inferred from these results that carbonic anhydrase is involved in calcification of regenerating spines of S. purpuratus.

Bio-active nanoemulsions enriched with gold nanoparticle, marigold extracts and lipoic acid: In vitro investigations.

PubMed

Guler, Emine; Barlas, F Baris; Yavuz, Murat; Demir, Bilal; Gumus, Z Pinar; Baspinar, Yucel; Coskunol, Hakan; Timur, Suna

2014-09-01

A novel and efficient approach for the preparation of enriched herbal formulations was described and their potential applications including wound healing and antioxidant activity (cell based and cell free) were investigated via in vitro cell culture studies. Nigella sativa oil was enriched with Calendula officinalis extract and lipoic acid capped gold nanoparticles (AuNP-LA) using nanoemulsion systems. The combination of these bio-active compounds was used to design oil in water (O/W) and water in oil (W/O) emulsions. The resulted emulsions were characterized by particle size measurements. The phenolic content of each nanoemulsion was examined by using both colorimetric assay and chromatographic analyses. Two different methods containing cell free chemical assay (1-diphenyl-2-picrylhydrazyl method) and cell based antioxidant activity test were used to evaluate the antioxidant capacities. In order to investigate the bio-activities of the herbal formulations, in vitro cell culture experiments, including cytotoxicity, scratch assay, antioxidant activity and cell proliferation were carried out using Vero cell line as a model cell line. Furthermore, to monitor localization of the nanoemulsions after application of the cell culture, the cell images were monitored via fluorescence microscope after FITC labeling. All data confirmed that the enriched N. sativa formulations exhibited better antioxidant and wound healing activity than N. sativa emulsion without any enrichment. In conclusion, the incorporation of AuNP-LA and C. officinalis extract into the N. sativa emulsions significantly increased the bio-activities. The present work may support further studies about using the other bio-active agents for the enrichment of herbal preparations to strengthen their activities. Copyright © 2014 Elsevier B.V. All rights reserved.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

PubMed

Beck, Raphaël; Pedrosa, Rozangela Curi; Dejeans, Nicolas; Glorieux, Christophe; Levêque, Philippe; Gallez, Bernard; Taper, Henryk; Eeckhoudt, Stéphane; Knoops, Laurent; Calderon, Pedro Buc; Verrax, Julien

2011-10-01

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

An Improved Small-Molecule Inhibitor of FtsZ with Superior In Vitro Potency, Drug-Like Properties, and In Vivo Efficacy

PubMed Central

Baker, Nicola; Bennett, James M.; Berry, Joanne; Collins, Ian; Czaplewski, Lloyd G.; Logan, Alastair; Macdonald, Rebecca; MacLeod, Leanne; Peasley, Hilary; Mitchell, Jeffrey P.; Nayal, Narendra; Yadav, Anju; Srivastava, Anil; Haydon, David J.

2013-01-01

The bacterial cell division protein FtsZ is an attractive target for small-molecule antibacterial drug discovery. Derivatives of 3-methoxybenzamide, including compound PC190723, have been reported to be potent and selective antistaphylococcal agents which exert their effects through the disruption of intracellular FtsZ function. Here, we report the further optimization of 3-methoxybenzamide derivatives towards a drug candidate. The in vitro and in vivo characterization of a more advanced lead compound, designated compound 1, is described. Compound 1 was potently antibacterial, with an average MIC of 0.12 μg/ml against all staphylococcal species, including methicillin- and multidrug-resistant Staphylococcus aureus and Staphylococcus epidermidis. Compound 1 inhibited an S. aureus strain carrying the G196A mutation in FtsZ, which confers resistance to PC190723. Like PC190723, compound 1 acted on whole bacterial cells by blocking cytokinesis. No interactions between compound 1 and a diverse panel of antibiotics were measured in checkerboard experiments. Compound 1 displayed suitable in vitro pharmaceutical properties and a favorable in vivo pharmacokinetic profile following intravenous and oral administration, with a calculated bioavailability of 82.0% in mice. Compound 1 demonstrated efficacy in a murine model of systemic S. aureus infection and caused a significant decrease in the bacterial load in the thigh infection model. A greater reduction in the number of S. aureus cells recovered from infected thighs, equivalent to 3.68 log units, than in those recovered from controls was achieved using a succinate prodrug of compound 1, which was designated compound 2. In summary, optimized derivatives of 3-methoxybenzamide may yield a first-in-class FtsZ inhibitor for the treatment of antibiotic-resistant staphylococcal infections. PMID:23114779

Genotoxicity testing approaches for the safety assessment of substances used in food contact materials prior to their authorization in the European Union.

PubMed

Bolognesi, Claudia; Castoldi, Anna F; Crebelli, Riccardo; Barthélémy, Eric; Maurici, Daniela; Wölfle, Detlef; Volk, Katharina; Castle, Laurence

2017-06-01

Food contact materials are all materials and articles intended to come directly or indirectly into contact with food. Before being included in the positive European "Union list" of authorized substances (monomers, other starting substances and additives) for plastic food contact materials, the European Food Safety Authority (EFSA) must assess their safety "in use". If relevant for risk, the safety of the main impurities, reaction and degradation products originating from the manufacturing process is also evaluated. Information on genotoxicity is always required irrespective of the extent of migration and the resulting human exposure, in view of the theoretical lack of threshold for genotoxic events. The 2008 EFSA approach, requiring the testing of food contact materials in three in vitro mutagenicity tests, though still acceptable, is now superseded by the 2011 EFSA Scientific Committee's recommendation for only two complementary tests including a bacterial gene mutation test and an in vitro micronucleus test, to detect two main genetic endpoints (i.e., gene mutations and chromosome aberrations). Follow-up of in vitro positive results depends on the type of genetic effect and on the substance's systemic availability. In this study, we provide an analysis of the data on genotoxicity testing gathered by EFSA on food contact materials for the period 1992-2015. We also illustrate practical examples of the approaches that EFSA took when evaluating "non standard" food contact chemicals (e.g., polymeric additives, oligomer or other reaction mixtures, and nanosubstances). Additionally, EFSA's experience gained from using non testing methods and/or future possibilities in this area are discussed. Environ. Mol. Mutagen. 58:361-374, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

In vitro to in vivo extrapolation of hepatic metabolism in fish: An inter-laboratory comparison of in vitro methods - presentation

EPA Science Inventory

Chemical biotransformation represents the largest source of uncertainty in chemical bioaccumulation assessments. Model-based estimates of chemical bioconcentration in fish may be greatly improved by including biotransformation rates, as measured in vitro. Substrate depletion assa...

Designing a Microfluidic Device with Integrated Ratiometric Oxygen Sensors for the Long-Term Control and Monitoring of Chronic and Cyclic Hypoxia

PubMed Central

Grist, Samantha M.; Schmok, Jonathan C.; Liu, Meng-Chi (Andy); Chrostowski, Lukas; Cheung, Karen C.

2015-01-01

Control of oxygen over cell cultures in vitro is a topic of considerable interest, as chronic and cyclic hypoxia can alter cell behaviour. Both static and transient hypoxic levels have been found to affect tumour cell behaviour; it is potentially valuable to include these effects in early, in vitro stages of drug screening. A barrier to their inclusion is that rates of transient hypoxia can be a few cycles/hour, which is difficult to reproduce in traditional in vitro cell culture environments due to long diffusion distances from control gases to the cells. We use a gas-permeable three-layer microfluidic device to achieve spatial and temporal oxygen control with biologically-relevant switching times. We measure the oxygen profiles with integrated, ratiometric optical oxygen sensors, demonstrate sensor and system stability over multi-day experiments, and characterize a pre-bleaching process to improve sensor stability. We show, with both finite-element modelling and experimental data, excellent control over the oxygen levels by the device, independent of fluid flow rate and oxygenation for the operating flow regime. We measure equilibration times of approximately 10 min, generate complex, time-varying oxygen profiles, and study the effects of oxygenated media flow rates on the measured oxygen levels. This device could form a useful tool for future long-term studies of cell behaviour under hypoxia. PMID:26287202

NO Dioxygenase Activity in Hemoglobins Is Ubiquitous In Vitro, but Limited by Reduction In Vivo

PubMed Central

Smagghe, Benoit J.; Trent, James T.; Hargrove, Mark S.

2008-01-01

Genomics has produced hundreds of new hemoglobin sequences with examples in nearly every living organism. Structural and biochemical characterizations of many recombinant proteins reveal reactions, like oxygen binding and NO dioxygenation, that appear general to the hemoglobin superfamily regardless of whether they are related to physiological function. Despite considerable attention to “hexacoordinate” hemoglobins, which are found in nearly every plant and animal, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is protection against NO. Here we have tested a comprehensive representation of hexacoordinate hemoglobins from plants (rice hemoglobin), animals (neuroglobin and cytoglobin), and bacteria (Synechocystis hemoglobin) for their abilities to scavenge NO compared to myoglobin. Our experiments include in vitro comparisons of NO dioxygenation, ferric NO binding, NO-induced reduction, NO scavenging with an artificial reduction system, and the ability to substitute for a known NO scavenger (flavohemoglobin) in E. coli. We conclude that none of these tests reveal any distinguishing predisposition toward a role in NO scavenging for the hxHbs, but that any hemoglobin could likely serve this role in the presence of a mechanism for heme iron re-reduction. Hence, future research to test the role of Hbs in NO scavenging would benefit more from the identification of cognate reductases than from in vitro analysis of NO and O2 binding. PMID:18446211

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

PubMed

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells

PubMed Central

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A.; Twiss, Jeffery L.

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5’ untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5’ UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality. PMID:27224031

Effect of deuteration on metabolism and clearance of Nerispirdine (HP184) and AVE5638.

PubMed

Schofield, Joseph; Derdau, Volker; Atzrodt, Jens; Zane, Patricia; Guo, Zuyu; van Horn, Robert; Czepczor, Valérie; Stoltz, Axelle; Pardon, Magalie

2015-07-01

Replacing hydrogen with deuterium as a means of altering ADME properties of drug molecules has recently enjoyed a renaissance, such that at least two deuterated chemical entities are currently in clinical development. Although most research in this area aims to increase the metabolic stability, and hence half-life of the active species, experience has shown that prediction of the in vivo behaviour of deuterated molecules is difficult and depends on multiple factors including the complexity of the metabolic scheme, the enzymes involved and hence the mechanism of the rate-determining step in the biotransformation. In an effort to elucidate some of these factors we examined the metabolic behaviour of two molecules from the Sanofi portfolio in a range of in vitro and in vivo systems. Although some key metabolic reactions of the acetylcholine release stimulator HP184 4 were slowed in vitro and in vivo when deuterium was present at the sites of metabolism, this did not translate to an increase in overall metabolic stability. By contrast, the tryptase inhibitor AVE5638 13 was much more metabolically stable in vitro in its deuterated form than when unlabelled. These results indicate that it could be of value to concentrate efforts in this area to molecules which are metabolised by a major pathway that involves enzymes of the amine oxidase family or other low-capacity enzyme families. Copyright © 2015 Elsevier Ltd. All rights reserved.

Induction of suppressor cells in vitro by Candida albicans.

PubMed

Cuff, C F; Rogers, C M; Lamb, B J; Rogers, T J

1986-06-01

Normal splenocytes cultured with Formalin-killed Candida albicans were shown to acquire significant suppressor cell activity in a period of 3 days. These cells were found to suppress both the phytohemagglutinin-induced mitogen response as well as the anti-sheep erythrocyte antibody response. Experiments were carried out to determine the nature of the suppressor cell population. Results showed that these cells were not susceptible to treatment with anti-Thy 1 antibody and complement. Panning experiments showed that the suppressor cells were not plastic-adherent or Mac-1 antigen-positive. The suppressor cells were, however, adherent to anti-mouse immunoglobulin (F(ab')2-fragment)-coated dishes. Additional experiments showed that the suppressor cell activity was susceptible to treatment with monoclonal anti-Lyb 2.1 antibody and complement. These results suggest that the suppressor cell induced in vitro by Candida is a member of the B-lymphocyte lineage.

Pestalpolyols A-D, Cytotoxic Polyketides from Pestalotiopsis sp. cr013.

PubMed

Li, Jing; Xie, Jin; Yang, Yin-He; Li, Xiao-Lian; Zeng, Ying; Zhao, Pei-Ji

2015-09-01

Four novel polyketides, named pestalpolyols A (1), B (2), C (3), and D (4), were isolated from solid fermentation products of Pestalotiopsis sp. cr013. Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry experiments, and the absolute configuration was confirmed by single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation. The inhibitory activities of compounds 1, 2, and 4 against five human tumor lines were tested in vitro, and showed IC50 values 2.3-31.2 µM. Georg Thieme Verlag KG Stuttgart · New York.

An Update on Oligosaccharides and Their Esters from Traditional Chinese Medicines: Chemical Structures and Biological Activities

PubMed Central

Chen, Xiang-Yang; Wang, Ru-Feng; Liu, Bin

2015-01-01

A great number of naturally occurring oligosaccharides and oligosaccharide esters have been isolated from traditional Chinese medicinal plants, which are used widely in Asia and show prominent curative effects in the prevention and treatment of kinds of diseases. Numerous in vitro and in vivo experiments have revealed that oligosaccharides and their esters exhibited various activities, including antioxidant, antidepressant, cytotoxic, antineoplastic, anti-inflammatory, neuroprotective, cerebral protective, antidiabetic, plant growth-regulatory, and immunopotentiating activities. This review summarizes the investigations on the distribution, chemical structures, and bioactivities of natural oligosaccharides and their esters from traditional Chinese medicines between 2003 and 2013. PMID:25861364

Phase separation and the formation of cellular bodies

NASA Astrophysics Data System (ADS)

Xu, Bin; Broedersz, Chase P.; Meir, Yigal; Wingreen, Ned S.

Cellular bodies in eukaryotic cells spontaneously assemble to form cellular compartments. Among other functions, these bodies carry out essential biochemical reactions. Cellular bodies form micron-sized structures, which, unlike canonical cell organelles, are not surrounded by membranes. A recent in vitro experiment has shown that phase separation of polymers in solution can explain the formation of cellular bodies. We constructed a lattice-polymer model to capture the essential mechanism leading to this phase separation. We used both analytical and numerical tools to predict the phase diagram of a system of two interacting polymers, including the concentration of each polymer type in the condensed and dilute phase.

Assessment of cardiac time intervals using high temporal resolution real-time spiral phase contrast with UNFOLDed-SENSE.

PubMed

Kowalik, Grzegorz T; Knight, Daniel S; Steeden, Jennifer A; Tann, Oliver; Odille, Freddy; Atkinson, David; Taylor, Andrew; Muthurangu, Vivek

2015-02-01

To develop a real-time phase contrast MR sequence with high enough temporal resolution to assess cardiac time intervals. The sequence utilized spiral trajectories with an acquisition strategy that allowed a combination of temporal encoding (Unaliasing by fourier-encoding the overlaps using the temporal dimension; UNFOLD) and parallel imaging (Sensitivity encoding; SENSE) to be used (UNFOLDed-SENSE). An in silico experiment was performed to determine the optimum UNFOLD filter. In vitro experiments were carried out to validate the accuracy of time intervals calculation and peak mean velocity quantification. In addition, 15 healthy volunteers were imaged with the new sequence, and cardiac time intervals were compared to reference standard Doppler echocardiography measures. For comparison, in silico, in vitro, and in vivo experiments were also carried out using sliding window reconstructions. The in vitro experiments demonstrated good agreement between real-time spiral UNFOLDed-SENSE phase contrast MR and the reference standard measurements of velocity and time intervals. The protocol was successfully performed in all volunteers. Subsequent measurement of time intervals produced values in keeping with literature values and good agreement with the gold standard echocardiography. Importantly, the proposed UNFOLDed-SENSE sequence outperformed the sliding window reconstructions. Cardiac time intervals can be successfully assessed with UNFOLDed-SENSE real-time spiral phase contrast. Real-time MR assessment of cardiac time intervals may be beneficial in assessment of patients with cardiac conditions such as diastolic dysfunction. © 2014 Wiley Periodicals, Inc.

Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

PubMed

Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

2017-01-01

Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

Use of piracetam improves sickle cell deformability in vitro and in vivo.

PubMed Central

Gini, E K; Sonnet, J

1987-01-01

Microsieving diluted suspensions of oxygenated sickle cell anaemia (HbSS) cells on polycarbonate filters shows that piracetam improves the red cell deformability in vitro. In vivo an oral intake of 160 mg/kg/day divided in four doses enhances the HbSS cell deformability as actively as it does in in vitro experiments. The drug is also able partially to restore the impaired deformability of physiologically deoxygenated HbSS cells. These findings are consistent with the results of clinical trials, which show that continuous treatment with piracetam reduces the incidence of vaso-occlusive crises in patients with sickle cell disease. PMID:3818978

Effect of vitro preservation on mechanical properties of brain tissue

NASA Astrophysics Data System (ADS)

Zhang, Wei; Liu, Yi-fan; Liu, Li-fu; Niu, Ying; Ma, Jian-li; Wu, Cheng-wei

2017-05-01

To develop the protective devices for preventing traumatic brain injuries, it requires the accurate characterization of the mechanical properties of brain tissue. For this, it necessary to elucidate the effect of vitro preservation on the mechanical performance of brain tissue as usually the measurements are carried out in vitro. In this paper, the thermal behavior of brain tissue preserved for various period of time was first investigated and the mechanical properties were also measured. Both reveals the deterioration with prolonged preservation duration. The observations of brain tissue slices indicates the brain tissue experiences karyorrhexis and karyorrhexis in sequence, which accounts for the deterioration phenomena.

In Vitro Biodegradation of Hepatotoxic Indospicine in Indigofera spicata and Its Degradation Derivatives by Camel Foregut and Cattle Rumen Fluids.

PubMed

Tan, Eddie T T; Al Jassim, Rafat; D'Arcy, Bruce R; Fletcher, Mary T

2017-08-30

The known accumulation of the hepatotoxin indospicine in tissues of camels and cattle grazing Indigofera pasture plants is unusual in that free amino acids would normally be expected to be degraded during the fermentation processes in these foregut fermenters. In this study, in vitro experiments were carried out to examine the degradability of indospicine of Indigofera spicata by camel and cattle foregut microbiota. In the first experiment, a 48 h in vitro incubation was carried out using foregut fluid samples that were collected from 15 feral camels and also a fistulated cow. Degradability of indospicine ranged between 97% and 99%, with the higher value of 99% for camels. A pooled sample of foregut fluids from three camels that were on a roughage diet was used in a second experiment to examine the time-dependent degradation of indospicine present in the plant materials. Results indicated that camels' foregut fluids have the ability to biodegrade ∼99% of the indospicine in I. spicata within 48 h of incubation and produced 2-aminopimelamic acid and 2-aminopimelic acid. The time-dependent degradation analysis showed rapid indospicine degradation (65 nmol/h) during the first 8-18 h of incubation followed by a slower degradation rate (12 nmol/h) between 18 and 48 h. Indospicine degradation products were also degraded toward the end of the experiment. The results of these in vitro degradation studies suggest that dietary indospicine may undergo extensive degradation in the foregut of the camel, resulting in trace levels after 48 h. The retention time for plant material in the camel foregut varies depending on feed quality, and the results of this study together with the observed accumulation of indospicine in camel tissues suggest that, although indospicine can be degraded by foregut fermentation, this degradation is not complete before the passage of the digesta into the intestine.

Utilization of a terrestrial cyanobacterium, Nostoc sp. HK-01, for space habitation

NASA Astrophysics Data System (ADS)

Kimura, Shunta; Tomita-Yokotani, Kaori; Arai, Mayumi; Yamashita, Masamichi; Katoh, Hiroshi; Ajioka, Reiko; Inoue, Kotomi

2016-07-01

A terrestrial cyanobacterium, Nostoc sp. HK-01 (hereafter HK-01), has several useful abilities for space habitation; photosynthesis, nitrogen fixation, and space environmental tolerances to vacuum, UV, gamma-ray, heavy particle beam, low and high temperature. Space environmental tolerances are important for transportation to Mars. HK-01 can grow on Martian regolith simulant (MRS) in vitro. Furthermore, HK-01 is useful as food. HK-01 may be utilized as oxygen supply, soil formation and food material for bio-chemical circulation in closed bio-ecosystems, including space habitation such as Mars. HK-01 was adopted as a biological material for the "TANPOPO" mission (JAXA et al.,), because of their high environmental tolerances. The "TANPOPO" mission is performing the space exposure experiments on the Japan Experimental Module (JEM) of the International Space Station (ISS). The results of these experiments will show the ability of HK-01 to survive in space.

Metabolites characterization of chamaechromone in vivo and in vitro by using ultra-performance liquid chromatography/Xevo G2 quadrupole time-of-flight tandem mass spectrometry.

PubMed

Lou, Yan; Zheng, Jinqi; Wang, Baohong; Zhang, Xingguo; Zhang, Xia; Zeng, Su

2014-01-01

Stellera chamaejasme L. (Thymelaeaceae) was a toxic perennial herb and widely used as pesticide and dermatological agents in China. Chamaechromone was a major component in the dried roots of Stellera chamaejasme with anti-HBV and insecticidal activity. Analysis of metabolic profile in vivo and in vitro plays a pivotal role to unravel how TCM works. And the metabolites of chamaechromone might influence the effects and toxicity of Stellera chamaejasme. Moreover, the metabolic routes of chamaechromone provide an important basis for toxicological safety evaluation. Until now, little is known about the metabolism of chamaechromone. The current study was designed to characterize the whole metabolic pathways of chamaechromone in vitro and in vivo. Twenty-four rats were randomly divided into four groups, including two oral administration groups (100mgkg(-1)), one intravenous injection group (5 mgkg(-1)), and one control group. The metabolites in rat urine and feces and bile were identified by UPLC/Q-TOF MS analysis and β-glucuronidase hydrolysis. Moreover, the possible metabolic mechanism was further confirmed by Phase I and Phase II metabolism and catechol-O-methyltransferase methylation in rat liver S9 fraction and degradation in rat intestinal bacteria. A total of 24 metabolites from chamaechromone were detected and identified in vivo and in vitro, 20 of which were novel. And the major metabolic processes were hydroxylation, methylation, glucuronation, acetylation, dehydroxylation and degradation. The present study revealed the whole metabolic pathways of chamaechromone in rat through both in vitro and in vivo experiments for the first time. And chamaechromone could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of chamaechromone. In addition, the results of this work have showed the feasibility of the UPLC/Q-TOF-MS approach for rapid and reliable characterization of metabolites. © 2013 Elsevier Ireland Ltd. All rights reserved.

Practical aspects of running DOE for improving growth media for in vitro plants

USDA-ARS?s Scientific Manuscript database

Experiments using DOE software to improve plant tissue culture growth medium are complicated and require complex setups. Once the experimental design is set and the treatment points calculated, media sheets and mixing charts must be developed. Since these experiments require three passages on the sa...

Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing β-amino acid residues - a lesson about the importance of animal experiments.

PubMed

Sparr, Christof; Purkayastha, Nirupam; Yoshinari, Tomohiro; Seebach, Dieter; Maschauer, Simone; Prante, Olaf; Hübner, Harald; Gmeiner, Peter; Kolesinska, Beata; Cescato, Renzo; Waser, Beatrice; Reubi, Jean Claude

2013-12-01

Neurotensin(8-13) (NTS(8-13)) analogs with C- and/or N-terminal β-amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1-6). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, 6a, into a crystallographically identified receptor NTSR1 (Fig.1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell-membrane homogenates, while, with NTSR1-exhibiting cancer tissues, affinities in the single-digit nanomolar range can be observed (Table 2). Most of the β-amino acid-containing NTS(8-13) analogs (Table 1 and Fig.2), including the (68) Ga complexes of the DOTA-substituted ones (6; Figs.2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two (68) Ga complexes (of 6a and 6b) in HT29 tumor-bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET-imaging experiments with the tumor-bearing mice (Fig.6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10-15 min) of the two (68) Ga complexes shows that they are rapidly cleaved in the animals (Fig.5). Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

β-Glucan from Saccharomyces cerevisiae Induces IFN-γ Production In Vivo in BALB/c Mice.

PubMed

Javmen, Artur; Nemeikaitė-Čėnienė, Aušra; Bratchikov, Maksim; Grigiškis, Saulius; Grigas, Fortūnatas; Jonauskienė, Irena; Zabulytė, Danguolė; Mauricas, Mykolas

2015-01-01

β-Glucan is one of the most abundant polymers in nature and has been established as an immunomodulator. This compound has notable physiological effects on mammalian immune systems, including anti-tumor and anti-infective activities and can activate the immune response. It is considered that the immune-stimulating activities of β-glucan can depend on physicochemical parameters, such as molecular size. Saccharomyces cerevisiae, also known as baker's yeast, is a frequently used source of β-glucan. The aim of the experiments was to investigate how different Saccharomyces cerevisiae β-glucan preparations with different molecular size affect interferon-gamma (IFN-γ) production in BALB/c mice. In vivo and in vitro BALB/c mouse models were used for the investigations. Different β-glucan preparations were orally administrated in the in vivo experiments. IFN-γ production in BALB/c mice was analyzed by enzyme-linked immunosorbent assay and measuring interferon-γ RNA concentration. The results showed that orally-administered β-glucan from S. cerevisiae enhanced IFN-γ production in BALB/c mice in the in vivo model, but not by mouse leukocytes in vitro. Moreover, water-soluble β-glucan enhanced IFN-γ production more effectively than did particulate β-glucan. IFN-γ plays an important role in immunity against viral and bacterial infections. Our experiments have shown that β-glucan preparations enhance IFN-γ production in BALB/c mice and can be potentially used for immune system stimulation in mammals. Current results may be used to develop soluble β-glucan nutritional supplements. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

In vitro and in vivo evaluation of a new nanocomposite, containing high density polyethylene, tricalcium phosphate, hydroxyapatite, and magnesium oxide nanoparticles.

PubMed

Pourdanesh, Fereydoun; Jebali, Ali; Hekmatimoghaddam, Seyedhossein; Allaveisie, Azra

2014-07-01

In this study, a new nanocomposite, which contained high density polyethylene (HDPE), tricalcium phosphate (Ca3(PO4)2) nanoparticles (TCP NPs), hydroxyapatite nanoparticles (HA NPs), and magnesium oxide nanoparticles (MgO NPs) was prepared. As in vitro experiment, human osteoblasts (HOB) cells were exposed to pristine HDPE and its nanocomposite for a period of 1, 4, and 7 days at 37 °C, and then different assays were carried out, including osteoblast cell proliferation, Trypan blue staining, cell viability, alkaline phosphatase (ALP), and cell adhesion. Antibacterial property of pristine HDPE and its nanocomposite was evaluated, and also their mechanical properties were measured after 2 and 4 months. As in vivo experiment, pristine HDPE and its nanocomposite were separately implanted on calvarium bone of rabbits, and tissue inflammation and osteogenesis were investigated after 2, 4, and 6 months. In case of HOB cells treated with HDPE or nanocomposite, as incubation time was increased, cell proliferation, live/dead ratio, and cell viability were decreased. But, the ALP activity and cell adhesion of HOB cells which treated with nanocomposite were raised after increase of incubation time. This study demonstrated that although the mechanical properties of nanocomposite were similar to HDPE sheet, but their antibacterial property was not similar. The in vivo experiment showed that both pristine HDPE and its nanocomposite had same inflammation responses. Interestingly, osteogenesis was observed after 2 months at bone/nanocomposite interface, and was highly increased after 4 and 6 months. It must be noted that such pattern was not seen at bone/HDPE interface. Copyright © 2014 Elsevier B.V. All rights reserved.

Curcumin synergistically increases effects of β-interferon and retinoic acid on breast cancer cells in vitro and in vivo by up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways.

PubMed

Ren, Min; Wang, Ying; Wu, Xiaodong; Ge, Suxia; Wang, Benzhong

2017-03-01

The study aimed to investigate the effects of combination treatment of curcumin and β-interferon (IFN-β)/retinoic acid (RA) on breast cancer cells, including cell viability, apoptosis and migration, and to determine the mechanisms related to GRIM-19 through STAT3-dependent and STAT3-independent pathways. The following groups were used for the in vitro experiment: control siRNA, GRIM-19 siRNA, IFN-β/RA and IFN-β/RA + curcumin. Cell viability is by the MTT method, cell apoptosis by flow cytometry and cell migration by wound healing experiment; GRIM-19, STAT3, survivin, Bcl-2, GADD153 and COX-2 expression was measured by Western blot. In vivo experiment, MCF-7 cells were subcutaneously injected into nude mice. GRIM-19 siRNA promoted MCF-7 cell proliferation and migration; inhibited cell apoptosis; and promoted the expression of STAT3, survivin, Bcl-2 and MMP-9. IFN-β/RA inhibited cell proliferation and migration; promoted cell apoptosis; up-regulated GRIM-19; and inhibited the expression of STAT3, survivin, Bcl-2 and MMP-9. Combination treatment of curcumin and IFN-β/RA had a stronger effect than that of the IFN-β/RA group. In addition, curcumin and IFN-β/RA combination inhibited the expression of COX-2 and up-regulated GADD153. Curcumin synergistically increases the effects of IFN-β/RA on breast cancer cells. The mechanism may be related to the up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways.

Somatic symptoms, sleep disturbance and psychological distress among women undergoing oocyte pick-up and in vitro fertilisation-embryo transfer.

PubMed

Lin, Ya-Hui; Chueh, Ke-Hsin; Lin, Jia-Ling

2016-06-01

This study investigated the relationship between somatic symptoms, sleep disturbance and psychological distress in women who underwent oocyte pick-up and in vitro fertilisation-embryo transfer. According to worldwide research, women receiving assisted reproductive technologies may suffer from somatic and psychological symptoms and even experience sleep disturbance. Apparently, the guilt of infecundity forces Asian women to conceal this scenario and delay the time at which they accept medical assistance and mental support. A longitudinal study. The subjects in this study were infertile female patients who received oocyte pick-up and in vitro fertilisation-embryo transfer therapies in a hospital in northern Taiwan. Data were collected via a structured questionnaire, including somatic symptoms, Pittsburgh Sleep Quality Index and a five-item brief symptom rating scale. Data were analysed using the McNemar's test, Wilcoxon Sign Rank and fully entered multiple regression with spss version 20.0 software. The mean age of 100 participants was 34·54 (SD = 3·94) years old. They experienced abdominal distention, breast engorgement, nausea, faintness, diarrhoea, sleep disturbance and psychological distress when they received in vitro fertilisation-embryo transfer; these results were apparently higher than those receiving oocyte pick-up. In addition, sleep disturbance was the most significant factor involved in psychological distress during oocyte pick-up and in vitro fertilisation-embryo transfer therapies. The most serious indicator of the women's psychological distress during oocyte pick-up and in vitro fertilisation-embryo transfer treatment is anxiety. Sleep disturbance was the most significant factor involved in the psychological distress of women having problems with conception. Assisted reproductive technologies nurses can assess women's psychological distress by caring for their sleep disturbance without directly exploring their mood state. Moreover, these medical personnel should understand infertile female patients' psychological distress is mainly associated with their sleep disturbance. Developing various strategies to improve both sleep quality and psychological distress for infertile female patients should be recognised in future studies. © 2016 John Wiley & Sons Ltd.

Ketoprofen-loaded polymer carriers in bigel formulation: an approach to enhancing drug photostability in topical application forms

PubMed Central

Andonova, Velichka; Peneva, Petya; Georgiev, George S; Toncheva, Vencislava T; Apostolova, Elisaveta; Peychev, Zhivko; Dimitrova, Stela; Katsarova, Mariana; Petrova, Nadia; Kassarova, Margarita

2017-01-01

The purpose of the study was to investigate the stability and biopharmaceutical characteristics of ketoprofen, loaded in polymeric carriers, which were included into a bigel in a semisolid dosage form. The polymer carriers with in situ-included ketoprofen were obtained by emulsifier-free emulsion polymerization of the monomers in aqueous medium or a solution of the polymers used. The morphological characteristics of the carriers, the in vitro release and the photochemical stability of ketoprofen were evaluated. The model with optimal characteristics was included in a bigel formulation. The bigel was characterized in terms of pH, rheological behavior, spreadability, and in vitro drug release. Acute skin toxicity, antinociceptive activity, anti-inflammatory activity, and antihyperalgesic effects of the prepared bigel with ketoprofen-loaded polymer carrier were evaluated. The carriers of ketoprofen were characterized by a high yield and drug loading. The particle size distribution varied widely according to the polymer used, and a sustained release was provided for up to 6 hours. The polymer mixture poly(vinyl acetate) and hydroxypropyl cellulose as a drug carrier, alone or included in the bigel composition, improved the photostability of the drug compared with unprotected ketoprofen. The bigel with ketoprofen-loaded particles provided sustained release of the drug and had optimal rheological parameters. In vivo experiments on the bigel showed no skin inflammation or irritation. Four hours after its application, a well-defined analgesic, anti-inflammatory, and antihyperalgesic effect was registered. The polymer mixture of poly(vinyl acetate) and hydroxypropyl cellulose as a carrier of ketoprofen and the bigel in which it was included provided an enhanced photostability and sustained drug release. PMID:28894363

Ketoprofen-loaded polymer carriers in bigel formulation: an approach to enhancing drug photostability in topical application forms.

PubMed

Andonova, Velichka; Peneva, Petya; Georgiev, George S; Toncheva, Vencislava T; Apostolova, Elisaveta; Peychev, Zhivko; Dimitrova, Stela; Katsarova, Mariana; Petrova, Nadia; Kassarova, Margarita

2017-01-01

The purpose of the study was to investigate the stability and biopharmaceutical characteristics of ketoprofen, loaded in polymeric carriers, which were included into a bigel in a semisolid dosage form. The polymer carriers with in situ-included ketoprofen were obtained by emulsifier-free emulsion polymerization of the monomers in aqueous medium or a solution of the polymers used. The morphological characteristics of the carriers, the in vitro release and the photochemical stability of ketoprofen were evaluated. The model with optimal characteristics was included in a bigel formulation. The bigel was characterized in terms of pH, rheological behavior, spreadability, and in vitro drug release. Acute skin toxicity, antinociceptive activity, anti-inflammatory activity, and antihyperalgesic effects of the prepared bigel with ketoprofen-loaded polymer carrier were evaluated. The carriers of ketoprofen were characterized by a high yield and drug loading. The particle size distribution varied widely according to the polymer used, and a sustained release was provided for up to 6 hours. The polymer mixture poly(vinyl acetate) and hydroxypropyl cellulose as a drug carrier, alone or included in the bigel composition, improved the photostability of the drug compared with unprotected ketoprofen. The bigel with ketoprofen-loaded particles provided sustained release of the drug and had optimal rheological parameters. In vivo experiments on the bigel showed no skin inflammation or irritation. Four hours after its application, a well-defined analgesic, anti-inflammatory, and antihyperalgesic effect was registered. The polymer mixture of poly(vinyl acetate) and hydroxypropyl cellulose as a carrier of ketoprofen and the bigel in which it was included provided an enhanced photostability and sustained drug release.

An in vitro experimental study of flow past aortic valve under varied pulsatile conditions

NASA Astrophysics Data System (ADS)

Zhang, Ruihang; Zhang, Yan

2017-11-01

Flow past aortic valve represents a complex fluid-structure interaction phenomenon that involves pulsatile, vortical, and turbulent conditions. The flow characteristics immediately downstream of the valve, such as the variation of pulsatile flow velocity, formation of vortices, distribution of shear stresses, are of particular interest to further elucidate the role of hemodynamics in various aortic diseases. However, the fluid dynamics of a realistic aortic valve is not fully understood. Particularly, it is unclear how the flow fields downstream of the aortic valve would change under varied pulsatile inlet boundary conditions. In this study, an in vitro experiment has been conducted to investigate the flow fields downstream of a silicone aortic valve model within a cardiovascular flow simulator. Phased-locked Particle Image Velocimetry measurements were performed to map the velocity fields and Reynolds normal and shear stresses at different phases in a cardiac cycle. Temporal variations of pressure across the valve model were measured using high frequency transducers. Results have been compared for different pulsatile inlet conditions, including varied frequencies (heart rates), magnitudes (stroke volumes), and cardiac contractile functions (shapes of waveforms).

Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids

PubMed Central

Nikolić, Marko Z; Caritg, Oriol; Jeng, Quitz; Johnson, Jo-Anne; Sun, Dawei; Howell, Kate J; Brady, Jane L; Laresgoiti, Usua; Allen, George; Butler, Richard; Zilbauer, Matthias; Giangreco, Adam; Rawlins, Emma L

2017-01-01

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated. DOI: http://dx.doi.org/10.7554/eLife.26575.001 PMID:28665271

Free radical scavenging abilities of polypeptide from Chlamys farreri

NASA Astrophysics Data System (ADS)

Han, Zhiwu; Chu, Xiao; Liu, Chengjuan; Wang, Yuejun; Mi, Sun; Wang, Chunbo

2006-09-01

We investigated the radical scavenging effect and antioxidation property of polypeptide extracted from Chlamys farreri (PCF) in vitro using chemiluminescence and electron spin resonance (ESR) methods. We examined the scavenging effects of PCF on superoxide anions (O{2/-}), hydroxyl radicals (OH·), peroxynitrite (ONOO-) and the inhibiting capacity of PCF on peroxidation of linoleic acid. Our experiment suggested that PCF could scavenge oxygen free radicals including superoxide anions (O{2/-}) (IC50=0.3 mg/ml), hydroxyl radicals (OH·) (IC50=0.2 μg/ml) generated from the reaction systems and effectively inhibit the oxidative activity of ONOO- (IC50=0.2 mg/ml). At 1.25 mg/ml of PCF, the inhibition ratio on lipid peroxidation of linoleic acid was 43%. The scavenging effect of PCF on O{2/-}, OH· and ONOO- free radicals were stronger than those of vitamin C but less on lipid peroxidation of linoleic acid. Thus PCF could scavenge free radicals and inhibit the peroxidation of linoleic acid in vitro. It is an antioxidant from marine products and potential for industrial production in future.

Nonlinear quasi-static finite element simulations predict in vitro strength of human proximal femora assessed in a dynamic sideways fall setup.

PubMed

Varga, Peter; Schwiedrzik, Jakob; Zysset, Philippe K; Fliri-Hofmann, Ladina; Widmer, Daniel; Gueorguiev, Boyko; Blauth, Michael; Windolf, Markus

2016-04-01

Osteoporotic proximal femur fractures are caused by low energy trauma, typically when falling on the hip from standing height. Finite element simulations, widely used to predict the fracture load of femora in fall, usually include neither mass-related inertial effects, nor the viscous part of bone׳s material behavior. The aim of this study was to elucidate if quasi-static non-linear homogenized finite element analyses can predict in vitro mechanical properties of proximal femora assessed in dynamic drop tower experiments. The case-specific numerical models of 13 femora predicted the strength (R(2)=0.84, SEE=540N, 16.2%), stiffness (R(2)=0.82, SEE=233N/mm, 18.0%) and fracture energy (R(2)=0.72, SEE=3.85J, 39.6%); and provided fair qualitative matches with the fracture patterns. The influence of material anisotropy was negligible for all predictions. These results suggest that quasi-static homogenized finite element analysis may be used to predict mechanical properties of proximal femora in the dynamic sideways fall situation. Copyright © 2015 Elsevier Ltd. All rights reserved.

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

USGS Publications Warehouse

Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

2016-01-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

PubMed

Hop, Cornelis E C A; Cole, Mark J; Davidson, Ralph E; Duignan, David B; Federico, James; Janiszewski, John S; Jenkins, Kelly; Krueger, Suzanne; Lebowitz, Rebecca; Liston, Theodore E; Mitchell, Walter; Snyder, Mark; Steyn, Stefan J; Soglia, John R; Taylor, Christine; Troutman, Matt D; Umland, John; West, Michael; Whalen, Kevin M; Zelesky, Veronica; Zhao, Sabrina X

2008-11-01

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Effects of chitosan on nutrient digestibility, methane emissions, and in vitro fermentation in beef cattle.

PubMed

Henry, D D; Ruiz-Moreno, M; Ciriaco, F M; Kohmann, M; Mercadante, V R G; Lamb, G C; DiLorenzo, N

2015-07-01

Chitosan was evaluated as a feed additive to mitigate in vivo CH4 emissions in beef cattle. Twenty-four crossbred heifers (BW = 318 ± 35 kg) were used in a randomized block design replicated in 2 periods. The design included a 2 × 3 factorial arrangement of treatments, which included diet (high concentrate [HC] or low concentrate [LC]) and 0.0, 0.5, or 1.0% of chitosan inclusion (DM basis). Diets were offered ad libitum and individual intake was recorded. An in vitro experiment to analyze chitosan’s effect on fermentation parameters and gas production kinetics was performed. A diet effect (P < 0.01) was observed for CH4 emissions expressed as grams/day, grams/kilogram of BW0.75, and grams/kilogram of DMI. Heifers consuming the LC diet produced 130 g of CH4/d vs. 45 g of CH4/d in those consuming the HC diet. Incubation fluid pH increased linearly (P < 0.05) when chitosan was included in HC substrates. In vitro CH4 production was not affected (P > 0.10) by chitosan in HC substrate; however, when incubated with the LC substrate, CH4 production increased quadratically (P < 0.01) as chitosan inclusion increased. A digestibility marker × diet interaction occurred (P < 0.05) for DM, OM, CP, NDF, and ADF digestibility. Diet × chitosan interactions (P < 0.05) occurred for DM, OM, NDF, and ADF digestibility when Cr2O3 was used. When TiO2 was used, diet × chitosan interactions (P < 0.05) were observed for NDF and ADF. However, using indigestible NDF as an internal marker, DM and OM digestibility were improved (P < 0.05) by 21 and 19%, respectively, when chitosan was included in LC diets. In conclusion, feeding up to 1% of chitosan (DM basis) to heifers consuming a LC diet increased apparent total tract digestibility of nutrients. Enteric CH4 emissions were not affected by chitosan feeding, regardless of type of diet, and heifers consuming a 36% concentrate diet produced 2.6 times more methane per day than those consuming an 85% concentrate diet.

A Study of Waste Management within the COL Florence A. Blanchfield Army Community Hospital, Fort Campbell, Kentucky.

DTIC Science & Technology

1981-08-01

besnoiti Borna disease virus Bovine infectious petechial fever virus Camel pox virus Ephemeral fever virus Fowl plague virus Goat pox virus Hog...Varicella virus Vole rickettsia Yellow fever virus, 17D vaccine strain 69 Class 3 Alastrun, smallpox, monkeypox, and whitepox, when used in vitro Arbovirus...animal inoculation experiments Vesicular stomatitis virus Yellow fever virus - wild when used in vitro Class 4 Alastrun, smallpox, monkeypox, and

Surface Enhanced Raman Spectroscopy for Monitoring Lactate and Glucose

DTIC Science & Technology

2005-07-01

lasers. We have successfully developed and tested these SERS active substances in vitro and in vivo in the subcutaneous space of a rat. Work continues in...using this system for detection in vitro and in vivo as specified in the original proposal. The specific aims, as proposed in the original "statement...assess performance. a. Control experiments. b. Use indwelling probes to quantitatively measure glucose levels in vivo . c. Use indwelling probes to

Fidelity of DNA Replication in Normal and Malignant Human Brest Cells.

DTIC Science & Technology

1995-08-31

cellular DNA replication machinery, we have initiated experiments that utilize a multiprotein DNA replication complex (MRC) isolated from breast cancer...gene in an in vitro DNA replication assay. By utilizing the target gene in a bacterial mutant selection assay we have begun to determine the...frequency with which mutational sequence errors occur as a result of the in vitro DNA replication mediated by the breast cancer cell MRC and the normal breast

New protopine alkaloids from Aristolochia constricta reduce morphine withdrawal in vitro.

PubMed

Capasso, A; De Tommasi, N; Rastrelli, L; De Simone, F

2000-12-01

The present study examines the effect of four new protopine alkaloids (1-4) isolated and purified from the aerial parts of Aristolochia constricta (Aristolichiaceae) on morphine withdrawal in vitro. The results of our experiments indicate that the pure compounds (1-4) significantly and in a concentration-dependent manner reduced the morphine withdrawal. The results of the present study suggest that these new protopine alkaloids may be potential anti-addictive agents.

Synthesis, characterization and in vitro biocompatibility assessment of a novel tripeptide hydrogelator, as a promising scaffold for tissue engineering applications.

PubMed

Pospišil, Tihomir; Ferhatović Hamzić, Lejla; Brkić Ahmed, Lada; Lovrić, Marija; Gajović, Srećko; Frkanec, Leo

2016-10-20

We have synthesized and characterized a self-assembling tripeptide hydrogelator Ac-l-Phe-l-Phe-l-Ala-NH2. A series of experiments showed that the hydrogel material could serve as a stabile and biocompatible physical support as it improves the survival of HEK293T cells in vitro, thus being a promising biomaterial for use in tissue engineering applications.

A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters.

PubMed

Pellacani, Claudia; Buschini, Annamaria; Furlini, Mariangela; Poli, Paola; Rossi, Carlo

2006-04-20

Since the 1980s, stricter water quality regulations have been promulgated in many countries throughout the world. We discuss the application of a battery of both in vivo and in vitro genotoxicity tests on lake water as a tool for a more complete assessment of surface water quality. The lake water concentrated by adsorption on C18 silica cartridges were used for the following in vitro biological assays: gene conversion, point mutation, mitochondrial DNA mutability assays on the diploid Saccharomyces cerevisiae D7 strain, with or without endogenous P450 complex induction; DNA damage on fresh human leukocytes by the comet. Toxicity testing on yeast and human cells was also performed. In vivo genotoxicity was determined by the comet assay on two well-established bio-indicator organisms of water quality (Cyprinus carpio erythrocytes and Dreissena polymorpha haemocytes) exposed in situ. The in vivo experiments and the water samplings were carried out during different campaigns to detect seasonal variations of both the water contents and physiological state of the animals. Temperature and oxygen level seasonal variations and different pollutant contents in the lake water appeared to affect the DNA migration in carp and zebra mussel cells. Seasonal variability of lake water quality was also evident in the in vitro genotoxicity and cytotoxicity tests, with regards to water pollutant quantity and quality (direct-acting compounds or indirect-acting compounds on yeast cells). However, the measured biological effects did not appear clearly related to the physical-chemical characteristics of lake waters. Therefore, together with the conventional chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs: their use could permit the quantification of mutagenic hazard in surface waters.

In Vitro and In Silico Antidiabetic and Antimicrobial Evaluation of Constituents from Kickxia ramosissima (Nanorrhinum ramosissimum)

PubMed Central

Amin, Adnan; Tuenter, Emmy; Foubert, Kenn; Iqbal, Jamhsed; Cos, Paul; Maes, Louis; Exarchou, Vassiliki; Apers, Sandra; Pieters, Luc

2017-01-01

Background and Aims: Kickxia ramosissima (Wall.) Janch (or Nanorrhinum ramosissimum (Wall.) Betsche is a well-known medicinal plant in Pakistan that is traditionally used in diabetic and inflammatory conditions. Because little information is available on its phytochemical composition, a range of constituents were isolated and evaluated in vitro in assays related to the traditional use. Methods: Dried whole plant material was extracted and chromatographically fractionated. Isolated constituents were evaluated in silico and in vitro in assays related to the traditional use against diabetes (inhibition of α-glucosidase activity; inhibition of advanced glycation endproducts) and in inflammatory conditions (inhibition of AAPH induced linoleic acid peroxidation, inhibition of 15-LOX, antimicrobial activity). Results: Phytochemical analysis of the extracts and fractions led to isolation of 7 compounds, including the iridoids kickxiasine (being a new compound), mussaenosidic acid, mussaenoside and linarioside; the flavonoids pectolinarigenin and pectolinarin; and 4-hydroxy-benzoic acid methyl ester. The iridoids showed weak antiglycation activity. The flavonoids, however, showed interesting results as pectolinarigenin was highly active compared to pectolinarin. In the α-glucosidase inhibition assay, only weak activity was observed for the iridoids. However, the flavonoid pectolinarigenin showed good activity, followed by pectolinarin. In the 15-LOX experiment, moderate inhibition was recorded for most compounds, the iridoids mussaenosidic acid and mussaenoside being the most active. In the AAPH assay, weak or no inhibition was recorded for all compounds. The in silico assays for the α-glucosidase and 15-LOX assays confirmed the results of respective in vitro assays. Pectolinarigenin showed moderate antimicrobial activity against Staphylococcus aureus, Plasmodium falciparum K1, and Trypanosoma cruzi, but it was not cytotoxic on a human MRC-5 cell line. Conclusion: Our findings may in part contribute to explain the traditional use of K. ramosissima. PMID:28507520

Properties of a novel series of inhibitors of rumen methanogenesis; in vitro and in vivo experiments including growth trials on 2,4-bis (trichloromethyl)-benzo [1, 3]dioxin-6-carboxylic acid.

PubMed

Davies, A; Nwaonu, H N; Stanier, G; Boyle, F T

1982-05-01

1. A procedure for measuring methane production by rumen contents incubated anaerobically in vitro is described. Assessments of methane production in vivo, in both sheep and cattle, were made by withdrawal of rumen contents and measuring their capacity to produce methane in vitro. 2. Many members of a series of 6-substituted 2,4-bis (trichloromethyl)-benzo[1,3]dioxins were potent inhibitors of methanogenesis by rumen contents in vitro. The most potent compound inhibited methane production by 70% or more at a concentration of 1 microgram/ml (approximately 2.5 mumol/l). 3. Two compounds, namely the 6-carboxylic acid (ICI 13409) and the 6-carboxamide (ICI 43586), caused a large inhibition of methanogenesis sustained for many hours, following a single intrarumen injection in sheep or cattle. Inhibition was maintained for long periods by single daily dosing directly into the rumen or by dietary administration. 4. In a 28-week growth trial in beef cattle inclusion of ICI 13409 in the concentrate element of the diet, at a level of 6 mg/kg body-weight, improved live-weight gain by 8.0% (P less than 0.05) with respect to untreated animals whilst reducing food intake by 5.0% (P less than 0.05). Smaller and not statistically-significant effects were seen with this compound at 3 mg/kg body-weight and with the antibiotic monensin (Romensin; Elanco PLC). All treatments significantly improved the retention of dietary energy into the carcass, offal and intestinal tracts of the trial animals and significantly reduced the quantity of methane eructed into expired gases.

Elucidation of Arctigenin Pharmacokinetics and Tissue Distribution after Intravenous, Oral, Hypodermic and Sublingual Administration in Rats and Beagle Dogs: Integration of In Vitro and In Vivo Findings.

PubMed

Li, Jie; Li, Xin; Ren, Yu-Shan; Lv, Yuan-Yuan; Zhang, Jun-Sheng; Xu, Xiao-Li; Wang, Xian-Zhen; Yao, Jing-Chun; Zhang, Gui-Min; Liu, Zhong

2017-01-01

Although arctigenin ( AG ) has diverse bioactivities, such as anti-oxidant, anti-inflammatory, anti-cancer, immunoregulatory and neuroprotective activities, its pharmacokinetics have not been systematically evaluated. The purpose of this work was to identify the pharmacokinetic properties of AG via various experiments in vivo and in vitro . In this research, rats and beagle dogs were used to investigate the PK (pharmacokinetics, PK) profiles of AG with different drug-delivery manners, including intravenous (i.v), hypodermic injection (i.h), and sublingual (s.l) administration. The data shows that AG exhibited a strong absorption capacity in both rats and beagle dogs (absorption rate < 1 h), a high absorption degree (absolute bioavailability > 100%), and a strong elimination ability ( t 1/2 < 2 h). The tissue distributions of AG at different time points after i.h showed that the distribution of AG in rat tissues is rapid (2.5 h to reach the peak) and wide (detectable in almost all tissues and organs). The AG concentration in the intestine was the highest, followed by that in the heart, liver, pancreas, and kidney. In vitro , AG were incubated with human, monkey, beagle dog and rat liver microsomes. The concentrations of AG were detected by UPLC-MS/MS at different time points (from 0 min to 90 min). The percentages of AG remaining in four species' liver microsomes were human (62 ± 6.36%) > beagle dog (25.9 ± 3.24%) > rat (15.7 ± 9%) > monkey (3.69 ± 0.12%). This systematic investigation of pharmacokinetic profiles of arctigenin (AG) in vivo and in vitro is worthy of further exploration.

Elucidation of Arctigenin Pharmacokinetics and Tissue Distribution after Intravenous, Oral, Hypodermic and Sublingual Administration in Rats and Beagle Dogs: Integration of In Vitro and In Vivo Findings

PubMed Central

Li, Jie; Li, Xin; Ren, Yu-Shan; Lv, Yuan-Yuan; Zhang, Jun-Sheng; Xu, Xiao-Li; Wang, Xian-Zhen; Yao, Jing-Chun; Zhang, Gui-Min; Liu, Zhong

2017-01-01

Although arctigenin (AG) has diverse bioactivities, such as anti-oxidant, anti-inflammatory, anti-cancer, immunoregulatory and neuroprotective activities, its pharmacokinetics have not been systematically evaluated. The purpose of this work was to identify the pharmacokinetic properties of AG via various experiments in vivo and in vitro. In this research, rats and beagle dogs were used to investigate the PK (pharmacokinetics, PK) profiles of AG with different drug-delivery manners, including intravenous (i.v), hypodermic injection (i.h), and sublingual (s.l) administration. The data shows that AG exhibited a strong absorption capacity in both rats and beagle dogs (absorption rate < 1 h), a high absorption degree (absolute bioavailability > 100%), and a strong elimination ability (t1/2 < 2 h). The tissue distributions of AG at different time points after i.h showed that the distribution of AG in rat tissues is rapid (2.5 h to reach the peak) and wide (detectable in almost all tissues and organs). The AG concentration in the intestine was the highest, followed by that in the heart, liver, pancreas, and kidney. In vitro, AG were incubated with human, monkey, beagle dog and rat liver microsomes. The concentrations of AG were detected by UPLC-MS/MS at different time points (from 0 min to 90 min). The percentages of AG remaining in four species’ liver microsomes were human (62 ± 6.36%) > beagle dog (25.9 ± 3.24%) > rat (15.7 ± 9%) > monkey (3.69 ± 0.12%). This systematic investigation of pharmacokinetic profiles of arctigenin (AG) in vivo and in vitro is worthy of further exploration. PMID:28659807

Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

NASA Technical Reports Server (NTRS)

Tixador, R.

1992-01-01

The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

Development and Validation of an in vitro Experimental GastroIntestinal Dialysis Model with Colon Phase to Study the Availability and Colonic Metabolisation of Polyphenolic Compounds.

PubMed

Breynaert, Annelies; Bosscher, Douwina; Kahnt, Ariane; Claeys, Magda; Cos, Paul; Pieters, Luc; Hermans, Nina

2015-08-01

The biological effects of polyphenols depend on their mechanism of action in the body. This is affected by bioconversion by colon microbiota and absorption of colonic metabolites. We developed and validated an in vitro continuous flow dialysis model with colon phase (GastroIntestinal dialysis model with colon phase) to study the gastrointestinal metabolism and absorption of phenolic food constituents. Chlorogenic acid was used as model compound. The physiological conditions during gastrointestinal digestion were mimicked. A continuous flow dialysis system simulated the one-way absorption by passive diffusion from lumen to mucosa. The colon phase was developed using pooled faecal suspensions. Several methodological aspects including implementation of an anaerobic environment, adapted Wilkins Chalgren broth medium, 1.10(8) CFU/mL bacteria suspension as inoculum, pH adaptation to 5.8 and implementation of the dialysis system were conducted. Validation of the GastroIntestinal dialysis model with colon phase system showed a good recovery and precision (CV < 16 %). Availability of chlorogenic acid in the small intestinal phase (37 ± 3 %) of the GastroIntestinal dialysis model with colon phase is comparable with in vivo studies on ileostomy patients. In the colon phase, the human faecal microbiota deconjugated chlorogenic acid to caffeic acid, 3,4-dihydroxyphenyl propionic acid, 4-hydroxybenzoic acid, 3- or 4-hydroxyphenyl acetic acid, 2-methoxy-4-methylphenol and 3-phenylpropionic acid. The GastroIntestinal dialysis model with colon phase is a new, reliable gastrointestinal simulation system. It permits a fast and easy way to predict the availability of complex secondary metabolites, and to detect metabolites in an early stage after digestion. Isolation and identification of these metabolites may be used as references for in vivo bioavailability experiments and for investigating their bioactivity in in vitro experiments. Georg Thieme Verlag KG Stuttgart · New York.

Development of In Vitro Systems for Switchgrass (Panicum virgatum) - Final Report for 1992 to 2002

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conger, B.V.

2003-01-16

Our project began on July 1, 1992, with the objective of developing systems that could be used in biotechnological approaches to switchgrass improvement. Within six months after initiation of the project, we had worked out protocols in which plants could be regenerated from callus cultures through both organogenesis and somatic embryogenesis. Documentation for both modes of regeneration was provided in our progress reports and in publications. One thousand regenerated plants were established in the field during the first year. We found that Alamo (lowland type) was much more amenable to in vitro culture, and plants could be regenerated much moremore » easily than from Cave-in-Rock (upland type). During the first three years of the project, we studied the influence of genotype, culture medium components, explant type, etc., on regeneration. As mentioned, we found that the lowland cultivars Alamo and Kanlow were much easier to regenerate than upland cultivars, such as Trailblazer, Blackwell, and Cave-in-Rock. For callus induction, we initially used mature caryopses, young leaf tissue, and portions of seedlings. We were successful in inducing callus and regenerating plants from all explants. Two other systems developed during the 4th to 6th year period of the project included multiple shoot formation initiated from germinated seedlings and regenerable suspension cultures. The latter were initiated from embryogenic calluses produced from in vitro developed inflorescences. An important factor for producing multiple shoots was the presence of thidiazuron in the medium. The shoots could be easily rooted and numerous plantlets produced. The last 3 to 4 years of the project focused on anther and microspore culture experiments to produce haploid plants and on genetic transformation. Although thousands of putative haploid plants were produced from a few anthers, they were very weak and difficult to keep alive. Chromosome counts revealed the gametic number in cells where it was possible to count chromosomes. The isolated microspore culture experiments were not successful.« less

Neoplastic transformation of SV40-immortalized human urinary tract epithelial cells by in vitro exposure to 3-methylcholanthrene.

PubMed

Reznikoff, C A; Loretz, L J; Christian, B J; Wu, S Q; Meisner, L F

1988-08-01

Normal human urinary tract epithelial cells (HUC) were neoplastically transformed in vitro using a step-wise strategy. First, a partially transformed non-virus-producing cell line was obtained after infection of HUC with simian virus 40 (SV40). This cell line (SV-HUC-1) was demonstrated to be clonal in origin, as 100% of cells contained at least five of seven marker chromosomes. Marker chromosomes were formed by balanced translocations resulting in a 'pseudodiploid' cell line. SV-HUC-1 showed altered growth properties in vitro (e.g. anchorage independent growth) but failed to form tumors in athymic nude mice, even after 3 years in culture (80 passages). In the studies reported here, SV-HUC-1 at early passages (P15-P19) were exposed to 3-methylcholanthrene (MCA) in three separate experiments. After a six-week post-treatment period of cell culture, cells were inoculated s.c. into athymic nude mice. In all experiments, MCA-treated SV-HUC-1 formed carcinomas in mice usually with a latent period of 5-8 weeks. These carcinomas showed heterogeneity with respect to histopathologies and growth properties in the mice and karyotypes. All the tumors retained SV-HUC-1 chromosome markers, but each independent transformant was aneuploid and contained unique new marker chromosomes. Chromosomes usually altered in tumor cells included numbers 3, 5, 6, 9, 11 and 13. Mutations in the ras family of cellular proto-oncogenes resulting in altered mobility of the p21 protein product were not detected in six cell lines established from independently derived tumors. It is not yet known whether other cellular proto-oncogenes are activated in these tumorigenic transformants. Neither control SV-HUC-1 (which were not exposed to MCA), nor early passage HUC exposed to MCA formed tumors when inoculated into mice. Thus, the tumorigenic transformation of HUC resulted from the combined actions of SV40 and MCA.

Low-energy Shock Wave Therapy Ameliorates Erectile Dysfunction in a Pelvic Neurovascular Injuries Rat Model.

PubMed

Li, Huixi; Matheu, Melanie P; Sun, Fionna; Wang, Lin; Sanford, Melissa T; Ning, Hongxiu; Banie, Lia; Lee, Yung-Chin; Xin, Zhongcheng; Guo, Yinglu; Lin, Guiting; Lue, Tom F

2016-01-01

Erectile dysfunction (ED) caused by pelvic injuries is a common complication of civil and battlefield trauma with multiple neurovascular factors involved, and no effective therapeutic approach is available. To test the effect and mechanisms of low-energy shock wave (LESW) therapy in a rat ED model induced by pelvic neurovascular injuries. Thirty-two male Sprague-Dawley rats injected with 5-ethynyl-2'-deoxyuridine (EdU) at newborn were divided into 4 groups: sham surgery (Sham), pelvic neurovascular injury by bilateral cavernous nerve injury and internal pudendal bundle injury (PVNI), PVNI treated with LESW at low energy (Low), and PVNI treated with LESW at high energy (High). After LESW treatment, rats underwent erectile function measurement and the tissues were harvested for histologic and molecular study. To examine the effect of LESW on Schwann cells, in vitro studies were conducted. The intracavernous pressure (ICP) measurement, histological examination, and Western blot (WB) were conducted. Cell cycle, Schwann cell activation-related markers were examined in in vitro experiments. LESW treatment improves erectile function in a rat model of pelvic neurovascular injury by leading to angiogenesis, tissue restoration, and nerve generation with more endogenous EdU(+) progenitor cells recruited to the damaged area and activation of Schwann cells. LESW facilitates more complete re-innervation of penile tissue with regeneration of neuronal nitric oxide synthase (nNOS)-positive nerves from the MPG to the penis. In vitro experiments demonstrated that LESW has a direct effect on Schwann cell proliferation. Schwann cell activation-related markers including p-Erk1/2 and p75 were upregulated after LESW treatment. LESW-induced endogenous progenitor cell recruitment and Schwann cell activation coincides with angiogenesis, tissue, and nerve generation in a rat model of pelvic neurovascular injuries. Copyright © 2016 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

Testing of Safety-Critical Software Embedded in an Artificial Heart

NASA Astrophysics Data System (ADS)

Cha, Sungdeok; Jeong, Sehun; Yoo, Junbeom; Kim, Young-Gab

Software is being used more frequently to control medical devices such as artificial heart or robotic surgery system. While much of software safety issues in such systems are similar to other safety-critical systems (e.g., nuclear power plants), domain-specific properties may warrant development of customized techniques to demonstrate fitness of the system on patients. In this paper, we report results of a preliminary analysis done on software controlling a Hybrid Ventricular Assist Device (H-VAD) developed by Korea Artificial Organ Centre (KAOC). It is a state-of-the-art artificial heart which completed animal testing phase. We performed software testing in in-vitro experiments and animal experiments. An abnormal behaviour, never detected during extensive in-vitro analysis and animal testing, was found.

The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids.

PubMed Central

El-Robh, Mohamed Samir; Busby, Stephen J W

2002-01-01

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters. PMID:12350222

Silymarin inhibits adipogenesis in the adipocytes in grass carp Ctenopharyngodon idellus in vitro and in vivo.

PubMed

Xiao, Peizhen; Yang, Zhou; Sun, Jian; Tian, Jingjing; Chang, Zhiguang; Li, Xuexian; Zhang, Baotong; Ye, Yuantu; Ji, Hong; Yu, Ermeng; Xie, Jun

2017-12-01

In this study, two experiments were performed to explore the function of silymarin in adipogenesis in grass carp (Ctenopharyngodon idellus) using in vitro and in vivo models. In experiment 1, differentiated grass carp pre-adipocytes were treated with silymarin for 6 days. Treatment with 100 μg mL -1 silymarin (SM100 group) significantly reduced triglyceride accumulation at day 6. The adipogenic gene expression levels of PPARγ, C/EBPα, SREBP1c, FAS, SCD1, and LPL, and the protein expression level of PPARγ were significantly down-regulated in the SM100 group. Additionally, the SM100 group had significantly lower reactive oxygen species production and reduced glutathione contents compared with the control in vitro. In experiment 2, the juvenile grass carp (mean body weight= 27.4 ± 0.17 g) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200 mg kg -1 silymarin (SM0, SM100, SM200) associated with either 4 or 8% lipid levels (low lipid, LL, and high lipid, HL, respectively) for 82 days. The results demonstrated that dietary silymarin supplementation significantly reduced the elevated intraperitoneal fat index in grass carp fed with high-lipid diets, and the gene expression of adipogenesis (PPARγ, FAS) when supplemented with dietary silymarin was notably lower than when no silymarin was supplemented under the high-lipid diets. Thus, our data suggest that silymarin suppressed lipid accumulation in grass carp both in vitro and in vivo, and the effect might be due to an influence on the expression of adipogenesis factors and ROS production partly associated with effects on antioxidant capability.

Ultrastructural organization of connective tissue microfibrils in the posterior chamber of the eye in vivo and in vitro.

PubMed

Inoue, S

1995-02-01

The ultrastructural organization of connective tissue microfibrils was studied in the mouse eye and also by means of in vitro experiments for reconstituting microfibrils. In the posterior chamber of the eye of the C57BL/6J mouse, 3 nm-wide ribbon-like double-tracked structures were present and were periodically associated on either side with 3.5 nm-wide particulate structures identified as pentosomes, the subunits of amyloid P component (AP). At certain sites, such composite structures were observed in various stages of helical winding, and in these helices, pentosomes were preferentially localized internally. In helices in the final stages of winding, the resulting rods appeared increasingly similar to those of microfibrils. In experiments in vitro, incubation of chondroitin sulfate proteoglycan (CSPG) in TRIS buffer, pH 7.4, at 35 degrees C for 1 h produced random aggregates of 3 nm-wide double-tracked structures similar to those observed in the eye. Co-incubation of CSPG and AP resulted in the formation of rod-like structures arranged parallel to one another in approximately 50 nm-thick sheet-like layers. These rods were ultrastructurally similar to microfibrils and were made up of helically wound, 3 nm-wide double-tracked structures containing pentosomes within their core. The results of in vivo as well as in vitro experiments suggest the possibility that the connective tissue microfibril is composed of helically wound, CSPG-containing, 3 nm-wide double-tracked structures periodically associated with pentosomes which, as the helix becomes progressively tighter, fit with one another at the core of the helix to form successive 8.5 nm-wide disks of AP segments.

WE-FG-BRA-01: Cancer Treatment Utilizing Photo-Activation of Psoralen with KV X-Rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oldham, M; Yoon, S; Meng, B

Purpose: This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with immunogenic anti-cancer potential. Psoralen therapies have been limited due to the requirement for psoralen activation by UVA light. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and reradiate (phosphoresce) at UV wavelengths. Methods: The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed tomore » X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry to investigate treatment induced apoptosis. Methylene blue staining, and WST assays were also used. X-PACT was then evaluated in an in-vivo pilot study on BALBc mice with syngeneic 4T1 tumors, including control arms for X-PACT components. Analysis focused on tumor growth delay. Results: A multivariable regression analysis of 36 independent in-vitro irradiation experiments demonstrated that X-PACT induces significant tumor cell apoptosis and cytotoxicity on all three tumor cell lines in-vitro (p<0.0001). Neither psoralen nor phosphor alone had a strongly significant effect. The in-vivo studies show a pronounced tumor growth delay when compared to controls (42% reduction at 25 days, p=0.0002). Conclusions: These studies demonstrate for the first time a therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation generate UVA light in-situ (including deep seated lesions) which in-turn photo-activates powerful anticancer therapeutics which may lead to short and long term therapeutic effect. This work was supported by Immunolight Llc.« less

Role of Dopamine Signaling in Drug Addiction.

PubMed

Chen, Wan; Nong, Zhihuan; Li, Yaoxuan; Huang, Jianping; Chen, Chunxia; Huang, Luying

2017-01-01

Addiction is a chronic, relapsing disease of the brain that includes drug-induced compulsive seeking behavior and consumption of drugs. Dopamine (DA) is considered to be critical in drug addiction due to reward mechanisms in the midbrain. In this article, we review the major animal models in addictive drug experiments in vivo and in vitro. We discuss the relevance of the structure and pharmacological function of DA receptors. To improve the understanding of the role of DA receptors in reward pathways, specific brain regions, including the Ventral tegmental area, Nucleus accumbens, Prefrontal cortex, and Habenula, are highlighted. These factors contribute to the development of novel therapeutic targets that act at DA receptors. In addiction, the development of neuroimaging method will increase our understanding of the mechanisms underlying drug addiction. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Rooting and acclimatization of micropropagated marubakaido apple rootstock using Adesmia latifolia rhizobia.

PubMed

Muniz, Aleksander Westphal; de Sá, Enilson Luiz; Dalagnol, Gilberto Luíz; Filho, João Américo

2013-01-01

In vitro rooting and the acclimatization of micropropagated rootstocks of apple trees is essential for plant development in the field. The aim of this work was to assess the use of rhizobia of Adesmia latifolia to promote rooting and acclimatization in micropropagated Marubakaido apple rootstock. An experiment involving in vitro rooting and acclimatization was performed with four strains of rhizobium and two controls, one with and the other without the addition of synthetic indoleacetic acid. The inoculated treatments involved the use of sterile inoculum and inoculum containing live rhizobia. The most significant effects on the rooting rate, primary-root length, number of roots, root length, fresh-shoot biomass, and fresh-root biomass were obtained by inoculation with strain EEL16010B and with synthetic indole acetic acid. However, there was no difference in the growth of apple explants in the acclimatization experiments. Strain EEL16010B can be used to induce in vitro rooting of the Marubakaido rootstock and can replace the use of synthetic indoleacetic acid in the rooting of this cultivar.

In vivo and in vitro changes in neurochemical parameters related to mercury concentrations from specific brain regions of polar bears (Ursus maritimus).

PubMed

Krey, Anke; Kwan, Michael; Chan, Hing Man

2014-11-01

Mercury (Hg) has been detected in polar bear brain tissue, but its biological effects are not well known. Relationships between Hg concentrations and neurochemical enzyme activities and receptor binding were assessed in the cerebellum, frontal lobes, and occipital lobes of 24 polar bears collected from Nunavik (Northern Quebec), Canada. The concentration-response relationship was further studied with in vitro experiments using pooled brain homogenate of 12 randomly chosen bears. In environmentally exposed brain samples, there was no correlative relationship between Hg concentration and cholinesterase (ChE) activity or muscarinic acetylcholine receptor (mAChR) binding in any of the 3 brain regions. Monoamine oxidase (MAO) activity in the occipital lobe showed a negative correlative relationship with total Hg concentration. In vitro experiments, however, demonstrated that Hg (mercuric chloride and methylmercury chloride) can inhibit ChE and MAO activities and muscarinic mAChR binding. These results show that Hg can alter neurobiochemical parameters but the current environmental Hg exposure level does have an effect on the neurochemistry of polar bears from northern Canada. © 2014 SETAC.

Multiscale modelling approaches for assessing cosmetic ingredients safety.

PubMed

Bois, Frédéric Y; Ochoa, Juan G Diaz; Gajewska, Monika; Kovarich, Simona; Mauch, Klaus; Paini, Alicia; Péry, Alexandre; Benito, Jose Vicente Sala; Teng, Sophie; Worth, Andrew

2017-12-01

The European Union's ban on animal testing for cosmetic ingredients and products has generated a strong momentum for the development of in silico and in vitro alternative methods. One of the focus of the COSMOS project was ab initio prediction of kinetics and toxic effects through multiscale pharmacokinetic modeling and in vitro data integration. In our experience, mathematical or computer modeling and in vitro experiments are complementary. We present here a summary of the main models and results obtained within the framework of the project on these topics. A first section presents our work at the organelle and cellular level. We then go toward modeling cell levels effects (monitored continuously), multiscale physiologically based pharmacokinetic and effect models, and route to route extrapolation. We follow with a short presentation of the automated KNIME workflows developed for dissemination and easy use of the models. We end with a discussion of two challenges to the field: our limited ability to deal with massive data and complex computations. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Reducing animal experimentation in foot-and-mouth disease vaccine potency tests.

PubMed

Reeve, Richard; Cox, Sarah; Smitsaart, Eliana; Beascoechea, Claudia Perez; Haas, Bernd; Maradei, Eduardo; Haydon, Daniel T; Barnett, Paul

2011-07-26

The World Organisation for Animal Health (OIE) Terrestrial Manual and the European Pharmacopoeia (EP) still prescribe live challenge experiments for foot-and-mouth disease virus (FMDV) immunogenicity and vaccine potency tests. However, the EP allows for other validated tests for the latter, and specifically in vitro tests if a "satisfactory pass level" has been determined; serological replacements are also currently in use in South America. Much research has therefore focused on validating both ex vivo and in vitro tests to replace live challenge. However, insufficient attention has been given to the sensitivity and specificity of the "gold standard"in vivo test being replaced, despite this information being critical to determining what should be required of its replacement. This paper aims to redress this imbalance by examining the current live challenge tests and their associated statistics and determining the confidence that we can have in them, thereby setting a standard for candidate replacements. It determines that the statistics associated with the current EP PD(50) test are inappropriate given our domain knowledge, but that the OIE test statistics are satisfactory. However, it has also identified a new set of live animal challenge test regimes that provide similar sensitivity and specificity to all of the currently used OIE tests using fewer animals (16 including controls), and can also provide further savings in live animal experiments in exchange for small reductions in sensitivity and specificity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

PubMed

Hoyt, Joseph R; Cheng, Tina L; Langwig, Kate E; Hee, Mallory M; Frick, Winifred F; Kilpatrick, A Marm

2015-01-01

Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens have recently emerged and caused widespread mortality in several vertebrate groups, including amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies. The skin microbiome is increasingly understood to play a large role in determining disease outcome. We isolated bacteria from the skin of four bat species, and co-cultured these isolates with P. destructans to identify bacteria that might inhibit or kill P. destructans. We then conducted two reciprocal challenge experiments in vitro with six bacterial isolates (all in the genus Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non-inhibitory control bacteria, and two isolates performed significantly better than others in suppressing P. destructans growth for at least 35 days. In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate. These results show that bacteria found naturally occurring on bats can inhibit the growth of P. destructans in vitro and should be studied further as a possible probiotic to protect bats from white-nose syndrome. In addition, the presence of these bacteria may influence disease outcomes among individuals, populations, and species.

Bacteria Isolated from Bats Inhibit the Growth of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome

PubMed Central

Hoyt, Joseph R.; Cheng, Tina L.; Langwig, Kate E.; Hee, Mallory M.; Frick, Winifred F.; Kilpatrick, A. Marm

2015-01-01

Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens have recently emerged and caused widespread mortality in several vertebrate groups, including amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies. The skin microbiome is increasingly understood to play a large role in determining disease outcome. We isolated bacteria from the skin of four bat species, and co-cultured these isolates with P. destructans to identify bacteria that might inhibit or kill P. destructans. We then conducted two reciprocal challenge experiments in vitro with six bacterial isolates (all in the genus Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non-inhibitory control bacteria, and two isolates performed significantly better than others in suppressing P. destructans growth for at least 35 days. In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate. These results show that bacteria found naturally occurring on bats can inhibit the growth of P. destructans in vitro and should be studied further as a possible probiotic to protect bats from white-nose syndrome. In addition, the presence of these bacteria may influence disease outcomes among individuals, populations, and species. PMID:25853558

Apelin: an endogenous peptide essential for cardiomyogenic differentiation of mesenchymal stem cells via activating extracellular signal-regulated kinase 1/2 and 5.

PubMed

Wang, Li; Zhu, Zhi-Ming; Zhang, Ning-Kun; Fang, Zhi-Rong; Xu, Xiao-Hong; Zheng, Nan; Gao, Lian-Ru

2016-05-01

Growing evidence has shown that apelin/APJ system functions as a critical mediator of cardiac development as well as cardiovascular function. Here, we investigated the role of apelin in the cardiomyogenic differentiation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord in vitro. In this research, we used RNA interference methodology and gene transfection technique to regulate the expression of apelin in Wharton's jelly-derived mesenchymal stem cells and induced cells with a effective cardiac differentiation protocol including 5-azacytidine and bFGF. Four weeks after induction, induced cells assumed a stick-like morphology and myotube-like structures except apelin-silenced cells and the control group. The silencing expression of apelin in Wharton's jelly-derived mesenchymal stem cells decreased the expression of several critical cardiac progenitor transcription factors (Mesp1, Mef2c, NKX2.5) and cardiac phenotypes (cardiac α-actin, β-MHC, cTnT, and connexin-43). Meanwhile, endogenous compensation of apelin contributed to differentiating into cells with characteristics of cardiomyocytes in vitro. Further experiment showed that exogenous apelin peptide rescued the cardiomyogenic differentiation of apelin-silenced mesenchymal stem cells in the early stage (1-4 days) of induction. Remarkably, our experiment indicated that apelin up-regulated cardiac specific genes in Wharton's jelly-derived mesenchymal stem cells via activating extracellular signal-regulated kinase (ERK) 1/2 and 5. © 2016 International Federation for Cell Biology.

[Experimental study of angiography using vascular interventional robot-2(VIR-2)].

PubMed

Tian, Zeng-min; Lu, Wang-sheng; Liu, Da; Wang, Da-ming; Guo, Shu-xiang; Xu, Wu-yi; Jia, Bo; Zhao, De-peng; Liu, Bo; Gao, Bao-feng

2012-06-01

To verify the feasibility and safety of new vascular interventional robot system used in vascular interventional procedures. Vascular interventional robot type-2 (VIR-2) included master-slave parts of body propulsion system, image navigation systems and force feedback system, the catheter movement could achieve under automatic control and navigation, force feedback was integrated real-time, followed by in vitro pre-test in vascular model and cerebral angiography in dog. Surgeon controlled vascular interventional robot remotely, the catheter was inserted into the intended target, the catheter positioning error and the operation time would be evaluated. In vitro pre-test and animal experiment went well; the catheter can enter any branch of vascular. Catheter positioning error was less than 1 mm. The angiography operation in animal was carried out smoothly without complication; the success rate of the operation was 100% and the entire experiment took 26 and 30 minutes, efficiency was slightly improved compared with the VIR-1, and the time what staff exposed to the DSA machine was 0 minute. The resistance of force sensor can be displayed to the operator to provide a security guarantee for the operation. No surgical complications. VIR-2 is safe and feasible, and can achieve the catheter remote operation and angiography; the master-slave system meets the characteristics of traditional procedure. The three-dimensional image can guide the operation more smoothly; force feedback device provides remote real-time haptic information to provide security for the operation.

Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen*

PubMed Central

Sharma, Aditya K.; Arora, Divya; Singh, Lalit K.; Gangwal, Aakriti; Sajid, Andaleeb; Molle, Virginie; Singh, Yogendra; Nandicoori, Vinay Kumar

2016-01-01

Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events. PMID:27758870

Effect of polyethylene glycol 4000 supplementation on the performance of indigenous Pedi goats fed different levels of Acacia nilotica leaf meal and ad libitum Buffalo grass hay.

PubMed

Motubatse, M R; Ng'ambi, J W; Norris, D; Malatje, M M

2008-04-01

In a first of two experiments, twenty yearling male Pedi goats weighing 21.3 +/- 0.5 kg live weight were used in a 37-day study in a 2 (levels of PEG 4000) x 2 (levels ofAcacia) Factorial arrangement in a Completely Randomised Design to determine the effect of the level of Acacia nilotica leaf meal supplementation plus 23 g polyethylene glycol 4000 on diet intake and digestibility, and growth rate of Pedi goats fed ad libitum Buffalo grass hay. Acacia nilotica leaf meal contained high amounts of total phenolics (2.04% DM) and low amounts of condensed tannins; both extracted (0.37% DM) and unextracted (1.83% DM). Supplementation with PEG 4000 increased (P < 0.05) crude protein intake as the level of Acacia nilotica leaf meal increased from 80 to 120 g. Similarly, treatment with PEG 4000 improved (P < 0.05) DM, OM and CP digestibilities when compared to 80 g Acacia nilotica leaf meal. Supplementation with PEG 4000 resulted in an increase (P < 0.05) in blood urea concentrations. Polyethylene glycol 4000 has the potential to improve the feeding value of A. nilotica leaf meal and can, therefore, be used in the feeding systems for ruminant animals. The second experiment determined the effect of A. nilotica leaf meal supplementation on in vitro digestibility of the diets similar to the actual ratios of the first experiment. Level of A. nilotica leaf meal supplementation plus 23 g PEG 4000 supplementation improved (P < 0.05) in vitro DM, OM and CP digestibilities where 120 g A. nilotica leaf meal was supplemented. Similarly, 23 g PEG 4000 supplementation also improved (P < 0.05) in vitro CP digestibility where 80 g A. nilotica leaf meal was supplemented. In vivo DM and OM digestibilities were best predicted from in vitro DM and OM digestibilities while in vivo CP was explained by in vitro OM and CP digestibilities. It is, therefore, concluded that in vitro DM and OM digestibilities have good capacity to predict in vivo DM and OM digestibilities while OM and CP digestibilities have good capacity to predict in vivo CP digestibility.

Phi-value analysis of apo-azurin folding: comparison between experiment and theory.

PubMed

Zong, Chenghang; Wilson, Corey J; Shen, Tongye; Wolynes, Peter G; Wittung-Stafshede, Pernilla

2006-05-23

Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.

New(ish) tools in the toolbox: Using in vitro bioassays for cumulative assessment of steroid hormone receptor active compounds in environmental samples

EPA Science Inventory

In vitro assays are currently a high priority tool within USEPA for screening chemicals and samples for biological activity. The work included in this abstract describes the usage and expertise in our research group for using in vitro assays to screen environmental samples for s...

In vitro chemo-sensitivity assay guided chemotherapy is associated with prolonged overall survival in cancer patients.

PubMed

Udelnow, Andrej; Schönfęlder, Manfred; Würl, Peter; Halloul, Zuhir; Meyer, Frank; Lippert, Hans; Mroczkowski, Paweł

2013-06-01

The overall survival (OS) of patients suffering From various tumour entities was correlated with the results of in vitro-chemosensitivity assay (CSA) of the in vivo applied drugs. Tumour specimen (n=611) were dissected in 514 patients and incubated for primary tumour cell culture. The histocytological regression assay was performed 5 days after adding chemotherapeutic substances to the cell cultures. n=329 patients undergoing chemotherapy were included in the in vitro/in vivo associations. OS was assessed and in vitro response groups compared using survival analysis. Furthermore Cox-regression analysis was performed on OS including CSA, age, TNM classification and treatment course. The growth rate of the primary was 73-96% depending on tumour entity. The in-vitro response rate varied with histology and drugs (e.g. 8-18% for methotrexate and 33-83% for epirubicine). OS was significantly prolonged for patients treated with in vitro effective drugs compared to empiric therapy (log-rank-test, p=0.0435). Cox-regression revealed that application of in vitro effective drugs, residual tumour and postoperative radiotherapy determined the death risk independently. When patients were treated with drugs effective in our CSA, OS was significantly prolonged compared to empiric therapy. CSA guided chemotherapy should be compared to empiric treatment by a prospective randomized trial.

In Vitro Exposure Systems and Dosimetry Assessment Tools ...

EPA Pesticide Factsheets

In 2009, the passing of The Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP) and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed “modified risk”. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference titled “In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products” to bring together stakeholders representing regulatory agencies, academia, and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapor exposure systems, as well as the various approaches and challenges to quantifying the complex exposures, in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were, 1) Tobacco Smoke And E-Cigarette Aerosols, 2) Air-Liquid Interface-In Vitro Exposure Systems, 3) Dosimetry Approaches For Particles And Vapors; In Vitro Dosimetry Determinations and 4) Exposure Microenvironment/Physiology Of Cells. The two and a half day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will re

Multichannel microformulators for massively parallel machine learning and automated design of biological experiments

NASA Astrophysics Data System (ADS)

Wikswo, John; Kolli, Aditya; Shankaran, Harish; Wagoner, Matthew; Mettetal, Jerome; Reiserer, Ronald; Gerken, Gregory; Britt, Clayton; Schaffer, David

Genetic, proteomic, and metabolic networks describing biological signaling can have 102 to 103 nodes. Transcriptomics and mass spectrometry can quantify 104 different dynamical experimental variables recorded from in vitro experiments with a time resolution approaching 1 s. It is difficult to infer metabolic and signaling models from such massive data sets, and it is unlikely that causality can be determined simply from observed temporal correlations. There is a need to design and apply specific system perturbations, which will be difficult to perform manually with 10 to 102 externally controlled variables. Machine learning and optimal experimental design can select an experiment that best discriminates between multiple conflicting models, but a remaining problem is to control in real time multiple variables in the form of concentrations of growth factors, toxins, nutrients and other signaling molecules. With time-division multiplexing, a microfluidic MicroFormulator (μF) can create in real time complex mixtures of reagents in volumes suitable for biological experiments. Initial 96-channel μF implementations control the exposure profile of cells in a 96-well plate to different temporal profiles of drugs; future experiments will include challenge compounds. Funded in part by AstraZeneca, NIH/NCATS HHSN271201600009C and UH3TR000491, and VIIBRE.

The isolated chicken eye test as a suitable in vitro method for determining the eye irritation potential of household cleaning products.

PubMed

Schutte, K; Prinsen, M K; McNamee, P M; Roggeband, R

2009-08-01

Eye irritation is an important endpoint in the safety evaluation of consumer products and their ingredients. Several in vitro methods have been developed and are used by different industry sectors to assess eye irritation. One such in vitro method in use for some time already is the isolated chicken eye test (ICE). This investigation focuses on assessing the ICE as a method to determine the eye irritation potential of household cleaning products, both for product safety assurance prior to marketing and for classification and labeling decisions. The ICE involves a single application of test substances onto the cornea of isolated chicken eyes. Endpoints are corneal swelling, corneal opacity and fluorescein retention. The ICE results were compared to historic LVET data in this study due to availability of such in vivo data and the ability to correlate LVET to human experience data on the outcome of accidental exposures to household cleaning products in general. The results of this study indicate that the ICE test is a useful in vitro method for evaluating the eye irritation/corrosion potential and establishing classification and labeling for household cleaning products. For new product formulations, it is best used as part of a weight-of-evidence approach and benchmarked against data from comparable formulations with known eye irritation/corrosion profiles and market experience.

Understanding and predicting the impact of critical dissolution variables for nifedipine immediate release capsules by multivariate data analysis.

PubMed

Mercuri, A; Pagliari, M; Baxevanis, F; Fares, R; Fotaki, N

2017-02-25

In this study the selection of in vivo predictive in vitro dissolution experimental set-ups using a multivariate analysis approach, in line with the Quality by Design (QbD) principles, is explored. The dissolution variables selected using a design of experiments (DoE) were the dissolution apparatus [USP1 apparatus (basket) and USP2 apparatus (paddle)], the rotational speed of the basket/or paddle, the operator conditions (dissolution apparatus brand and operator), the volume, the pH, and the ethanol content of the dissolution medium. The dissolution profiles of two nifedipine capsules (poorly soluble compound), under conditions mimicking the intake of the capsules with i. water, ii. orange juice and iii. an alcoholic drink (orange juice and ethanol) were analysed using multiple linear regression (MLR). Optimised dissolution set-ups, generated based on the mathematical model obtained via MLR, were used to build predicted in vitro-in vivo correlations (IVIVC). IVIVC could be achieved using physiologically relevant in vitro conditions mimicking the intake of the capsules with an alcoholic drink (orange juice and ethanol). The multivariate analysis revealed that the concentration of ethanol used in the in vitro dissolution experiments (47% v/v) can be lowered to less than 20% v/v, reflecting recently found physiological conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Urinary biomarkers of trimethoprim bioactivation in vivo following therapeutic dosing in children.

PubMed

van Haandel, Leon; Goldman, Jennifer L; Pearce, Robin E; Leeder, J Steven

2014-02-17

The antimicrobial trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for the treatment of skin and soft-tissue infections in the outpatient setting. Despite its therapeutic benefits, TMP-SMX has been associated with a number of adverse drug reactions, which have been primarily attributed to the formation of reactive metabolites from SMX. Recently, in vitro experiments have demonstrated that TMP may form reactive intermediates as well. However, evidence of TMP bioactivation in patients has not yet been demonstrated. In this study, we performed in vitro trapping experiments with N-acetyl-l-cysteine (NAC) to determine stable markers of reactive TMP intermediates, focusing on eight potential markers (NAC-TMP adducts), some of which were previously identified in vitro. We developed a specific and sensitive assay involving liquid chromatography followed by tandem mass spectrometry for measurement of these adducts in human liver microsomal samples and expanded the methodology toward the detection of these analytes in human urine. Urine samples from four patients receiving TMP-SMX treatment were analyzed, and all samples demonstrated the presence of six NAC-TMP adducts, which were also detected in vitro. These adducts are consistent with the formation of imino-quinone-methide and para-quinone-methide reactive intermediates in vivo. As a result, the TMP component of TMP-SMX should be considered as well when evaluating adverse drug reactions to TMP-SMX.

Effects of gas composition in headspace and bicarbonate concentrations in media on gas and methane production, degradability, and rumen fermentation using in vitro gas production techniques.

PubMed

Patra, Amlan Kumar; Yu, Zhongtang

2013-07-01

Headspace gas composition and bicarbonate concentrations in media can affect methane production and other characteristics of rumen fermentation in in vitro gas production systems, but these 2 important factors have not been evaluated systematically. In this study, these 2 factors were investigated with respect to gas and methane production, in vitro digestibility of feed substrate, and volatile fatty acid (VFA) profile using in vitro gas production techniques. Three headspace gas compositions (N2+ CO2+ H2 in the ratio of 90:5:5, CO2, and N2) with 2 substrate types (alfalfa hay only, and alfalfa hay and a concentrate mixture in a 50:50 ratio) in a 3×2 factorial design (experiment 1) and 3 headspace compositions (N2, N2 + CO2 in a 50:50 ratio, and CO2) with 3 bicarbonate concentrations (80, 100, and 120 mM) in a 3×3 factorial design (experiment 2) were evaluated. In experiment 1, total gas production (TGP) and net gas production (NGP) was the lowest for CO2, followed by N2, and then the gas mixture. Methane concentration in headspace gas after fermentation was greater for CO2 than for N2 and the gas mixture, whereas total methane production (TMP) and net methane production (NMP) were the greatest for CO2, followed by the gas mixture, and then N2. Headspace composition did not affect in vitro digestibility or the VFA profile, except molar percentages of propionate, which were greater for CO2 and N2 than for the gas mixture. Methane concentration in headspace gas, TGP, and NGP were affected by the interaction of headspace gas composition and substrate type. In experiment 2, increasing concentrations of CO2 in the headspace decreased TGP and NGP quadratically, but increased the concentrations of methane, NMP, and in vitro fiber digestibility linearly, and TMP quadratically. Fiber digestibility, TGP, and NGP increased linearly with increasing bicarbonate concentrations in the medium. Concentrations of methane and NMP were unaffected by bicarbonate concentration, but TMP tended to increase due to increasing bicarbonate concentration. Although total VFA concentration and molar percentage of butyrate were unchanged, the molar percentage of acetate, and acetate-to-propionate ratio decreased, whereas the molar percentage of propionate increased quadratically with increasing bicarbonate concentration. This study demonstrated for the first time that headspace composition, especially CO2 content, and bicarbonate concentration in media could significantly influence gas and methane production, and rumen fermentation in gas production techniques. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

MR-compatible polyetheretherketone-based guide wire assisting MR-guided stenting of iliac and supraaortic arteries in swine: feasibility study.

PubMed

Kos, Sebastian; Huegli, Rolf; Hofmann, Eugen; Quick, Harald H; Kuehl, Hilmar; Aker, Stephanie; Kaiser, Gernot M; Borm, Paul J A; Jacob, Augustinus L; Bilecen, Deniz

2009-01-01

The purpose of this study was to demonstrate first magnetic resonance (MR)-guided stenting of iliac and supraaortic arteries using a polyetheretherketone-based (PEEK) MR-compatible guide wire. In vitro and animal experiments were performed in a short magnet wide-bore scanner (1.5 Tesla, Espree, Siemens Healthcare, Erlangen, Germany). For all experiments, a 0.035'' MR-compatible guide wire prototoype was used. This wire had a compound core of PEEK with reinforcing fibres, a soft and atraumatic tip and a hydrophilic coating. For its passive visualization, paramagnetic markings were attached. All experiments were performed through a vascular introducer sheath under MR-guidance. In vitro repetitive selective over the wire catheterizations of either the right carotid artery and the left subclavian artery were performed. In vivo, selective catheterization and over-the-wire stenting of the brachiocephalic trunk and the left subclavian artery were performed. The common iliac arteries were catheterized retrogradely (left) and cross-over (right). Angioplasty and stenting were performed over-the-wire. All procedures were successful. Visibility of the PEEK-based guide-wire was rated good in vitro and acceptable in vivo. Guide wire pushability and endovascular device support were good. The PEEK-based MR-compatible guide wire is well visible and usable under MR-guidance. It supports over-the-wire treatment of iliac arteries and supraaortic arteries.

In Vitro Fertilization (IVF)

MedlinePlus

... or eggs are implanted in your uterus. One cycle of IVF takes about two weeks. IVF is ... Specific steps of an in vitro fertilization (IVF) cycle carry risks, including: Multiple births. IVF increases the ...

Advances in pharmacological strategies for the prevention of cataract development

PubMed Central

Gupta, S K; Selvan, V Kalai; Agrawal, S S; Saxena, Rohit

2009-01-01

Cataractous-opacification of the lens is one of the leading causes of blindness in India. The situation can be managed by surgical removal of the cataractous lens. Various pharmacological strategies have been proposed for the prevention and treatment of cataract. Information on possible benefits of putative anticataract agents comes from a variety of approaches, ranging from laboratory experiments, both in vitro and in vivo, to epidemiological studies in patients. This review deals with the various mechanisms, and possible pharmacological interventions for the prevention of cataract. The article also reviews research on potential anticataractous agents, including aldose reductase inhibitors, glutathione boosters, antiglycating agents, vitamins and various drugs from indigenous sources. PMID:19384010

Antimicrobial effects of an antiperspirant formulation containing aqueous aluminum chloride hexahydrate.

PubMed

Hölzle, E; Neubert, U

1982-01-01

To document deodorant efficacy the antimicrobial activity of a gelatinous antiperspirant formulation of aqueous aluminum chloride hexahydrate was investigated. In vitro assays demonstrated highly bactericidal activity on microorganisms comprising the resident axillary skin flora, including micrococcaceae and aerobic diphtheroid bacteria. Gram-negative bacteria and yeast were partially inhibited. In vivo experiments utilizing occlusive patches on forearm skin and bacterial sampling of the axilla showed pronounced bacteriostasis and persistence of aluminum chloride on the skin. Inhibition of microbial growth lasted more than 3 days after a single treatment of the axilla. Following repeated open applications to the volar aspect of the forearm, the skin remained virtually sterile for 3 days.

Dietary conjugated linoleic acid and long-chain n-3 fatty acids in mammary and prostate cancer protection: a review.

PubMed

Heinze, Verónica M; Actis, Adriana B

2012-02-01

The role of dietary fatty acids on cancer is still controversial. To examine the current literature on the protective role of conjugated linoleic acid (CLA) and marine long-chain fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and the risk of breast and prostate cancer, data from 41 case-control and cohort studies and relevant in vitro and animal experiments were included in this 2000-2010 revision. Epidemiological studies on CLA intake or its tissue concentration related to breast and prostate tumorigenesis are not conclusive; EPA and DHA intake have shown important inverse associations just in some studies. Additional research on the analysed association is required.

Microfluidic platform for neurotransmitter sensing based on cyclic voltammetry and dielectrophoresis for in vitro experiments.

PubMed

Mathault, Jessy; Zamprogno, Pauline; Greener, Jesse; Miled, Amine

2015-08-01

This paper presents a new microfluidic platform that can simultaneously measure and locally modulate neurotransmitter concentration in a neuron network. This work focuses on the development of a first prototype including a potentiostat and electrode functionalization to detect several neurotransmitter's simultaneously. We tested dopamine as proof of concept to validate functionality. The system is based on 320 bidirectional electrode array for dielectrophoretic manipulation and cyclic voltammetry. Each electrode is connected to a mechanical multiplexer in order to reduce noise interference and fully isolate the electrode. The multiplexing rate is 476 kHz and each electrode can drive a signal with an amplitude of 60 V pp for dielectrophoretic manipulation.

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis.

PubMed

Li, S Kevin; Higuchi, William I; Zhu, Honggang; Kern, Steven E; Miller, David J; Hastings, Matthew S

2003-09-04

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Fujin; Department of Urinary Surgery, Huai'an Hospital to Xuzhou Medical University, Huai'an, Jiangsu; Ma, Song

Lactate dehydrogenase-A(LDH-A) is an important rate-limiting enzyme in the Warburg effect. Survival analysis indicated poor clinical outcomes in MIBC with high LDH-A expression. The results of in vitro experiment indicated that LDH-A promotes MIBC cells proliferation, invasion and migration. The positive relationship between LDH-A expression and CSC/EMT markers was confirmed both in invasive bladder cell line and in 136 MIBC specimens. Thus, we conclude that LDH-A may be a promising target for MIBC. - Highlights: • Survival analysis indicated poor clinical outcomes in MIBC with high LDH-A expression. • IHC analysis of 136 MIBC specimens revealed increased LDH-A is correlated withmore » positive Oct4 and negative E-cadherin. • In vitro experiments demonstrated LDH-A promotes MIBC progression by positive regulation of EMT/CSC.« less

Surgical manipulation of mammalian embryos in vitro.

PubMed

Naruse, I; Keino, H; Taniguchi, M

1997-04-01

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

PubMed Central

Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

2016-01-01

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

SkinEthic Laboratories, a company devoted to develop and produce in vitro alternative methods to animal use.

PubMed

de Brugerolle, Anne

2007-01-01

SkinEthic Laboratories is a France-based biotechnology company recognised as the world leader in tissue engineering. SkinEthic is devoted to develop and produce reliable and robust in vitro alternative methods to animal use in cosmetic, chemical and pharmaceutical industries. SkinEthic models provide relevant tools for efficacy and safety screening tests in order to support an integrated decision-making during research and development phases. Some screening tests are referenced and validated as alternatives to animal use (Episkin), others are in the process of validation under ECVAM and OECD guidelines. SkinEthic laboratories provide a unique and joined experience of more than 20 years from Episkin SNC and SkinEthic SA. Their unique cell culture process allows in vitro reconstructed human tissues with well characterized histology, functionality and ultrastructure features to be mass produced. Our product line includes skin models: a reconstructed human epidermis with a collagen layer, Episkin, reconstructed human epidermis without or with melanocytes (with a tanning degree from phototype II to VI) and a reconstructed human epithelium, i.e. cornea, and other mucosa, i.e. oral, gingival, oesophageal and vaginal. Our philosophy is based on 3 main commitments: to support our customers by providing robust and reliable models, to ensure training and education in using validated protocols, allowing a large array of raw materials, active ingredients and finished products in solid, liquid, powder, cream or gel form to be screened, and, to provide a dedicated service to our partners.

Hazard assessment through hybrid in vitro / in silico approach: The case of zearalenone.

PubMed

Ehrlich, Veronika A; Dellafiora, Luca; Mollergues, Julie; Dall'Asta, Chiara; Serrant, Patrick; Marin-Kuan, Maricel; Lo Piparo, Elena; Schilter, Benoit; Cozzini, Pietro

2015-01-01

Within the framework of reduction, refinement and replacement of animal experiments, new approaches for identification and characterization of chemical hazards have been developed. Grouping and read across has been promoted as a most promising alternative approach. It uses existing toxicological information on a group of chemicals to make predictions on the toxicity of uncharacterized ones. In the present work, the feasibility of applying in vitro and in silico techniques to group chemicals for read across was studied using the food mycotoxin zearalenone (ZEN) and metabolites as a case study. ZEN and its reduced metabolites are known to act through activation of the estrogen receptor α (ERα). The ranking of their estrogenic potencies appeared highly conserved across test systems including binding, in vitro and in vivo assays. This data suggests that activation of ERα may play a role in the molecular initiating event (MIE) and be predictive of adverse effects and provides the rationale to model receptor-binding for hazard identification. The investigation of receptor-ligand interactions through docking simulation proved to accurately rank estrogenic potencies of ZEN and reduced metabolites, showing the suitability of the model to address estrogenic potency for this group of compounds. Therefore, the model was further applied to biologically uncharacterized, commercially unavailable, oxidized ZEN metabolites (6α-, 6β-, 8α-, 8β-, 13- and 15-OH-ZEN). Except for 15-OH-ZEN, the data indicate that in general, the oxidized metabolites would be considered a lower estrogenic concern than ZEN and reduced metabolites.

Anticancer agent ABT-737 possesses anti-atopic dermatitis activity via blockade of caspase-1 in atopic dermatitis in vitro and in vivo models.

PubMed

Jeong, Hyun-Ja; Ryu, Ka-Jung; Kim, Hyung-Min

2018-06-29

Previous studies reported that depletion of Bcl-2 has a protective effect against allergic diseases. Furthermore, recently our study showed that anticancer drug has antiallergic inflammatory effect. An anticancer agent ABT-737 is an inhibitor of Bcl-2 and has an anti-inflammatory effect. However, the antiallergic inflammatory activity of ABT-737 is still unknown. Here, we aimed to explore the anti-atopic dermatitis (AD) activity and the mechanism of ABT-737 in AD models. HaCaT cells were used for in vitro experiments. To evaluate the effect of ABT-737 in vivo model, BalB/c mice were orally administered ABT-737 for 6 weeks in 2,4-dinitrofluorobenzene (DNFB)-induced AD-like murine model. Major assays were enzyme-linked immunosorbent assay, reverse transcription-PCR, caspase-1 assay, histamine assay, and H&E staining. ABT-737 significantly decreased thymic stromal lymphopoietin (TSLP) secretion and caspase-1 activity in activated HaCaT cells. In DNFB-induced AD mice, oral administration of ABT-737 alleviated clinical severity and scratching behavior. ABT-737 decreased levels of AD-related biomarkers including IgE, histamine, TSLP, and inflammatory cytokines. In addition, ABT significantly reduced caspase-1 activity in skin lesions of AD mice. ABT-737 elicited an anti-AD activity via suppression of caspase-1 activation in AD in vitro and in vivo models. Therefore, this study provides important information regarding the use of anticancer drugs for controlling allergic inflammatory diseases.

Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

PubMed

Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

2016-05-19

Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

Assessment of the effect of condensed (acacia and quebracho) and hydrolysable (chestnut and valonea) tannins on rumen fermentation and methane production in vitro.

PubMed

Hassanat, Fadi; Benchaar, Chaouki

2013-01-01

Tannins added to animal diets may have a positive effect on energy and protein utilisation in the rumen. The objective of this study was to examine the impact of different sources and concentrations (20, 50, 100, 150 and 200 g kg⁻¹ dry matter (DM)) of condensed (acacia and quebracho) and hydrolysable (chestnut and valonea) tannins on rumen microbial fermentation in vitro. The experiment also included a negative control with no tannins (control) and a positive control with monensin (10 mg L⁻¹). In vitro gas production and total volatile fatty acid (VFA) concentration decreased as tannin concentration increased. Addition of acacia, chestnut or valonea tannins at ≥ 50 g kg⁻¹ or quebracho tannins at ≥ 100 g kg⁻¹ resulted in a decrease (up to 40%) in methane (CH₄) production compared with the control. Valonea tannins were the only tannin source that reduced (-11%) CH₄ production at 50 g kg⁻¹ without affecting VFA concentration. Tannin treatments reduced ammonia (NH₃) and branched-chain VFA concentrations, indicating a reduction in ruminal protein degradation. Monensin reduced CH₄ production (-37%) and NH₃ concentration (-20%) without affecting total VFA concentration. Supplying acacia, chestnut or valonea tannins at 50 g kg⁻¹ has the potential to reduce CH₄ production and ruminal protein degradation with minimum detrimental effects on efficiency of ruminal fermentation. Copyright © 2012 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.

Plasmodium falciparum in vitro continuous culture conditions: A comparison of parasite susceptibility and tolerance to anti-malarial drugs throughout the asexual intra-erythrocytic life cycle.

PubMed

Duffy, Sandra; Avery, Vicky M

2017-12-01

The continuous culture of Plasmodium falciparum is often seen as a means to an end, that end being to probe the biology of the parasite in question, and ultimately for many in the malaria drug discovery arena, to identify means of killing the parasite in order to treat malaria. In vitro continuous culture of Plasmodium falciparum is a fundamental requirement when undertaking malaria research where the primary objectives utilise viable parasites of a desired lifecycle stage. This investigation, and resulting data, compared the impact culturing Plasmodium falciparum long term (4 months) in different environmental conditions had on experimental outcomes and thus conclusions. The example presented here focused specifically on the effect culture conditions had on the in vitro tolerance of Plasmodium falciparum to standard anti-malarial drugs, including artemisinin and lumefantrine. Historical data from an independent experiment for 3D7-ALB (5% O 2 ) was also compared with that obtained from this study. We concluded that parasites cultured for several months in media supplemented with a serum substitute such as Albumax II ® or within hyperoxic conditions (21% O 2 ), demonstrate highly variable responses to artemisinin and lumefantrine but not all anti-malarial drugs, when compared to those cultured in human serum in combination with Albumax II ® under normoxic conditions (5% O 2 ) for the parasite. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Variation in Septoria musiva and Implications for Disease Resistance Screening

Treesearch

K.T. Ward; M.E. Ostry

2005-01-01

A set of isolates of Septoria musiva differed in aggressiveness in hybrid poplar leaf disk and stem assays and culture growth in vitro. Clone x isolate interactions were observed in one of the stem assay experiments, but not in the leaf disk assay experiments. Random amplified polymorphic DNA (RAPD) analyses were performed using 52 isolates of

The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance.

PubMed

Scarth, James P; Spencer, Holly A; Timbers, Sarah E; Hudson, Simon C; Hillyer, Lynn L

2010-01-01

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document.In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm. © 2010 John Wiley & Sons, Ltd.

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.

PubMed

Hinrichs, K; Love, C C; Brinsko, S P; Choi, Y H; Varner, D D

2002-07-01

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.

Protective effects of Erigeron breviscapus Hand.– Mazz. (EBHM) extract in retinal neurodegeneration models

PubMed Central

Zhu, Jingyuan; Chen, Li; Qi, Yun; Feng, Jing; Zhu, Li; Bai, Yujing

2018-01-01

Purpose To investigate the neuroprotective effects of scutellarin, an active component of the multifunctional traditional Chinese herb Erigeron breviscapus (vant.) Hand.-Mazz. (EBHM), which has been used as a neuroprotective therapy for cerebrovascular diseases. We performed the experiments using in vitro and in vivo models of retinal neurodegeneration. Methods In the in vitro experiments, we exposed BV-2 cells to low oxygen levels in an incubator for 24 and 48 h to generate hypoxia models. We then treated these cells with scutellarin at concentrations of 2, 10, and 50 µM. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA). The levels of the components of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome signaling pathway, including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved caspase-1, interleukin-18 (IL-18), and IL-1β were analyzed using western blots and ELISAs. In the in vivo study, we raised the intraocular pressure of Brown Norway rats to 60 mmHg for 30 min to generate a high intraocular pressure (HIOP) model, that is, an acute glaucoma model. The rats were then treated with scutellarin via oral gavage for 2 consecutive weeks. The relevant components of the NLRP3 inflammasome signaling pathway were analyzed with western blots and ELISAs. Retinal ganglion cells (RGCs) were retrogradely labeled using 4% Fluoro-Gold, and then the numbers of cells were calculated. Retinal microglial cells were labeled using immunofluorescence, and then the morphological changes were observed. Results In the in vitro cell viability experiments, 50 µM scutellarin statistically significantly enhanced the viability rate when compared to 2 µM and 10 µM scutellarin (hypoxia + 50 µM EBHM group: 94.01±2.130% and 86.02±2.520% after 24 and 48 h, respectively; hypoxia model group: 74.98±3.860% and 64.41±4.890% after 24 and 48 h, respectively; for all when compared to normal control, p<0.001). Scutellarin inhibited the expression of NLRP3 in vitro (the hypoxia + EBHM group/normal control group ratio versus the hypoxia model group/normal control group ratio: 2.30±0.12 versus 4.06±0.19, p<0.01) and in vivo (the HIOP + EBHM group/normal control group ratio versus the HIOP model group/normal control ratio: 3.39±0.42 versus 6.07±0.22, p<0.01). Scutellarin administration also reduced the upregulation of ASC, cleaved caspase-1, IL-18, and IL-1β in vitro and in vivo. In the in vivo study, the RGC survival rate was statistically significantly improved following scutellarin administration (p<0.001 versus the HIOP group), and the number of impaired retinal microglial cells was statistically significantly reduced following scutellarin treatment when compared with the HIOP model group. Conclusions EBHM extract scutellarin exhibits protective effects in retinal hypoxia models by inhibiting NLRP3 inflammasome-mediated inflammatory reactions. Thus, EBHM extract scutellarin may be an appropriate therapeutic option for disorders related to retinal neurodegeneration, such as glaucoma.

Protective effects of Erigeron breviscapus Hand.- Mazz. (EBHM) extract in retinal neurodegeneration models.

PubMed

Zhu, Jingyuan; Chen, Li; Qi, Yun; Feng, Jing; Zhu, Li; Bai, Yujing; Wu, Huijuan

2018-01-01

To investigate the neuroprotective effects of scutellarin, an active component of the multifunctional traditional Chinese herb Erigeron breviscapus (vant.) Hand.-Mazz. (EBHM), which has been used as a neuroprotective therapy for cerebrovascular diseases. We performed the experiments using in vitro and in vivo models of retinal neurodegeneration. In the in vitro experiments, we exposed BV-2 cells to low oxygen levels in an incubator for 24 and 48 h to generate hypoxia models. We then treated these cells with scutellarin at concentrations of 2, 10, and 50 µM. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA). The levels of the components of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome signaling pathway, including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved caspase-1, interleukin-18 (IL-18), and IL-1β were analyzed using western blots and ELISAs. In the in vivo study, we raised the intraocular pressure of Brown Norway rats to 60 mmHg for 30 min to generate a high intraocular pressure (HIOP) model, that is, an acute glaucoma model. The rats were then treated with scutellarin via oral gavage for 2 consecutive weeks. The relevant components of the NLRP3 inflammasome signaling pathway were analyzed with western blots and ELISAs. Retinal ganglion cells (RGCs) were retrogradely labeled using 4% Fluoro-Gold, and then the numbers of cells were calculated. Retinal microglial cells were labeled using immunofluorescence, and then the morphological changes were observed. In the in vitro cell viability experiments, 50 µM scutellarin statistically significantly enhanced the viability rate when compared to 2 µM and 10 µM scutellarin (hypoxia + 50 µM EBHM group: 94.01±2.130% and 86.02±2.520% after 24 and 48 h, respectively; hypoxia model group: 74.98±3.860% and 64.41±4.890% after 24 and 48 h, respectively; for all when compared to normal control, p<0.001). Scutellarin inhibited the expression of NLRP3 in vitro (the hypoxia + EBHM group/normal control group ratio versus the hypoxia model group/normal control group ratio: 2.30±0.12 versus 4.06±0.19, p<0.01) and in vivo (the HIOP + EBHM group/normal control group ratio versus the HIOP model group/normal control ratio: 3.39±0.42 versus 6.07±0.22, p<0.01). Scutellarin administration also reduced the upregulation of ASC, cleaved caspase-1, IL-18, and IL-1β in vitro and in vivo. In the in vivo study, the RGC survival rate was statistically significantly improved following scutellarin administration (p<0.001 versus the HIOP group), and the number of impaired retinal microglial cells was statistically significantly reduced following scutellarin treatment when compared with the HIOP model group. EBHM extract scutellarin exhibits protective effects in retinal hypoxia models by inhibiting NLRP3 inflammasome-mediated inflammatory reactions. Thus, EBHM extract scutellarin may be an appropriate therapeutic option for disorders related to retinal neurodegeneration, such as glaucoma.

Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.

PubMed

Albert, Ina; Hefti, Martin; Luginbuehl, Vera

2014-11-01

The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2  =  19%) and physiological (pO2  =  9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.

Surface modification of nano-silica on the ligament advanced reinforcement system for accelerated bone formation: primary human osteoblasts testing in vitro and animal testing in vivo

NASA Astrophysics Data System (ADS)

Li, Mengmeng; Wang, Shiwen; Jiang, Jia; Sun, Jiashu; Li, Yuzhuo; Huang, Deyong; Long, Yun-Ze; Zheng, Wenfu; Chen, Shiyi; Jiang, Xingyu

2015-04-01

The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ~21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction.The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ~21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01439e

Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

PubMed

Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

2005-01-01

This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

Oocyte quality and estradiol supplementation affect in vitro maturation success in the white-tailed deer (Odocoileus virginianus).

PubMed

Siriaroonrat, B; Comizzoli, P; Songsasen, N; Monfort, S L; Wildt, D E; Pukazhenthi, B S

2010-01-01

White-tailed deer oocyte biology is not well documented. The objective of this study was to determine (1) the influence of estradiol (E(2)) supplementation on meiotic resumption and the ability to "rescue" poorer quality (lower grade) oocytes and (2) the kinetics of oocyte nuclear maturation in vitro in the white-tailed deer. In Experiment 1, immature oocytes harvested during hunting-culling operations were cultured for 24h in the presence or absence of E(2). Incubation in 1mug/mL E(2) promoted nuclear maturation (to telophase I, TI; or to metaphase II, MII) in a higher proportion of Grade 1 oocytes ( approximately 77%; P<0.05) compared with that in Grade 2 or Grade 3 counterparts ( approximately 51%). For Grades 2 and 3 oocytes, there was no advantage (P>0.05) for E(2) supplementation in reaching TI/MII. In Experiment 2, Grade 1 oocytes were cultured in the presence of E(2) and nuclear status evaluated at 0, 3, 6, 12, and 24h of in vitro incubation. At 0h,>70% of oocytes already had undergone germinal vesicle breakdown. After 12h, approximately 70% of oocytes had reached metaphase I of nuclear maturation, with approximately 75% achieving TI/MII by 24h in vitro. In summary, adding E(2) to an in vitro maturation (IVM) culture system for white-tailed deer was advantageous, but only for the highest quality oocytes, with approximately 75% achieving nuclear maturation. In contrast, E(2) supplement did not benefit lower-grade oocytes, half of which will reach MII, with the other half failing. Under the described culture conditions, good-quality white-tailed deer oocytes achieve nuclear maturation over a time duration comparable with that reported in other ungulates.

Role of Protein Synthesis Initiation Factors in Dietary Soy Isoflavone-Mediated Effects on Breast Cancer Progression

DTIC Science & Technology

2014-08-01

experiments of this award and published in the high-impact peer-reviewed journal: Journal of Biological Chemistry (13), was that the described increase...of genistein and other flavonoids in human breast cancer cells in vitro, Nutr Cancer, 27: 31-40, 1997. 13 de la,P.C., Otero-Franqui,E., Martinez...properties of genistein and other flavonoids in human breast cancer cells in vitro, Nutr Cancer, 27: 31-40, 1997. 3 Okabe,Y., Shimazu,T. and Tanimoto,H

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

PubMed

Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

2016-08-01

Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

Buffer-regulated biocorrosion of pure magnesium.

PubMed

Kirkland, Nicholas T; Waterman, Jay; Birbilis, Nick; Dias, George; Woodfield, Tim B F; Hartshorn, Richard M; Staiger, Mark P

2012-02-01

Magnesium (Mg) alloys are being actively investigated as potential load-bearing orthopaedic implant materials due to their biodegradability in vivo. With Mg biomaterials at an early stage in their development, the screening of alloy compositions for their biodegradation rate, and hence biocompatibility, is reliant on cost-effective in vitro methods. The use of a buffer to control pH during in vitro biodegradation is recognised as critically important as this seeks to mimic pH control as it occurs naturally in vivo. The two different types of in vitro buffer system available are based on either (i) zwitterionic organic compounds or (ii) carbonate buffers within a partial-CO(2) atmosphere. This study investigated the influence of the buffering system itself on the in vitro corrosion of Mg. It was found that the less realistic zwitterion-based buffer did not form the same corrosion layers as the carbonate buffer, and was potentially affecting the behaviour of the hydrated oxide layer that forms on Mg in all aqueous environments. Consequently it was recommended that Mg in vitro experiments use the more biorealistic carbonate buffering system when possible.

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus.

PubMed

Hong, S K; Matsumoto, A; Horinouchi, S; Beppu, T

1993-01-01

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

Integration of QSAR and in vitro toxicology.

PubMed Central

Barratt, M D

1998-01-01

The principles of quantitative structure-activity relationships (QSAR) are based on the premise that the properties of a chemical are implicit in its molecular structure. Therefore, if a mechanistic hypothesis can be proposed linking a group of related chemicals with a particular toxic end point, the hypothesis can be used to define relevant parameters to establish a QSAR. Ways in which QSAR and in vitro toxicology can complement each other in development of alternatives to live animal experiments are described and illustrated by examples from acute toxicological end points. Integration of QSAR and in vitro methods is examined in the context of assessing mechanistic competence and improving the design of in vitro assays and the development of prediction models. The nature of biological variability is explored together with its implications for the selection of sets of chemicals for test development, optimization, and validation. Methods are described to support the use of data from in vivo tests that do not meet today's stringent requirements of acceptability. Integration of QSAR and in vitro methods into strategic approaches for the replacement, reduction, and refinement of the use of animals is described with examples. PMID:9599692

In Vitro Cardiomyogenic Potential of Human Amniotic Fluid Stem Cells

PubMed Central

Guan, Xuan; Delo, Dawn M.; Atala, Anthony; Soker, Shay

2010-01-01

Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including the inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells, and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy by testing the in vitro differentiation capabilities. Undifferentiated hAFS cells express several cardiac genes, including the transcription factor mef2, the gap junction connexin43, and H- and N-cadherin. A 24-hour incubation with 5-aza-2′–deoxycytidine (5-AZA-dC) induced hAFS cell differentiation along the cardiac lineage. Evidence for this differentiation included morphological changes, up-regulation of cardiac-specific genes (cardiac troponin I and cardiac troponin T) and redistribution of connexin43, as well as down-regulation of the stem cell marker SRY-box 2 (sox2). When co-cultured with neonatal rat cardiomyocytes (NRCs), hAFS cells formed both mechanical and electrical connections with the NRCs. Dye transfer experiments showed that calcein dye could be transferred from NRCs to hAFS cells through cellular connections. The gap junction connexin 43 likely involved in the communication between the two cell types, because 12-O-Tetradecanoylphorbol 13-acetate (TPA) could partially block cellular crosstalk. We conclude that hAFS cells can be differentiated into a cardiomyocyte-like phenotype and can establish functional communication with NRCs. Thus, hAFS cells may potentially be used for cardiac cell therapy. PMID:20687122

In vitro cardiomyogenic potential of human amniotic fluid stem cells.

PubMed

Guan, Xuan; Delo, Dawn M; Atala, Anthony; Soker, Shay

2011-03-01

Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy, by testing the in vitro differentiation capabilities. Undifferentiated hAFS cells express several cardiac genes, including the transcription factor mef2, the gap junction connexin43, and H- and N-cadherin. A 24 h incubation with 5-aza-2'-deoxycytidine (5-AZA-dC) induced hAFS cell differentiation along the cardiac lineage. Evidence for this differentiation included morphological changes, upregulation of cardiac-specific genes (cardiac troponin I and cardiac troponin T) and redistribution of connexin43, as well as downregulation of the stem cell marker SRY-box 2 (sox2). When co-cultured with neonatal rat cardiomyocytes (NRCs), hAFS cells formed both mechanical and electrical connections with the NRCs. Dye transfer experiments showed that calcein dye could be transferred from NRCs to hAFS cells through cellular connections. The gap junction connexin43 likely involved in the communication between the two cell types, because 12-O-tetradecanoylphorbol 13-acetate (TPA) could partially block cellular crosstalk. We conclude that hAFS cells can be differentiated into a cardiomyocyte-like phenotype and can establish functional communication with NRCs. Thus, hAFS cells may potentially be used for cardiac cell therapy. Copyright © 2010 John Wiley & Sons, Ltd.

EPA National Center for Computational Toxicology UPDATE (ICCVAM public forum)

EPA Science Inventory

A presentation to the ICCVAM Public Forum on several new and exciting activities at NCCT, including Chemical library update, Chemistry Dashboard, Retrofitting in vitro assays with metabolic competence and In vitro PK.

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius).

PubMed

Wani, N A; Skidmore, J A

2010-08-01

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 microg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 +/- 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 +/- 4.1) and 26-27 h (82.1 +/- 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 +/- 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 +/- 2.1 vs. 60.9 +/- 6.6) and developed to blastocyst stages (52.4 +/- 4.1 vs. 30.5 +/- 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation. In conclusion, about 80-90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro. Copyright 2010 Elsevier Inc. All rights reserved.

Influence of a constant and variable magnetic field on the coagulation of human blood in vitro and in vivo

NASA Technical Reports Server (NTRS)

Degen, I. L.; Plaksenko, V. Y.

1974-01-01

The influence of constant and varying magnetic fields on the coagulation of the blood was studied in experiments performed in vitro and vivo. In the in vitro tests it was found that a constant magnetic field with a strength of 100 or 200 oersteds influences the coagulation of the blood, retarding it in some cases and speeding up the coagulation time in others. In the in vivo studies, both retarding and accelerating effects were likewise observed with respect to the coagulation of the blood, but the nature of the change was a function of the background. A normalizing effect of the magnetic field on the coagulation of the blood was observed.

The role of the social worker in the in-vitro fertilization program.

PubMed

Greenfeld, D; Mazure, C; Haseltine, F; DeCherney, A

1984-01-01

The role of the clinical social worker in the In-Vitro fertilization Program is to help provide patients with an environment that includes realistic expectation and emphasizes the emotional spectrum of euphoria, anxiety and dysphoria that can accompany the demanding protocol. The literature supports the need for counseling and supportive psychotherapy in the infertility clinic but has not dealt specifically with the psychological demands of In-Vitro fertilization. This paper addresses the emotional stress of in-vitro fertilization and emphasizes the role of social worker as counselor, educator and guide.

Toxicity assessment of tobacco products in vitro.

PubMed

Manuppello, Joseph R; Sullivan, Kristie M

2015-03-01

Driven by new regulatory demands to demonstrate risk reduction, the toxicity assessment of tobacco products increasingly employs innovative in vitro methods, including biphasic cell and tissue cultures exposed to whole cigarette smoke at the air-liquid interface, cell transformation assays, and genomic analyses. At the same time, novel tobacco products are increasingly compared to traditional cigarettes. This overview of in vitro toxicology studies of tobacco products reported in the last five years provides evidence to support the prioritisation of in vitro over in vivo methods by industry and their recommendation by regulatory authorities. 2015 FRAME.

Role of Combination Antimicrobial Therapy for Vancomycin-Resistant Enterococcus faecium Infections: Review of the Current Evidence.

PubMed

Yim, Juwon; Smith, Jordan R; Rybak, Michael J

2017-05-01

Enterococcus species are the second most common cause of nosocomial infections in the United States and are particularly concerning in critically ill patients with preexisting comorbid conditions. Rising resistance to antimicrobials that were historically used as front-line agents for treatment of enterococcal infections, such as ampicillin, vancomycin, and aminoglycosides, further complicates the treatment of these infections. Of particular concern are Enterococcus faecium strains that are associated with the highest rate of vancomycin resistance. The introduction of antimicrobial agents with specific activity against vancomycin-resistant Enterococcus (VRE) faecium including daptomycin, linezolid, quinupristin-dalfopristin, and tigecycline did not completely resolve this clinical dilemma. In this review, the mechanisms of action and resistance to currently available anti-VRE antimicrobial agents including newer agents such as oritavancin and dalbavancin will be presented. In addition, novel combination therapies including β-lactams and fosfomycin, and the promising results from in vitro, animal studies, and clinical experience in the treatment of VRE faecium will be discussed. © 2017 Pharmacotherapy Publications, Inc.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

PubMed

Behrsing, Holger; Hill, Erin; Raabe, Hans; Tice, Raymond; Fitzpatrick, Suzanne; Devlin, Robert; Pinkerton, Kent; Oberdörster, Günter; Wright, Chris; Wieczorek, Roman; Aufderheide, Michaela; Steiner, Sandro; Krebs, Tobias; Asgharian, Bahman; Corley, Richard; Oldham, Michael; Adamson, Jason; Li, Xiang; Rahman, Irfan; Grego, Sonia; Chu, Pei-Hsuan; McCullough, Shaun; Curren, Rodger

2017-07-01

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide. 2017 FRAME.

In Vitro Determination of Bicarbonate Dosage to Alkalinize Local Anesthetics to Physiologic pH

DTIC Science & Technology

1997-05-01

as "an unpleasant sensory and emotional experience associated with actual damage or injury to a body tissue" (Mersky, 1986, p.SI). However, this...definition does not adequately express the unique, mutable and subjective experience that those who suffer pain encounter. Milton alludes to the...emotional experience of pain in Paradise Lost when he writes "Pain is perfect miserie, the worst of evils, and excessive, overturns all patience" (Milton

In vitro fertilisation treatment and factors affecting success.

PubMed

Huang, Jack Yu Jen; Rosenwaks, Zev

2012-12-01

The efficacy of assisted reproductive technologies has improved significantly over the past decades. The main indications for in vitro fertilisation include tubal obstruction, severe male-factor infertility, severe endometriosis, ovulatory dysfunction, diminished ovarian reserve, and infertility of unexplained cause. In vitro fertilisation has also become an effective treatment option for couples wishing to undergo pre-implantation genetic diagnosis or screening, and for those wishing to cryopreserve their oocytes or embryos for preservation of fertility. The management of women in late reproductive age poses a major challenge; the optimum in vitro fertilisation treatment for poor responders remains elusive. The success of in vitro fertilisation treatment can be optimised by taking an individualised, patient-centered approach to controlled ovarian hyperstimulation. Key components involve selection of an appropriate controlled ovarian protocol, close-cycle monitoring, adjustment of gonadotropin dosage to avoid hyper-response, and individualised timing of human chorionic gonadotropin injection. Future directions of assisted reproductive technologies include development of non-invasive embryo selection methods, use of transcriptomics, proteomics, metabolomics, and time-lapse imaging technologies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antioxidant Activity and Hepatoprotective Potential of Quercetin 7-Rhamnoside In Vitro and In Vivo.

PubMed

Huang, Zhi-Qiang; Chen, Pan; Su, Wei-Wei; Wang, Yong-Gang; Wu, Hao; Peng, Wei; Li, Pei-Bo

2018-05-16

Hypericum japonicum is traditionally used as a folk medicine to treat cholestasis and hepatitis. Quercetin 7-rhamnoside (Q7R) is one of the main flavonoid components of Hypericum japonicum and has been rarely studied. The aim of the present study was to evaluate the antioxidant activity and hepatoprotective potential of Q7R. In the in vitro experiments, DPPH, ABTS and ferric reducing antioxidant power (FRAP) assays were first performed to assess the antioxidant properties of Q7R, and then a H₂O₂-induced oxidative damage cellular model was used to determine the cytoprotective and antioxidant properties of Q7R in human liver L-02 cells. In the in vivo experiment, the hepatoprotective activity of Q7R was evaluated by carbon tetrachloride (CCl₄)-induced liver damage model in mice. The results of the three in vitro assays (DPPH, ABTS and FRAP) demonstrated that Q7R significantly exhibited antioxidant activity. The cell experiment results showed that Q7R possessed cytoprotective and antioxidant effects on H₂O₂-treated L-02 cells. In the in vivo experiments, Q7R suppressed the up-regulation of serum activities of ALT, AST, LDH and triglyceride (TG) levels with dose-dependency. Q7R down-regulated the production of MDA and increased the hepatic GSH content and antioxidant enzymes CAT activities. Hepatic morphological analysis was also performed to confirm the biochemical changes. In summary, these results suggested that Q7R could be considered as a potential source of natural antioxidants, and may become a promising candidate for the treatment of liver injury in the future.

Use of in-vitro experimental results to model in-situ experiments: bio-denitrification under geological disposal conditions.

PubMed

Masuda, Kaoru; Murakami, Hiroshi; Kurimoto, Yoshitaka; Kato, Osamu; Kato, Ko; Honda, Akira

2013-01-01

Some of the low level radioactive wastes from reprocessing of spent nuclear fuels contain nitrates. Nitrates can be present in the form of soluble salts and can be reduced by various reactions. Among them, reduction by metal compounds and microorganisms seems to be important in the underground repository. Reduction by microorganism is more important in near field area than inside the repository because high pH and extremely high salt concentration would prevent microorganism activities. In the near field, pH is more moderate (pH is around 8) and salt concentration is lower. However, the electron donor may be limited there and it might be the control factor for microorganism's denitrification activities. In this study, in-vitro experiments of the nitrate reduction reaction were conducted using model organic materials purported to exist in underground conditions relevant to geological disposal. Two kinds of organic materials were selected. A super plasticizer was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Nitrates were reduced almost to N2 gas in the existence of super plasticizer. In the case of humic acids, although nitrates were reduced, the rate was much lower and, in this case, dead organism was used as an electron donor instead of humic acids. A reaction model was developed based on the in-vitro experiments and verified by running simulations against data obtained from in-situ experiments using actual groundwaters and microorganisms. The simulation showed a good correlation with the experimental data and contributes to the understanding of microbially mediated denitrification in geological disposal systems.

Performance assessment of an opto-fluidic phantom mimicking porcine liver parenchyma

NASA Astrophysics Data System (ADS)

Akl, Tony J.; King, Travis J.; Long, Ruiqi; McShane, Michael J.; Nance Ericson, M.; Wilson, Mark A.; Coté, Gerard L.

2012-07-01

An implantable, optical oxygenation and perfusion sensor to monitor liver transplants during the two-week period following the transplant procedure is currently being developed. In order to minimize the number of animal experiments required for this research, a phantom that mimics the optical, anatomical, and physiologic flow properties of liver parenchyma is being developed as well. In this work, the suitability of this phantom for liver parenchyma perfusion research was evaluated by direct comparison of phantom perfusion data with data collected from in vivo porcine studies, both using the same prototype perfusion sensor. In vitro perfusion and occlusion experiments were performed on a single-layer and on a three-layer phantom perfused with a dye solution possessing the absorption properties of oxygenated hemoglobin. While both phantoms exhibited response patterns similar to the liver parenchyma, the signal measured from the multilayer phantom was three times higher than the single layer phantom and approximately 21 percent more sensitive to in vitro changes in perfusion. Although the multilayer phantom replicated the in vivo flow patterns more closely, the data suggests that both phantoms can be used in vitro to facilitate sensor design.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

PubMed

Hiroishi, S; Sugie, K; Yoshida, T; Morimoto, J; Taniguchi, Y; Imai, S; Kurebayashi, J

2001-06-26

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

[Study thought of material basis of secondary development of major traditional Chinese medicine varieties on basis of combination of in vivo and in vitro experiments].

PubMed

Cheng, Xu-Dong; Jia, Xiao-Bin; Feng, Liang; Jiang, Jun

2013-12-01

The secondary development of major traditional Chinese medicine varieties is one of important links during the modernization, scientification and standardization of traditional Chinese medicines. How to accurately and effectively identify the pharmacodynamic material basis of original formulae becomes the primary problem in the secondary development, as well as the bottleneck in the modernization development of traditional Chinese medicines. On the basis of the existing experimental methods, and according to the study thought that the multi-component and complex effects of traditional Chinese medicine components need to combine multi-disciplinary methods and technologies, we propose the study thought of the material basis of secondary development of major traditional Chinese medicine varieties based on the combination of in vivo and in vitro experiments. It is believed that studies on material basis needs three links, namely identification, screening and verification, and in vivo and in vitro study method corresponding to each link is mutually complemented and verified. Finally, the accurate and reliable material basis is selected. This thought provides reference for the secondary development of major traditional Chinese medicine varieties and studies on compound material basis.

Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting amyloid beta formation: in silico and in vitro study

NASA Astrophysics Data System (ADS)

Thai, Nguyen Quoc; Tseng, Ning-Hsuan; Vu, Mui Thi; Nguyen, Tin Trung; Linh, Huynh Quang; Hu, Chin-Kun; Chen, Yun-Ru; Li, Mai Suan

2016-08-01

Combining Lipinski's rule with the docking and steered molecular dynamics simulations and using the PubChem data base of about 1.4 million compounds, we have obtained DNA dyes Hoechst 34580 and Hoechst 33342 as top-leads for the Alzheimer's disease. The binding properties of these ligands to amyloid beta (Aβ) fibril were thoroughly studied by in silico and in vitro experiments. Hoechst 34580 and Hoechst 33342 prefer to locate near hydrophobic regions with binding affinity mainly governed by the van der Waals interaction. By the Thioflavin T assay, it was found that the inhibition constant IC50 ≈ 0.86 and 0.68 μM for Hoechst 34580 and Hoechst 33342, respectively. This result qualitatively agrees with the binding free energy estimated using the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulations with the AMBER-f99SB-ILDN force field and water model TIP3P. In addition, DNA dyes have the high capability to cross the blood brain barrier. Thus, both in silico and in vitro experiments have shown that Hoechst 34580 and 33342 are good candidates for treating the Alzheimer's disease by inhibiting Aβ formation.

Prediction of percutaneous absorption in human using three-dimensional human cultured epidermis LabCyte EPI-MODEL.

PubMed

Hikima, Tomohiro; Kaneda, Noriaki; Matsuo, Kyouhei; Tojo, Kakuji

2012-01-01

The objective of this study is to establish a relationship of the skin penetration parameters between the three-dimensional cultured human epidermis LabCyte EPI-MODEL (LabCyte) and hairless mouse (HLM) skin penetration in vitro and to predict the skin penetration and plasma concentration profile in human. The skin penetration experiments through LabCyte and HLM skin were investigated using 19 drugs that have a different molecular weight and lipophilicity. The penetration flux for LabCyte reached 30 times larger at maximum than that for HLM skin. The human data can be estimated from the in silico approach with the diffusion coefficient (D), the partition coefficient (K) and the skin surface concentration (C) of drugs by assuming the bi-layer skin model for both LabCyte and HLM skin. The human skin penetration of β-estradiol, prednisolone, testosterone and ethynylestradiol was well agreed between the simulated profiles and in vitro experimental data. Plasma concentration profiles of β-estradiol in human were also simulated and well agreed with the clinical data. The present alternative method may decrease human or animal skin experiment for in vitro skin penetration.

Endogenous boldenone-formation in cattle: alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed.

PubMed

Verheyden, K; Noppe, H; Zorn, H; Van Immerseel, F; Vanden Bussche, J; Wille, K; Bekaert, K; Janssen, C R; De Brabander, H F; Vanhaecke, L

2010-04-01

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature. 2010 Elsevier Ltd. All rights reserved.

Transcutol containing vesicles for topical delivery of minoxidil.

PubMed

Mura, Simona; Manconi, Maria; Valenti, Donatella; Sinico, Chiara; Vila, Amparo Ofelia; Fadda, Anna Maria

2011-04-01

The aim of this work was to evaluate the ability of Transcutol (Trc) to produce elastic vesicles with soy lecithin (SL) and study the influence of the obtained vesicles on in vitro (trans)dermal delivery of minoxidil. To this purpose, so-called penetration enhancer-containing vesicles (PEVs) were prepared using Trc aqueous solutions (5-10-20-30% v/v) as hydrophilic phase. SL liposomes, without Trc, were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, deformability, and rheological behavior. The influence of the obtained PEVs on (trans)dermal delivery of minoxidil was studied by in vitro diffusion experiments through pig skin. Results showed that all prepared PEVs were able to give good entrapment efficiency (E%≈67) similar to that of conventional liposomes. Trc-containing PEVs showed to be more deformable than liposomes only when minoxidil was loaded in 5 and 10% Trc-containing vesicles. Rheological studies showed that PEVs have higher fluidity than conventional liposomes. All PEVs showed a higher stability than liposomes as shown by studying zeta potential and size distribution during three months. Results of in vitro diffusion experiments showed that Trc-containing PEVs are able to deliver minoxidil to deep skin layers without any transdermal permeation.

In Vitro Degradation and Fermentation of Three Dietary Fiber Sources by Human Colonic Bacteria

PubMed Central

Bliss, Donna Z.; Weimer, Paul J.; Jung, Hans-Joachim G.; Savik, Kay

2013-01-01

Although clinical benefits of dietary fiber supplementation seem to depend partially on the extent of fiber degradation and fermentation by colonic bacteria, little is known about the effect of supplemental fiber type on bacterial metabolism. In an experiment using a non-adapted human bacterial population from three normal subjects, extent of in vitro fermentation was greater for gum arabic (GA) than for psyllium (PSY), which was greater than that for carboxymethylcellulose (CMC). In a separate experiment, in vitro incubation with feces from 52 subjects with fecal incontinence, before and after random assignment to and consumption of one of three fiber (GA, PSY, or CMC) supplements or a placebo for 20-21d, indicated that prior consumption of a specific fiber source did not increase its degradation by fecal bacteria. Results suggest that the colonic microbial community enriched on a particular fiber substrate can rapidly adapt to the presentation of a new fiber substrate. Clinical implications of the findings are that intake of a fiber source by humans is not expected to result in bacterial adaptation that would require continually larger and eventually intolerable amounts of fiber to achieve therapeutic benefits. PMID:23556460

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

PubMed

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro degradation and fermentation of three dietary fiber sources by human colonic bacteria.

PubMed

Bliss, Donna Z; Weimer, Paul J; Jung, Hans-Joachim G; Savik, Kay

2013-05-15

Although clinical benefits of dietary fiber supplementation seem to depend partially on the extent of fiber degradation and fermentation by colonic bacteria, little is known about the effect of supplemental fiber type on bacterial metabolism. In an experiment using a nonadapted human bacterial population from three normal subjects, the extent of in vitro fermentation was greater for gum arabic (GA) than for psyllium (PSY), which was greater than that for carboxymethylcellulose (CMC). In a separate experiment, in vitro incubation with feces from 52 subjects with fecal incontinence, before and after random assignment to and consumption of one of three fiber (GA, PSY, or CMC) supplements or a placebo for 20-21 days, indicated that prior consumption of a specific fiber source did not increase its degradation by fecal bacteria. Results suggest that the colonic microbial community enriched on a particular fiber substrate can rapidly adapt to the presentation of a new fiber substrate. Clinical implications of the findings are that intake of a fiber source by humans is not expected to result in bacterial adaptation that would require continually larger and eventually intolerable amounts of fiber to achieve therapeutic benefits.

Hydrostatic pressure affects in vitro maturation of oocytes and follicles and increases granulosa cell death.

PubMed

Rashidi, Zahra; Azadbakht, Mehri; Amini, Ali; Karimi, Isac

2014-01-01

This study examines the effects of hydrostatic pressure on in vitro maturation (IVM) of oocytes derived from in vitro grown follicles. In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM) under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student's t test. The percentage of metaphase II oocytes (MII) increased in hydrostatic pressuretreated follicles compared to controls (p<0.05). Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05). Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05). Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.

In vitro assessment of six aspiration catheters using a distal protection filter.

PubMed

Fujimura, Tatsuhiro; Okamura, Takayuki; Ando, Miyuki; Uchida, Kousuke; Tone, Takashi; Yonezawa, Fumio; Yano, Masafumi

2016-01-01

We assessed performance of 6 aspiration catheters for distal embolization using a distal protection filter in an in vitro experiment. In acute myocardial infarction, a distal protection filter is used for lesions likely to induce a distal embolism. Which aspiration cathether is most effective when used with a distal protection filter remains still unclear. A 0.5-cm3 bolus of gelatin as a model of stagnant pools of coronary plaque debris was captured in the distal protection filter and aspirated by 6 aspiration catheters. We measured and compared the length of the suspended embolus matter. Among the 6 catheters evaluated, the use of the Export Advance catheter (Medtronic) resulted in significantly shorter lengths of the suspended embolus matter compared to the use of the TVAC II (Nipro), Thrombuster III SL (Kaneka), and Rebirth Pro (Goodman) catheters (p < 0.01). The residual embolus matter in all cases had drained distally to the distal protection filter when the filter was retrieved. The use of the Export Advance catheter showed better performance using a distal protection filter in this in vitro experiment, and its use might be more effective in preventing distal embolisms in combination with a distal protection filter.

Current and future antimicrobial treatment of gonorrhoea - the rapidly evolving Neisseria gonorrhoeae continues to challenge.

PubMed

Unemo, Magnus

2015-08-21

Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to all drugs previously and currently recommended for empirical monotherapy of gonorrhoea. In vitro resistance, including high-level, to the last option ceftriaxone and sporadic failures to treat pharyngeal gonorrhoea with ceftriaxone have emerged. In response, empirical dual antimicrobial therapy (ceftriaxone 250-1000 mg plus azithromycin 1-2 g) has been introduced in several particularly high-income regions or countries. These treatment regimens appear currently effective and should be considered in all settings where local quality assured AMR data do not support other therapeutic options. However, the dual antimicrobial regimens, implemented in limited geographic regions, will not entirely prevent resistance emergence and, unfortunately, most likely it is only a matter of when, and not if, treatment failures with also these dual antimicrobial regimens will emerge. Accordingly, novel affordable antimicrobials for monotherapy or at least inclusion in new dual treatment regimens, which might need to be considered for all newly developed antimicrobials, are essential. Several of the recently developed antimicrobials deserve increased attention for potential future treatment of gonorrhoea. In vitro activity studies examining collections of geographically, temporally and genetically diverse gonococcal isolates, including multidrug-resistant strains particularly with resistance to ceftriaxone and azithromycin, are important. Furthermore, understanding of effects and biological fitness of current and emerging (in vitro induced/selected and in vivo emerged) genetic resistance mechanisms for these antimicrobials, prediction of resistance emergence, time-kill curve analysis to evaluate antibacterial activity, appropriate mice experiments, and correlates between genetic and phenotypic laboratory parameters, and clinical treatment outcomes, would also be valuable. Subsequently, appropriately designed, randomized controlled clinical trials evaluating efficacy, ideal dose, toxicity, adverse effects, cost, and pharmacokinetic/pharmacodynamics data for anogenital and, importantly, also pharyngeal gonorrhoea, i.e. because treatment failures initially emerge at this anatomical site. Finally, in the future treatment at first health care visit will ideally be individually-tailored, i.e. by novel rapid phenotypic AMR tests and/or genetic point of care AMR tests, including detection of gonococci, which will improve the management and public health control of gonorrhoea and AMR. Nevertheless, now is certainly the right time to readdress the challenges of developing a gonococcal vaccine.

Cytotoxicity assessment of modified bioactive glasses with MLO-A5 osteogenic cells in vitro.

PubMed

Modglin, Vernon C; Brown, Roger F; Jung, Steven B; Day, Delbert E

2013-05-01

The primary objective of this study was to evaluate in vitro responses of MLO-A5 osteogenic cells to two modifications of the bioactive glass 13-93. The modified glasses, which were designed for use as cell support scaffolds and contained added boron to form the glasses 13-93 B1 and 13-93 B3, were made to accelerate formation of a bioactive hydroxyapatite surface layer and possibly enhance tissue growth. Quantitative MTT cytotoxicity tests revealed no inhibition of growth of MLO-A5 cells incubated with 13-93 glass extracts up to 10 mg/ml, moderate inhibition of growth with 13-93 B1 glass extracts, and noticeable inhibition of growth with 13-93 B3 glass extracts. A morphology-based biocompatibility test was also performed and yielded qualitative assessments of the relative biocompatibilities of glass extracts that agree with those obtained by the quantitative MTT test. However, as a proof of concept experiment, when MLO-A5 cells were seeded onto 13-93 B3 scaffolds in a dynamic in vitro environment, cell proliferation occurred as evidenced by qualitative and quantitative MTT labeling of scaffolds. Together these results demonstrate the in vitro toxicity of released borate ion in static experiments; however borate ion release can be mitigated in a dynamic environment similar to the human body where microvasculature is present. Here we argue that despite toxicity in static environments, boron-containing 13-93 compositions may warrant further study for use in tissue engineering applications.

Diamond Nanoparticles Modify Curcumin Activity: In Vitro Studies on Cancer and Normal Cells and In Ovo Studies on Chicken Embryo Model

PubMed Central

Strojny, Barbara; Grodzik, Marta; Sawosz, Ewa; Winnicka, Anna; Kurantowicz, Natalia; Jaworski, Sławomir; Kutwin, Marta; Urbańska, Kaja; Hotowy, Anna; Wierzbicki, Mateusz; Chwalibog, André

2016-01-01

Curcumin has been studied broadly for its wide range of biological activities, including anticancer properties. The major problem with curcumin is its poor bioavailability, which can be improved by the addition of carriers, such as diamond nanoparticles (DN). They are carbon allotropes, and are therefore biocompatible and easily taken up by cells. DN are non-toxic and have antiangiogenic properties with potential applications in cancer therapy. Their large surface makes them promising compounds in a drug delivery system for bioactive agents, as DN create bio-complexes in a fast and simple process of self-organisation. We investigated the cytotoxicity of such bio-complexes against liver cancer cells and normal fibroblasts, revealing that conjugation of curcumin with DN significantly improves its activity. The experiment performed in a chicken embryo model demonstrated that neither curcumin nor DN nor bio-complexes affect embryo development, even though DN can form deposits in tissues. Preliminary results confirmed the applicability of DN as an efficient carrier of curcumin, which improves its performance against cancer cells in vitro, yet is not toxic to an organism, which makes the bio-complex a promising anticancer agent. PMID:27736939

Developments in Methods for Measuring the Intestinal Absorption of Nanoparticle-Bound Drugs

PubMed Central

Liu, Wei; Pan, Hao; Zhang, Caiyun; Zhao, Liling; Zhao, Ruixia; Zhu, Yongtao; Pan, Weisan

2016-01-01

With the rapid development of nanotechnology, novel drug delivery systems comprising orally administered nanoparticles (NPs) have been paid increasing attention in recent years. The bioavailability of orally administered drugs has significant influence on drug efficacy and therapeutic dosage, and it is therefore imperative that the intestinal absorption of oral NPs be investigated. This review examines the various literature on the oral absorption of polymeric NPs, and provides an overview of the intestinal absorption models that have been developed for the study of oral nanoparticles. Three major categories of models including a total of eight measurement methods are described in detail (in vitro: dialysis bag, rat gut sac, Ussing chamber, cell culture model; in situ: intestinal perfusion, intestinal loops, intestinal vascular cannulation; in vivo: the blood/urine drug concentration method), and the advantages and disadvantages of each method are contrasted and elucidated. In general, in vitro and in situ methods are relatively convenient but lack accuracy, while the in vivo method is troublesome but can provide a true reflection of drug absorption in vivo. This review summarizes the development of intestinal absorption experiments in recent years and provides a reference for the systematic study of the intestinal absorption of nanoparticle-bound drugs. PMID:27455239

In vitro immune functions in thiamine-replete and -depleted lake trout (Salvelinus namaycush).

PubMed

Ottinger, Christopher A; Honeyfield, Dale C; Densmore, Christine L; Iwanowicz, Luke R

2014-05-01

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP--biologically inactive form) to thiamine pyrophosphate (TPP--biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state. Published by Elsevier Ltd.

In vitro immune functions in thiamine-replete and -depleted lake trout (Salvelinus namaycush)

USGS Publications Warehouse

Ottinger, Christopher A.; Honeyfield, Dale C.; Densmore, Christine L.; Iwanowicz, Luke R.

2014-01-01

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP – biologically inactive form) to thiamine pyrophosphate (TPP – biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state.

An in vitro screening cascade to identify neuroprotective antioxidants in ALS

PubMed Central

Barber, Siân C.; Higginbottom, Adrian; Mead, Richard J.; Barber, Stuart; Shaw, Pamela J.

2009-01-01

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success. PMID:19439221

Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coli.

PubMed

Tran, An X; Dong, Changjiang; Whitfield, Chris

2010-10-22

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.

Photothermal monitoring of interaction of carcinoma cells with cytostatic drugs in vitro

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri; Hanna, Ehab; Cannon, Martin

2003-06-01

Background/problem. Monitoring of tumor response to cancer chemotherapy and dose optimization for specific patients are the key factors for successful application of anti-tumor drugs. Using patient's tumor cells for preliminary in vitro drug screening may allow optimal selection of drug type and dose. Method. Single cell state was studied with photothermal microscope. Carcinoma cells were irradiated at 427 nm with 8 ns laser pulse with energy 30 - 40 μJ. Cell photothermal (PT) response amplitude and shape from each cell were analyzed and amount of cells that produced specific PT response was used as PT parameter. Parallel experiment included cell viability control. Results were obtained for two cytotoxic chemotherapy agents -- Platinol-aq and Adrucil. Incubation of cell suspensions for 90 min at 20 and 37°C caused changes in cell PT parameters. Reaction of carcinoma cells to the drug was very similar to reaction of hepatocytes to respiratory chain inhibition and reaction of RBC to osmotic pressure decrease. PT effect was found to be dose-dependent. PT method allows detecting drug-induced changes before cell death or morphological changes and therefore can be fast and sensitive modality for control of chemotherapy.

Fiber optic micro sensor for the measurement of tendon forces

PubMed Central

2012-01-01

A fiber optic sensor developed for the measurement of tendon forces was designed, numerically modeled, fabricated, and experimentally evaluated. The sensor incorporated fiber Bragg gratings and micro-fabricated stainless steel housings. A fiber Bragg grating is an optical device that is spectrally sensitive to axial strain. Stainless steel housings were designed to convert radial forces applied to the housing into axial forces that could be sensed by the fiber Bragg grating. The metal housings were fabricated by several methods including laser micromachining, swaging, and hydroforming. Designs are presented that allow for simultaneous temperature and force measurements as well as for simultaneous resolution of multi-axis forces. The sensor was experimentally evaluated by hydrostatic loading and in vitro testing. A commercial hydraulic burst tester was used to provide uniform pressures on the sensor in order to establish the linearity, repeatability, and accuracy characteristics of the sensor. The in vitro experiments were performed in excised tendon and in a dynamic gait simulator to simulate biological conditions. In both experimental conditions, the sensor was found to be a sensitive and reliable method for acquiring minimally invasive measurements of soft tissue forces. Our results suggest that this sensor will prove useful in a variety of biomechanical measurements. PMID:23033868

Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach.

PubMed

Wilson, Charlotte M; Loach, Diane; Lawley, Blair; Bell, Tracey; Sims, Ian M; O'Toole, Paul W; Zomer, Aldert; Tannock, Gerald W

2014-10-01

Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Insulinotrophic and insulin-like effects of a high molecular weight aqueous extract of Pterocarpus marsupium Roxb. hardwood.

PubMed

Mohankumar, Suresh K; O'Shea, Tim; McFarlane, James R

2012-05-07

Pterocarpus marsupium Roxb. (PM) is an Ayurvedic traditional medicine well known for its antidiabetic potential. To fractionate the antidiabetic constituent(s) of the aqueous of extract of PM hardwood (PME). Bio-assay methods including, insulin secretion from mouse pancreas and glucose uptake by mouse skeletal muscle, were used to determine and fractionate the antidiabetic activity of PME. Results obtained from the in vitro experiments were then verified by examining the effect of PME on glucose clearance in normoglycemic, non-diabetic sheep in vivo. Exposure of mouse pancreatic and muscle tissues to PME stimulated the insulin secretion and glucose uptake, respectively, in a concentration-dependent manner. PME-mediated muscle glucose uptake was not potentiated in the presence of insulin indicating that PME acts via pathways which are utilized by insulin. Bio-assay-guided fractionation of PME yielded a high molecular weight fraction which had potent antidiabetic properties in vitro, and in in vivo. Our findings, we believe for the first time, provide novel insights for the antidiabetic constituents of PM and demonstrate that a high molecular weight constituent(s) of PM has potent insulinotrophic and insulin-like properties. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species

PubMed Central

Kastman, Erik K.; Kamelamela, Noelani; Norville, Josh W.; Cosetta, Casey M.; Dutton, Rachel J.

2016-01-01

ABSTRACT Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ. Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes. PMID:27795388

Folic acid-conjugated soybean protein-based nanoparticles mediate efficient antitumor ability in vitro.

PubMed

Yao, Weijing; Zha, Qian; Cheng, Xu; Wang, Xin; Wang, Jun; Tang, Rupei

2016-11-23

In this study, soy protein isolate was hydrolyzed by compound enzymes to give aqueous soy protein with low molecular weights. Folic acid modified and free soy protein nanoparticles were successfully prepared by a desolvation method as target-specific drug delivery, respectively. Ultraviolet spectrophotometry demonstrated that folic acid was successfully grafted onto soy protein. The shape and size of folic acid modified soy protein nanoparticles were detected by transmission electron microscopy, scanning electron microscope, and dynamic light scattering. In addition, a series of characteristics including kinetic stability, pH stability, and time stability were also performed. Doxorubicin was successfully loaded into folic acid modified soy protein nanoparticles, and the encapsulation and loading efficiencies were 96.7% and 23%, respectively. Doxorubicin-loaded folic acid modified soy protein nanoparticles exhibited faster drug release rate than soy protein nanoparticles in PBS solution (pH = 5). The tumor penetration and antitumor experiments were done using three-dimensional multicellular tumor spheroids as the in vitro model. The results proved that folic acid modified soy protein nanoparticles display higher penetration and accumulation than soy protein nanoparticles, therefore possessing efficient growth inhibitory ability against multicellular tumor spheroids. © The Author(s) 2016.

Red blood cell (RBC) suspensions in confined microflows: Pressure-flow relationship.

PubMed

Stauber, Hagit; Waisman, Dan; Korin, Netanel; Sznitman, Josué

2017-10-01

Microfluidic-based assays have become increasingly popular to explore microcirculation in vitro. In these experiments, blood is resuspended to a desired haematocrit level in a buffer solution, where frequent choices for preparing RBC suspensions comprise notably Dextran and physiological buffer. Yet, the rational for selecting one buffer versus another is often ill-defined and lacks detailed quantification, including ensuing changes in RBC flow characteristics. Here, we revisit RBC suspensions in microflows and attempt to quantify systematically some of the differences emanating between buffers. We measure bulk flow rate (Q) of RBC suspensions, using PBS- and Dextran-40, as a function of the applied pressure drop (ΔP) for two hematocrits (∼0% and 23%). Two distinct microfluidic designs of varying dimensions are employed: a straight channel larger than and a network array similar to the size of individual RBCs. Using the resulting pressure-flow curves, we extract the equivalent hydrodynamic resistances and estimate the relative viscosities. These efforts are a first step in rigorously quantifying the influence of the 'background' buffer on RBC flows within microfluidic devices and thereby underline the importance of purposefully selecting buffer suspensions for microfluidic in vitro assays. Copyright © 2017. Published by Elsevier Ltd.

Diamond Nanoparticles Modify Curcumin Activity: In Vitro Studies on Cancer and Normal Cells and In Ovo Studies on Chicken Embryo Model.

PubMed

Strojny, Barbara; Grodzik, Marta; Sawosz, Ewa; Winnicka, Anna; Kurantowicz, Natalia; Jaworski, Sławomir; Kutwin, Marta; Urbańska, Kaja; Hotowy, Anna; Wierzbicki, Mateusz; Chwalibog, André

2016-01-01

Curcumin has been studied broadly for its wide range of biological activities, including anticancer properties. The major problem with curcumin is its poor bioavailability, which can be improved by the addition of carriers, such as diamond nanoparticles (DN). They are carbon allotropes, and are therefore biocompatible and easily taken up by cells. DN are non-toxic and have antiangiogenic properties with potential applications in cancer therapy. Their large surface makes them promising compounds in a drug delivery system for bioactive agents, as DN create bio-complexes in a fast and simple process of self-organisation. We investigated the cytotoxicity of such bio-complexes against liver cancer cells and normal fibroblasts, revealing that conjugation of curcumin with DN significantly improves its activity. The experiment performed in a chicken embryo model demonstrated that neither curcumin nor DN nor bio-complexes affect embryo development, even though DN can form deposits in tissues. Preliminary results confirmed the applicability of DN as an efficient carrier of curcumin, which improves its performance against cancer cells in vitro, yet is not toxic to an organism, which makes the bio-complex a promising anticancer agent.

Characterization of monomeric DNA-binding protein Histone H1 in Leishmania braziliensis.

PubMed

Carmelo, Emma; González, Gloria; Cruz, Teresa; Osuna, Antonio; Hernández, Mariano; Valladares, Basilio

2011-08-01

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Nikolov, Ivan; Skutella, Thomas; Just, Lothar

2007-01-01

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

In Vitro Activity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions and In Vivo Efficacy in a Murine Model of Oral Candidiasis

PubMed Central

Chaturvedi, Ashok K.; Rozental, Sonia

2015-01-01

The generation of a new antifungal against Candida albicans biofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potent in vitro activity against multiple fluconazole-susceptible and -resistant C. albicans clinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibits C. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library of C. albicans transcription factor mutants provided additional insight into the activity of miltefosine against C. albicans growing under planktonic and biofilm conditions. Finally, we demonstrate the in vivo efficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis. PMID:26416861

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ismaiel, Afrah A.K.; Espinosa-Oliva, Ana M.; Santiago, Martiniano

Metformin is a widely used oral antidiabetic drug with known anti-inflammatory properties due to its action on AMPK protein. This drug has shown a protective effect on various tissues, including cortical neurons. The aim of this study was to determine the effect of metformin on the dopaminergic neurons of the substantia nigra of mice using the animal model of Parkinson's disease based on the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an inhibitor of the mitochondrial complex I. In vivo and in vitro experiments were used to study the activation of microglia and the damage of the dopaminergic neurons. Our results show that metforminmore » reduced microglial activation measured both at cellular and molecular levels. Rather than protecting, metformin exacerbated dopaminergic damage in response to MPTP. Our data suggest that, contrary to other brain structures, metformin treatment could be deleterious for the dopaminergic system. Hence, metformin treatment may be considered as a risk factor for the development of Parkinson's disease. - Highlights: • Metformin treatment decreases microglial activation in the MPTP model of Parkinson's disease. • Metformin treatment increases the neurodegeneration in the MPTP model of Parkinson's disease, both in vivo and vitro. • Metformin treatment could be a risk factor for the development of Parkinson's disease.« less

Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1.

PubMed

Roberts, Kenneth P; Wamstad, Joseph A; Ensrud, Kathy M; Hamilton, David W

2003-08-01

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

Lactobacillus reuteri 100-23 Modulates Urea Hydrolysis in the Murine Stomach

PubMed Central

Wilson, Charlotte M.; Loach, Diane; Lawley, Blair; Bell, Tracey; Sims, Ian M.; O'Toole, Paul W.; Zomer, Aldert

2014-01-01

Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut. PMID:25063664

Evaluation of biocompatibility of various ceramic powders with human fibroblasts in vitro.

PubMed

Li, J; Liu, Y; Hermansson, L; Söremark, R

1993-01-01

Cell reaction to powders of ceramics was studied in vitro. Cultured human fibroblasts were exposed to different types of ceramic powders: zirconia (ZP), alumina (A), tricalcium phosphate (TCP) and hydroxyapatite (HA), at various concentrations. The cell viability at the different exposure times was measured by the colony formation (expressed as colony forming efficiency, CFE), neutral red uptake (NR) and colorimetric tetrazolium (MTT) reduction. Alumina and hydroxyapatite showed no cytotoxic effects at studied doses (1-500 mug/ml) while zirconia and tricalcium phosphate inhibited cell viability, with 50% of CFE reduction at the concentration of about 50 mug/ml. In order to study the cytotoxic mechanism of zirconia powder, two further experiments were included, viz. the cellular response to the sintered zirconia ceramic powders (CZP) which were obtained by crushing the sintered ceramic material; and the measurement of the degradation of zirconia ceramic plate in the different solutions, i.e., either in saline or in 0.02 M lactic acid (pH 2.72). Similar cell reactions were obtained for the CZP and ZP by using MTT and NR assays. Slow releases of ions from zirconia ceramic plate, yttrium in both solutions and zirconium and yttrium in lactic acid, were detected.

Regulating NH4+, N0-, and K+ concentration and proportions improves in vitro tissue growth.

USDA-ARS?s Scientific Manuscript database

A mixture-amount experiment that simultaneously varied both the ratios and total ionic concentration (from 20-100 mM) of NH4+, NO3+, and K+ was used to maximize sweet orange callus growth cv. ‘Hamlin.’ These experiments were free of ion confounding effects, i.e. ions added via pH adjustments and sa...

[Controlling effect of antagonist bioorganic fertilizer on tomato root-knot nematode].

PubMed

Zhu, Zhen; Chen, Fang; Xiao, Tong-jian; Wang, Xiao-hui; Ran, Wei; Yang, Xing-ming; Shen, Qi-rong

2011-04-01

Indoor in vitro culture experiment and greenhouse pot experiment were conducted to evaluate the capabilities of three bacterial strains XZ-173 (Bacillus amyloliquefaciens), SL-25 (B. gibsonii), and KS-62 (Paenibacillus polymyxa) that can hydrolyze collagen protein in controlling tomato root-knot nematode. In the in vitro culture experiment, suspensions of XZ-173, SL-25, and KS-62 induced a mortality rate of 75.9%, 66.7%, and 50.0% to the second-stage junior nematode within 24 h, and decreased the egg hatching rate to 17.8%, 28.9% and 37.6% after 7-day incubation, respectively, in contrast to the 17.4% mortality rate and 53.6% egg hatching rate in the control (sterilized water). In the greenhouse pot experiment, the bioorganic fertilizer mixed with equal parts of fermented XZ-173, SL-25, and KS-62 gained the best result, with the root-knot nematode population in rhizosphere soil decreased by 84.0% as compared with the control. The bioorganic fertilizer also decreased the numbers of galls and eggs on tomato roots significantly, and increased the underground and aboveground biomass of tomato. Therefore, antagonist bioorganic fertilizer has promising potential in controlling root-knot nematode.

[Orthogonal experiments for optimizing the formulation and preparation conditions of temozolomide solid lipid nanoparticles].

PubMed

Dou, Mingjin; Huang, Guihua; Xi, Yanwei; Zhang, Na

2008-10-01

TMZ-SLN were prepared by emulsification-low temperature solidification method with stearic acid. The formulation and the preparation conditions were optimized by orthogonal experiments using entrapment efficiency as the evaluation index. The morphology was detected by transmission electron microscope. The Zeta potentials and the particle size distribution were evaluated by Laser Doppler Anemometry. The entrapment efficiencies and the drug release characteristics in vitro were assessed. The result showed that TMZ-SLN were concinnous and spherical in shape. The mean diameter (d(av) ) was 65.0 +/- 6.2 nm and the Zeta potential was -37.2 mV. The average entrapment efficiency was 58.9% +/- 1.21 %. The drug release behavior in vitro conformed to Higuchi Equation. The formation of a new material phase was testified by analysis of differential scanning calorimetry.

Seismomorphogenesis: a novel approach to acclimatization of tissue culture regenerated plants.

PubMed

Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

2014-12-01

Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and sometimes difficult to handle subsequently increasing their risk to damage and disease; hence acclimatization of these plantlets is the most important step in tissue culture techniques. An experiment was conducted under in vitro conditions to study the effects of shaking duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14 days) on Sansevieria trifasciata L. as a model plant. Results showed that shaking improved handling, total plant height, and leaf characteristics of the model plant. Forty-eight hours after 14 days of shaking treatments with increasing shaking time, leaf length decreased but proline content of leaf increased. However, 6 months after starting the experiment different results were observed. In explants that received 16 min of shaking treatment, leaf length and area and photosynthesis rate were increased compared with control plantlets. Six months after starting the experiment, control plantlets had 12.5 % mortality; however, no mortality was observed in other treated explants. The results demonstrated that shaking improved the explants' root length and number and as a simple, cost-effective, and non-chemical novel approach may be substituted for other prevalent acclimatization techniques used for tissue culture regenerated plantlets. Further studies with sensitive plants are needed to establish this hypothesis.

Innovative formulations for the delivery of levothyroxine to the skin.

PubMed

Padula, Cristina; Nicoli, Sara; Santi, Patrizia

2009-05-08

The aim of this work was to realize innovative transdermal formulations containing sodium levothyroxine in view of topical administration. Permeation experiments were performed in vitro, using rabbit ear skin as barrier. At the end of the permeation experiments levothyroxine retained in the skin was extracted and quantified by HPLC. Formulations tested were microemulsions and transdermal films. Microemulsions containing isopropyl myristate and isobutanol were shown to be able to increase levothyroxine solubility by the inclusion in reverse micelles. However, the inclusion in reversed micelles reduced the drug release to a significant extent, and consequently skin retention, compared to aqueous solutions. When the microemulsion was included in the transdermal film, drug retention was increased, probably for the enhancer effect of its excipients. The transdermal film proposed in this work could be an interesting alternative to semisolid formulations for the ease of use and the control in the amount of active applied. Additional benefit can be obtained if the film is used in occlusive conditions.

A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, S.; Labanca, I.; Rech, I.

2014-10-15

Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments.more » However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.« less

Recent publications in medical microbiology and immunology: a retrospective.

PubMed

Doerr, H W; Cinatl, J

2012-02-01

A look back is done to some clinical and basic research activities recently published in medical microbiology and immunology. The review covers clinical experiences and in vitro experiments to understand the emergency, pathogenicity, epidemic spread, and vaccine-based prevention of avian and swine-origin flu. Some new developments and concepts in diagnosis, (molecular) epidemiology, and therapy of AIDS, viral hepatitis C, and herpesvirus-associated diseases are outlined. Regulation of immune system has been discussed in a special issue 2010 including some aspects of CNS affections (measles). Mycobacterial infection and its prevention by modern recombinant vaccines have reached new interest, as well as new concepts of vaccination and prophylaxis against several other bacteria. Adaptation to host niches enables immune escape (example brucella) and determines virulence (example N. meningitidis). Chlamydia pneumoniae, previously considered to trigger atherosclerosis, is hypothetically associated to Alzheimer disease, while CMV, another putative trigger of atherosclerosis, gains evidence of oncomodulation in CNS tumor diseases. In terms of globalization, exotic virus infections are increasingly imported from southern countries.

Manipulation of novel nano-prodrug composed of organic pigment-based hybrid network and its optical uses.

PubMed

Zhou, Zhan; Zheng, Yuhui; Cheng, Cheng Zhang; Wen, Jiajia; Wang, Qianming

2017-01-01

Here we developed the first case of pyropheophorbide-a-loaded PEGylated-hybrid carbon nanohorns (CNH-Pyro) to study tumor targeting therapy. During incubation with living cells, CNH-Pyro exhibited very intense red emissions. The intracellular imaging results were carried out by flow cytometry based on four different kinds of cell lines (including three adherent cell lines and one suspension cell line). Compared with free pyropheophorbide-a, CNH-Pyro demonstrated enhanced photodynamic tumor ablation efficiency during in vitro experiments due to improved biocompatibility of the hybrid nanomaterial and the photothermal therapy effect derived from carbon-network structure. Trypan blue staining experiments supported that the cell fate was dependent on the synergistic effects of both CNH-Pyro and laser irradiations. These results indicated that the chlorin-entrapped carbon nanohorns could provide powerful delivery vehicles for increasing photodynamic efficacy and possess early identification of the disease. Copyright © 2016 Elsevier B.V. All rights reserved.

The Convallis Rule for Unsupervised Learning in Cortical Networks

PubMed Central

Yger, Pierre; Harris, Kenneth D.

2013-01-01

The phenomenology and cellular mechanisms of cortical synaptic plasticity are becoming known in increasing detail, but the computational principles by which cortical plasticity enables the development of sensory representations are unclear. Here we describe a framework for cortical synaptic plasticity termed the “Convallis rule”, mathematically derived from a principle of unsupervised learning via constrained optimization. Implementation of the rule caused a recurrent cortex-like network of simulated spiking neurons to develop rate representations of real-world speech stimuli, enabling classification by a downstream linear decoder. Applied to spike patterns used in in vitro plasticity experiments, the rule reproduced multiple results including and beyond STDP. However STDP alone produced poorer learning performance. The mathematical form of the rule is consistent with a dual coincidence detector mechanism that has been suggested by experiments in several synaptic classes of juvenile neocortex. Based on this confluence of normative, phenomenological, and mechanistic evidence, we suggest that the rule may approximate a fundamental computational principle of the neocortex. PMID:24204224

Non surgical laser and light in the treatment of chronic diseases: a review based on personal experiences

NASA Astrophysics Data System (ADS)

Longo, L.

2010-11-01

Since many years some effects of non surgical laser and light on biological tissue have been demonstrated, in vitro and in vivo. This review is based on the results obtained by me and my colleagues/follower in Italy. Aim of our study is to verify the anti-inflammatory and regenerative effects of non surgical laser and light therapy on patients with chronic diseases not good treatable with traditional therapies, as diabetes, and central nervous system injuries. In addition, many clinical data have emerged from double-blind trials on laser treatment of rheumatic diseases and in sports medicine. So, we would like to do a review on the state of the art of non surgical laser treatment in medicine, included aesthetic laser and light therapy field. We discuss the indications and limitations of aesthetic laser medicine, as concluded from the data analysis of the published literature and from over thirty years of personal experiences.

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo.

PubMed

Torella, Daniele; Iaconetti, Claudio; Catalucci, Daniele; Ellison, Georgina M; Leone, Angelo; Waring, Cheryl D; Bochicchio, Angela; Vicinanza, Carla; Aquila, Iolanda; Curcio, Antonio; Condorelli, Gianluigi; Indolfi, Ciro

2011-09-30

MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Pulsed cavitational ultrasound for non-invasive chordal cutting guided by real-time 3D echocardiography.

PubMed

Villemain, Olivier; Kwiecinski, Wojciech; Bel, Alain; Robin, Justine; Bruneval, Patrick; Arnal, Bastien; Tanter, Mickael; Pernot, Mathieu; Messas, Emmanuel

2016-10-01

Basal chordae surgical section has been shown to be effective in reducing ischaemic mitral regurgitation (IMR). Achieving this section by non-invasive mean can considerably decrease the morbidity of this intervention on already infarcted myocardium. We investigated in vitro and in vivo the feasibility and safety of pulsed cavitational focused ultrasound (histotripsy) for non-invasive chordal cutting guided by real-time 3D echocardiography. Experiments were performed on 12 sheep hearts, 5 in vitro on explanted sheep hearts and 7 in vivo on beating sheep hearts. In vitro, the mitral valve (MV) apparatus including basal and marginal chordae was removed and fixed on a holder in a water tank. High-intensity ultrasound pulses were emitted from the therapeutic device (1-MHz focused transducer, pulses of 8 µs duration, peak negative pressure of 17 MPa, repetition frequency of 100 Hz), placed at a distance of 64 mm under 3D echocardiography guidance. In vivo, after sternotomy, the same therapeutic device was applied on the beating heart. We analysed MV coaptation and chordae by real-time 3D echocardiography before and after basal chordal cutting. After sacrifice, the MV apparatus were harvested for anatomical and histological post-mortem explorations to confirm the section of the chordae. In vitro, all chordae were completely cut after a mean procedure duration of 5.5 ± 2.5 min. The procedure duration was found to increase linearly with the chordae diameter. In vivo, the central basal chordae of the anterior leaflet were completely cut. The mean procedure duration was 20 ± 9 min (min = 14, max = 26). The sectioned chordae was visible on echocardiography, and MV coaptation remained normal with no significant mitral regurgitation. Anatomical and histological post-mortem explorations of the hearts confirmed the section of the chordae. Histotripsy guided by 3D echo achieved successfully to cut MV chordae in vitro and in vivo in beating heart. We hope that this technique will open the door in the near future to the non-invasive treatment of functional IMR. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

PubMed

Barbieri, Federica; Thellung, Stefano; Ratto, Alessandra; Carra, Elisa; Marini, Valeria; Fucile, Carmen; Bajetto, Adriana; Pattarozzi, Alessandra; Würth, Roberto; Gatti, Monica; Campanella, Chiara; Vito, Guendalina; Mattioli, Francesca; Pagano, Aldo; Daga, Antonio; Ferrari, Angelo; Florio, Tullio

2015-04-07

Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

Saint John's wort: An in vitro analysis of P-glycoprotein induction due to extended exposure

PubMed Central

Perloff, Michael D; von Moltke, Lisa L; Störmer, Elke; Shader, Richard I; Greenblatt, David J

2001-01-01

Chronic use of Saint John's wort (SJW) has been shown to lower the bioavailability for a variety of co-administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P-glycoprotein (P-gp), may explain decreased bioavailability. The present study characterized the response of P-gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300 μg ml−1 of methanol-extracted SJW and 0.03 to 3 μM HYP, representing low to high estimates of intestinal concentrations. In induction experiments, LS-180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P-gp was quantified using Western blot analysis. P-gp expression was strongly induced by SJW (400% increase at 300 μg ml−1) and by HYP (700% at 3 μM) in a dose-dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P-gp substrate, that was reversed with acute verapamil, a P-gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco-2 cell monolayers, SJW and HYP caused moderate inhibition of P-gp-attributed transport at the maximum concentrations tested. SJW and HYP significantly induced P-gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P-gp substrate drugs when co-administered with SJW. PMID:11739235

Saccharomyces boulardii CNCM I-745 Restores intestinal Barrier Integrity by Regulation of E-cadherin Recycling.

PubMed

Terciolo, Chloé; Dobric, Aurélie; Ouaissi, Mehdi; Siret, Carole; Breuzard, Gilles; Silvy, Françoise; Marchiori, Bastien; Germain, Sébastien; Bonier, Renaté; Hama, Adel; Owens, Roisin; Lombardo, Dominique; Rigot, Véronique; André, Frédéric

2017-08-01

Alteration in intestinal permeability is the main factor underlying the pathogenesis of many diseases affecting the gut, such as inflammatory bowel disease [IBD]. Characterization of molecules targeting the restoration of intestinal barrier integrity is therefore vital for the development of alternative therapies. The yeast Saccharomyces boulardii CNCM I-745 [Sb], used to prevent and treat antibiotic-associated infectious and functional diarrhea, may have a beneficial effect in the treatment of IBD. We analyzed the impact of Sb supernatant on tissue integrity and components of adherens junctions using cultured explants of colon from both IBD and healthy patients. To evaluate the pathways by which Sb regulates the expression of E-cadherin at the cell surface, we developed in vitro assays using human colonic cell lines, including cell aggregation, a calcium switch assay, real-time measurement of transepithelial electrical resistance [TEER] and pulse-chase experiments. We showed that Sb supernatant treatment of colonic explants protects the epithelial morphology and maintains E-cadherin expression at the cell surface. In vitro experiments revealed that Sb supernatant enhances E-cadherin delivery to the cell surface by re-routing endocytosed E-cadherin back to the plasma membrane. This process, involving Rab11A-dependent recycling endosome, leads to restoration of enterocyte adherens junctions, in addition to the overall restoration and strengthening of intestinal barrier function. These findings open new possibilities of discovering novel options for prevention and therapy of diseases that affect intestinal permeability. Copyright © 2017 European Crohn's and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library

PubMed Central

Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G.; Zhang, Ying

2016-01-01

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10–20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even when pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease. PMID:27242757

A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library.

PubMed

Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G; Zhang, Ying

2016-01-01

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even when pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

Isolation, culture, and imaging of human fetal pancreatic cell clusters.

PubMed

Lopez, Ana D; Kayali, Ayse G; Hayek, Alberto; King, Charles C

2014-05-18

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

Evaluation of the genotoxic potential of Mangifera indica L. extract (Vimang), a new natural product with antioxidant activity.

PubMed

Rodeiro, I; Cancino, L; González, J E; Morffi, J; Garrido, G; González, R M; Nuñez, A; Delgado, R

2006-10-01

Mangifera indica L. extract (Vimang) consists of a defined mixture of components (polyphenols, terpenoids, steroids, fatty acids and microelements). It contains a variety of polyphenols, phenolic esters, flavan-3-ols and a xanthone (mangiferin), as main component. This extract has antioxidant action, antitumor and immunemodulatory effects proved in experimental models in both in vitro and in vivo assays. The present study was performed to investigate the genotoxicity potential activity of Vimang assessed through different tests: Ames, Comet and micronucleus assays. Positive and negative controls were included in each experimental series. Histidine requiring mutants of Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 strains for point-mutation tests and in vitro micronucleus assay in primary human lymphocytes with and without metabolic activation were performed. In addition, genotoxic effects were evaluated on blood peripheral lymphocytes of NMRI mice of both sexes, which were treated during 2 days with intraperitoneal doses of M. indica L. extract (50-150 mg/kg). The observed results permitted to affirm that Vimang (200-5,000 microg/plate) did not increase the frequency of reverse mutations in the Ames test in presence or not of metabolic activation. Results of Comet assay showed that the extract did not induce single strand breaks or alkali-labile sites on blood peripheral lymphocytes of treated animals compared with controls. On the other hand, the results of the micronucleus studies (in vitro and in vivo) showed Vimang induces cytotoxic activity, determined as cell viability or PCE/NCE ratio, but neither increased the frequency of micronucleated binucleate cells in culture of human lymphocytes nor in mice bone marrow cells under our experimental conditions. The positive control chemicals included in each experiment induced the expected changes. The present results indicate that M. indica L. extract showed evidences of light cytotoxic activity but did not induce a mutagenic or genotoxic effects in the battery of assays used.

EFFECTS OF ELECTROMAGNETICALLY SIGNALIZED MEDIA ON HOST-PATHOGEN INTERACTION.

PubMed

D'Hallewin, G; Venditti, T; Cubaiu, L; Ladu, G; Renati, P

2014-01-01

Up to date, limited data are available about electromagnetic phase signaling effects on host-pathogen interactions during the postharvest of horticultural commodities. Inspired by the last striking works on water physics, quantum signaling through phase transfer and its impact on biological and histological structures, we studied the effect of different electromagnetic signals on pome blue mold (Penicillium expansum) pathogenesis. Tags with different electromagnetic-signals (EmS) were used to generate 3 Coherent Electro Dynamic (CED) environments. Artificially wounded 'Coscia' pears, placed onto 3 EmS tags (QF, QA and QR), were employed for the in vivo experiment. Whereas, a set of wounded-fruit placed onto an un-electromagnetic-signalized tag (QN) or kept without tag were used as blank or control, respectively. Inoculation was performed 2 or 24 h post-wounding with P. expansum conidia. The same tags placed under Petri dishes containing dot-inoculated PDA served for the in vitro experiment. Both experiments performed at 25 degrees C endured 7 days. The percentage of infected wounds was calculated and the radial growth measured in vitro. Concerning the in vivo experiment, 100% of control and blank fruit inoculated 2 h post-wounding was infected after 5 days, while, 97% after 7 days, when inoculation occurred 24 h post-wounding. Compared to control and blank, the pathogenesis in fruit placed on the EmS tags resulted inhibited, and when fruit was inoculated 2 h post-wounding, the infection degree on QF, QA and QR tags resulted 19, 52 and 64%, respectively. The degree for the same EmS tags was significantly lower when fruit was inoculated 24 h post-wounding (9, 32 and 42%, respectively). The in vitro experiment evidenced a notable inhibition of the radial growth by all EmS tags in comparison to control and blank (51 mm), while the QF tag provided the greatest inhibition (12 mm).

Catecholamines and in vitro growth of pathogenic bacteria: enhancement of growth varies greatly among bacterial species

NASA Technical Reports Server (NTRS)

Belay, Tesfaye; Aviles, Hernan; Vance, Monique; Fountain, Kimberly; Sonnenfeld, Gerald

2003-01-01

The purpose of this study was to examine the effects of catecholamines on in vitro growth of a range of bacterial species, including anaerobes. Bacteria tested included: Porphyromonas gingivalis, Bacteriodes fragilis, Shigella boydii, Shigella sonnie, Enterobacter Sp, and Salmonella choleraesuis. The results of the current study indicated that supplementation of bacterial cultures in minimal medium with norepinephrine or epinephrine did not result in increased growth of bacteria. Positive controls involving treatment of Escherichia coli with catecholamines did result in increased growth of that bacterial species. The results of the present study extend previous observations that showed differential capability of catecholamines to enhance bacterial growth in vitro.

Approaches and considerations for the assessment of immunotoxicity for environmental chemicals: a workshop summary.

PubMed

Boverhof, Darrell R; Ladics, Greg; Luebke, Bob; Botham, Jane; Corsini, Emanuela; Evans, Ellen; Germolec, Dori; Holsapple, Michael; Loveless, Scott E; Lu, Haitian; van der Laan, Jan Willem; White, Kimber L; Yang, Yung

2014-02-01

As experience is gained with toxicology testing and as new assays and technologies are developed, it is critical for stakeholders to discuss opportunities to advance our overall testing strategies. To facilitate these discussions, a workshop on practices for assessing immunotoxicity for environmental chemicals was held with the goal of sharing perspectives on immunotoxicity testing strategies and experiences, developmental immunotoxicity (DIT), and integrated and alternative approaches to immunotoxicity testing. Experiences across the chemical and pharmaceutical industries suggested that standard toxicity studies, combined with triggered-based testing approaches, represent an effective and efficient approach to evaluate immunotoxic potential. Additionally, discussions on study design, critical windows, and new guideline approaches and experiences identified important factors to consider before initiating DIT evaluations including assay choice and timing and the impact of existing adult data. Participants agreed that integrating endpoints into standard repeat-dose studies should be considered for fulfilling any immunotoxicity testing requirements, while also maximizing information and reducing animal use. Participants also acknowledged that in vitro evaluation of immunosuppression is complex and may require the use of multiple assays that are still being developed. These workshop discussions should contribute to developing an effective but more resource and animal efficient approach for evaluating chemical immunotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Setup for investigating gold nanoparticle penetration through reconstructed skin and comparison to published human skin data

NASA Astrophysics Data System (ADS)

Labouta, Hagar I.; Thude, Sibylle; Schneider, Marc

2013-06-01

Owing to the limited source of human skin (HS) and the ethical restrictions of using animals in experiments, in vitro skin equivalents are a possible alternative for conducting particle penetration experiments. The conditions for conducting penetration experiments with model particles, 15-nm gold nanoparticles (AuNP), through nonsealed skin equivalents are described for the first time. These conditions include experimental setup, sterility conditions, effective applied dose determination, skin sectioning, and skin integrity check. Penetration at different exposure times (two and 24 h) and after tissue fixation (fixed versus unfixed skin) are examined to establish a benchmark in comparison to HS in an attempt to get similar results to HS experiments presented earlier. Multiphoton microscopy is used to detect gold luminescence in skin sections. λex=800 nm is used for excitation of AuNP and skin samples, allowing us to determine a relative index for particle penetration. Despite the observed overpredictability of penetration into skin equivalents, they could serve as a first fast screen for testing the behavior of nanoparticles and extrapolate their penetration behavior into HS. Further investigations are required to test a wide range of particles of different physicochemical properties to validate the skin equivalent-human skin particle penetration relationship.

[Study on solid dispersion of precipitated calcium carbonate-based oleanolic acid].

PubMed

Yan, Hong-mei; Zhang, Zhen-hai; Jia, Xiao-bin; Jiang, Yan-rong; Sun, E

2015-05-01

Oleanolic acid-precipitated calcium carbonate solid dispersion was prepared by using solvent evaporation method. The microscopic structure and physicochemical properties of solid dispersion were analyzed using differential scanning calorimetry and scanning electron microscopy (SEM). And its in vitro release also was investigated. The properties of the precipitated calcium carbonate was studied which was as a carrier of oleanolic acid solid dispersion. Differential scanning calorimetry analysis suggested that oleanolic acid may be present in solid dispersion as amorphous substance. The in vitro release determination results of oleanolic acid-precipitated calcium carbonate (1: 5) solid dispersion showed accumulated dissolution rate of.oleanolic acid was up to 90% at 45 min. Accelerating experiment showed that content and in vitro dissolution of oleanolic acid solid dispersion did not change after storing over 6 months. The results indicated that in vitro dissolution of oleanolic acid was improved greatly by the solid dispersion with precipitated calcium carbonate as a carrier. The solid dispersion is a stabilizing system which has actual applied value.

Polyploidization facilitates biotechnological in vitro techniques in the genus Cucumis.

PubMed

Skálová, Dagmar; Ondřej, Vladan; Doležalová, Ivana; Navrátilová, Božena; Lebeda, Aleš

2010-01-01

Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed.

Polyploidization Facilitates Biotechnological In Vitro Techniques in the Genus Cucumis

PubMed Central

Skálová, Dagmar; Ondřej, Vladan; Doležalová, Ivana; Navrátilová, Božena; Lebeda, Aleš

2010-01-01

Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed. PMID:21234406

Monte Carlo simulations guided by imaging to predict the in vitro ranking of radiosensitizing nanoparticles

PubMed Central

Retif, Paul; Reinhard, Aurélie; Paquot, Héna; Jouan-Hureaux, Valérie; Chateau, Alicia; Sancey, Lucie; Barberi-Heyob, Muriel; Pinel, Sophie; Bastogne, Thierry

2016-01-01

This article addresses the in silico–in vitro prediction issue of organometallic nanoparticles (NPs)-based radiosensitization enhancement. The goal was to carry out computational experiments to quickly identify efficient nanostructures and then to preferentially select the most promising ones for the subsequent in vivo studies. To this aim, this interdisciplinary article introduces a new theoretical Monte Carlo computational ranking method and tests it using 3 different organometallic NPs in terms of size and composition. While the ranking predicted in a classical theoretical scenario did not fit the reference results at all, in contrast, we showed for the first time how our accelerated in silico virtual screening method, based on basic in vitro experimental data (which takes into account the NPs cell biodistribution), was able to predict a relevant ranking in accordance with in vitro clonogenic efficiency. This corroborates the pertinence of such a prior ranking method that could speed up the preclinical development of NPs in radiation therapy. PMID:27920524

Monte Carlo simulations guided by imaging to predict the in vitro ranking of radiosensitizing nanoparticles.

PubMed

Retif, Paul; Reinhard, Aurélie; Paquot, Héna; Jouan-Hureaux, Valérie; Chateau, Alicia; Sancey, Lucie; Barberi-Heyob, Muriel; Pinel, Sophie; Bastogne, Thierry

This article addresses the in silico-in vitro prediction issue of organometallic nanoparticles (NPs)-based radiosensitization enhancement. The goal was to carry out computational experiments to quickly identify efficient nanostructures and then to preferentially select the most promising ones for the subsequent in vivo studies. To this aim, this interdisciplinary article introduces a new theoretical Monte Carlo computational ranking method and tests it using 3 different organometallic NPs in terms of size and composition. While the ranking predicted in a classical theoretical scenario did not fit the reference results at all, in contrast, we showed for the first time how our accelerated in silico virtual screening method, based on basic in vitro experimental data (which takes into account the NPs cell biodistribution), was able to predict a relevant ranking in accordance with in vitro clonogenic efficiency. This corroborates the pertinence of such a prior ranking method that could speed up the preclinical development of NPs in radiation therapy.

In vitro evaluation of electrospun PLGA/PLLA/PDLLA blend fibers loaded with naringin for guided bone regeneration.

PubMed

Guo, Zhenzhao; Wu, Shuai; Li, Hong; Li, Qiyan; Wu, Gang; Zhou, Changren

2018-03-30

The present study was to evaluate fiber mesh loaded with naringin via electrospinning to guide bone regeneration in vitro. The naringin-loaded fiber mesh was prepared via elctrospinning of PLGA, PLLA, PDLLA blending solution with naringin. SEM showed that naringin decreased the fiber's diameter according to the concentration of naringin. After 20 days' degradation in PBS, the drug-loaded fiber meshes still kept their stability with about 10% decrease in tensile strength. In vitro release experiments showed a sustained and steady naringin releasing profile with little initial burst releasing. Compared to the mats without naringin, the fiber mats loaded with naringin showed the most pronounced enhancement of cell growth when MC3T3-E1 cells were cultured on the fiber mats. The blend fiber loaded with naringin has optimized physical properties and sustained release profile in vitro. The study presents a promising fibrous mesh material for guided bone regeneration therapy.

Using discrete multi-physics for detailed exploration of hydrodynamics in an in vitro colon system.

PubMed

Alexiadis, A; Stamatopoulos, K; Wen, W; Batchelor, H K; Bakalis, S; Barigou, M; Simmons, M J H

2017-02-01

We developed a mathematical model that describes the motion of viscous fluids in the partially-filled colon caused by the periodic contractions of flexible walls (peristalsis). In-vitro data are used to validate the model. The model is then used to identify two fundamental mechanisms of mass transport: the surfing mode and the pouring mode. The first mechanism is faster, but only involves the surface of the liquid. The second mechanism causes deeper mixing, and appears to be the main transport mechanism. Based on the gained understanding, we propose a series of measures that can improve the reliability of in-vitro models. The tracer in PET-like experiments, in particular, should not be injected in the first pocket, and its viscosity should be as close as possible to that of the fluid. If these conditions are not met, the dynamics of the tracer and the fluid diverge, compromising the accuracy of the in-vitro data. Copyright © 2017 Elsevier Ltd. All rights reserved.

Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo.

PubMed

Shi, Ming; Zhang, Ling; Gu, Hong-Tao; Jiang, Feng-Qin; Qian, Lu; Yu, Ming; Chen, Guo-Jiang; Luo, Qun; Shen, Bei-Fen; Guo, Ning

2007-10-01

To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin 2 (IL-2) fusion protein (HFI) in vitro and in vivo. Fusion protein HFI was constructed. The efficacy of HFI in mediating tumor cell lysis was determined by colorimetric lactate dehydrogenase release assays. The antitumor activity of HFI was evaluated in tumor xenograft models. The fusion protein was folded as a homodimer formed by covalently linking Fc portions and it retained ErbB2 specificity and IL-2 biological activity. HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and improved the therapeutic efficacy of IL-2 in experiments in vivo. The genetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high efficiency both in mediating ADCC in vitro and significant antitumor activity in tumor xenograft models.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays.

PubMed

Martins, Flaviano S; Miranda, Ieso C; Rosa, Carlos A; Nicoli, Jacques R; Neves, Maria J

2008-01-01

Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

In vitro and in vivo biological activities of anthocyanins from Nitraria tangutorun Bobr. fruits.

PubMed

Ma, Tao; Hu, Na; Ding, Chenxi; Zhang, Qiulong; Li, Wencong; Suo, Yourui; Wang, Honglun; Bai, Bo; Ding, Chenxu

2016-03-01

Anthocyanins are the main compounds in Nitraria tangutorun Bobr. The enrichment and purification of anthocyanins on macroporous resins were investigated. Regarding anthocyanin purification, static adsorption and desorption were studied. The optimal experimental conditions were the following: resin type: X-5; static adsorption time: 6h; desorption solution: ethanol-water-HCl (80:19:1, V/V/V; pH 1); desorption time: 40min. Furthermore, the in vitro and in vivo biological activities of the anthocyanins were evaluated. The anthocyanins showed ideal scavenging effects on free radicals in vitro, especially on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl free radical (OH). In the animal experiment, blood lipid metabolism of hyperlipidemia rats was regulated by anthocyanin contents. The superoxide dismutase (SOD) activity and the total antioxidant capacity (TAC) of hyperlipidemia rats were also improved by anthocyanins. These results showed that anthocyanins from N. tangutorun Bobr. fruits had potential biological activities in vivo as well as in vitro. Copyright © 2015. Published by Elsevier Ltd.

Module assemblage technology for floating systems: in vitro flotation and in vivo gastro-retention.

PubMed

Strusi, Orazio Luca; Sonvico, Fabio; Bettini, Ruggero; Santi, Patrizia; Colombo, Gaia; Barata, Pedro; Oliveira, Ana; Santos, Delfim; Colombo, Paolo

2008-07-14

The aim of this research was to study, in vitro by resultant-weight measurement and in vivo by gamma-scintigraphy experiments in humans, the floatation behavior of systems obtained by modules assembled in void configuration. The assembled system technology allowed the manufacturing of a system characterized by the presence of an internal void space that provided an apparent density lower than water. The gastro-retention times of floating assembled systems were determined in comparison with non-floating systems having the same mass and composition. In vitro the floatation of the system started immediately after immersion in water and lasted for more than 5 h. The in vivo studies confirmed that the in vitro floating ability of void configuration was maintained also in the human stomach where the system stayed for periods of time ranging from 2.5 to 5.0 h, depending on the food regimen and the sex of the subject. Reiterate eating and drinking further prolonged the stomach residence time.

[Electrohydraulic intracorporeal lithotripsy of salivary calculi. In vitro and animal experiment studies].

PubMed

Iro, H; Zenk, J; Hosemann, W G; Benzel, W

1993-08-01

Extracorporeal lithotripsy is now used routinely for the treatment of salivary duct stones. The question arose whether electrohydraulic intracorporeal lithotripsy, which is applied in urology and gastroenterology, might also be useful in the treatment of this disease. Before its possible clinical application the influence of electrohydraulic intracorporeal shock waves on salivary stones in vitro and any influence on the tissue in the head and neck region (in vivo) had to be investigated. In vitro experiments. Fifty-eight salivary stones and 11 extirpated human submandibular glands were treated by three different electrohydraulic devices. Animal experiments. Electrohydraulic shock waves were applied to the dilated Stensen's duct and other tissues (muscle, parotid gland, facial nerve) of six rabbits. Of 58 salivary stones, 53 (91%) were fragmented, 39 (67%) with a remaining size of less than 1.5 mm and 14 (24%) more than 1.5 mm. In 5 cases (9%) no effects were seen at all. Fragmentation occurred independent of the mineralogical components of the stone and independent of the different lithotriptors used. The smaller the probe diameter and the larger the stone, the more shock waves were needed to achieve fragmentation. With the smaller probes the stones could not be fragmented completely. In human submandibular glands, extensive tissue lesions could be evidenced macroscopically and histologically after application of electrohydraulic shock waves in vitro. Application of electrohydraulic shock waves to the dilated parotid gland duct of rabbits led to perforations of the duct after 1-5 single pulses. Lesions of nerves and blood vessels could also be observed within the duct environment. This occurred with all of the different electrohydraulic devices, probe diameters and intensities used. In our opinion the damage produced is probably the result of both the direct effect of the plasma as well as the resultant stress wave. In view of the severe damage caused to different tissues, use of the described method for the treatment of stones in the narrow human salivary ducts should be greatly restricted.

Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm.

PubMed

Shahzad, Qaisar; Mehmood, Muhammad Usman; Khan, Hamayun; ul Husna, Asma; Qadeer, Saima; Azam, Asima; Naseer, Zahid; Ahmad, Ejaz; Safdar, Muhammad; Ahmad, Mushtaq

2016-04-01

Two experiments were conducted to evaluate the effect of royal jelly (RJ) on post-thaw sperm quality, in vitro and in vivo fertility rate of cryopreserved buffalo bull sperm. The semen was collected from three mature regular donor buffalo bulls, ejaculates were pooled and semen evaluated initially. In Experiment 1, the ejaculates were extended in tris-citric acid diluter supplemented with different RJ concentrations (0, 0.05, 0.1, 0.2, 0.3 or 0.4%). The diluted semen was cooled to 4°C, packaged into 0.5 mL straws and frozen using standard procedure. The straws were thawed and assessed for sperm progressive motility, viability, plasma membrane, acrosome, and chromatin integrity. The results indicated that sperm progressive motility was significantly greater (P<0.05) in 0.05, 0.1, 0.2 and 0.3% RJ than 0.4% RJ supplemented and control groups. The sperm viability, plasma membrane and acrosome integrity were significantly improved (P<0.05) in 0.1% RJ supplemented group the compared to other treatment groups. In Experiment 2, cryopreserved sperm with 0.1% RJ supplementation and control (without RJ supplementation) were used to observe the in vitro fertilizing potential and in vivo fertility. In vitro fertilization method was applied to assess the cleavage rate; whereas, AI was performed in buffalo during in vivo fertility trial. The buffaloes were inseminated 12h after standing estrus and pregnancy diagnosis was performed through ultrasonography. The results revealed that the cleavage rate was higher (P<0.05) in 0.1% RJ as compared to control group. However, the pregnancy rate was similar (P>0.05) between 0.1% RJ supplemented and control groups. It is concluded that supplementation of RJ in freezing extender can improve the cryosurvival rate and in vitro fertilizing capacity of buffalo bull sperm. Copyright © 2016 Elsevier B.V. All rights reserved.

Concentration-Dependent Inhibitory Effect of Baicalin on the Plasma Protein Binding and Metabolism of Chlorzoxazone, a CYP2E1 Probe Substrate, in Rats In Vitro and In Vivo

PubMed Central

Gao, Na; Zou, Dan; Qiao, Hai-Ling

2013-01-01

Some of the components found in herbs may be inhibitors or inducers of cytochrome P450 enzymes, which may therefore result in undesired herb-drug interactions. As a component extracted from Radix Scutellariae, the direct effect of baicalin on cytochrome P450 has not been investigated sufficiently. In this study, we investigated concentration-dependent inhibitory effect of baicalin on the plasma protein binding and metabolism of chlorzoxazone (CZN), a model CYP2E1 probe substrate, in rats in vitro and in vivo. Animal experiment was a randomized, three-period crossover design. Significant changes in pharmacokinetic parameters of CZN such as Cmax, t1/2 and Vd were observed after treatment with baicalin in vivo (P<0.05). Cmax decreased by 25% and 33%, whereas t1/2 increased by 34% and 53%, Vd increased by 37% and 50% in 225 mg/kg and 450 mg/kg baicalin-treated rats, respectively. The AUC and CL of CZN were not affected (P>0.05). Correlation analysis showed that the changes in CZN concentrations and baicalin concentrations were in good correlation (r>0.99). In vitro experiments, baicalin decreased the formation of 6-OH-chlorzoxazone in a concentration-dependent manner and exhibited a competitive inhibition in rat liver microsomes, with a Ki value of 145.8 µM. The values of Cmax/Ki were 20 and 39 after treatment with baicalin (225 and 450 mg/kg), respectively. Protein binding experiments in vivo showed that the plasma free-fraction (fu) of CZN increased 2.6-fold immediately after baicalin treatment (450 mg/kg) and in vitro showed that baicalin (125–2500 mg/L) increased the unbound CZN from 1.63% to 3.58%. The results indicate that pharmacokinetic changes in CZN are induced by inhibitory effect of baicalin on the plasma protein binding of CZN and CYP2E1 activity. PMID:23301016

Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality.

PubMed

Dovolou, Eleni; Periquesta, Eva; Messinis, Ioannis E; Tsiligianni, Theodora; Dafopoulos, Konstantinos; Gutierrez-Adan, Alfonso; Amiridis, Georgios S

2014-03-01

Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P < 0.05). In experiment 3, ghrelin significantly suppressed blastocysts yield. Significant differences were detected in various relative mRNA abundance, giving an overall final notion that embryos produced in the presence of ghrelin were of better quality than controls. Our results imply a specific role of ghrelin in early embryonic development; however, the specific mode of its action needs further investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

[Study of the interrelations of ethmozine, cordarone and phenycaberan with heparin].

PubMed

Tolstopiatov, B I

1981-01-01

Cordarone, etmozin and phenycaberan form complexes with heparin. Etmozin and phenycaberan form complexes insoluble in an aqueous medium and exhibit a pronounced antiheparin action in in-vitro experiments. Cordarone and heparin form a complex which is soluble in an aqueous medium. This complex potentiates the biological activity of the anticoagulant. In experiments on rabbits cordarone and phenycaberan increase plasma tolerance to heparin followed by its lowering as compared with controls in experiments with phenycaberan. Etmozin decreases plasma tolerance to heparin.

Preventing Hypothermia: Comparison of Current Devices Used by the U.S. Army with an In Vitro Warmed Crystalloid Fluid Model

DTIC Science & Technology

2010-04-01

the start of the experiment and remained so throughout the rest of the experimental period (Fig 4, Table 2). Mean core temperature observed was 36.72...removal [4, 9]. At temperatures less than 32ºC the body experiences a severe decrease in intrinsic metabolic rate. At less than 24ºC endocrine...in the field are thus forced to base their procurement decisions on either personal anecdotal experience or manufacturer claims of performance or

Growth and differentiation of mammalian embryonic tissues exposed to hypergravity in vivo and in vitro

NASA Technical Reports Server (NTRS)

Duke, J.; Janer, L.; Moore, J.

1985-01-01

Decreased cartilage areas in embryonic limbs developing under excess g in vitro, is reported, as well as delayed skeletal development in embryos and fetuses exposed to excess g in utero. 12.5-day mouse limb buds were cultured at 2.6 g, and fixed at two days and six days of culture. In vivo experiments used alizarin-stained 18-day fetuses exposed to 2.3 g. In all studies, cartilage areas were determined using a digitized tablet. Form factor analysis determined that the main effect of in vitro centrifugation was a reduction in length of the limb elements, probably due to the precocious chondrogenesis seen in the upper regions of centrifuged limbs. Similar reductions in length of ossified areas was seen in the in utero studies.

In Vitro Dosimetry of Silver Nanoparticles

EPA Science Inventory

An important issue for interpreting in vitro nanomaterial testing is quantifying the dose absorbed by target cells. Considerations include the concentration added to the culture and the proportion of the applied dose that is absorbed by the target cells. Rapid and efficient techn...

Agent-Based Modeling of Cancer Stem Cell Driven Solid Tumor Growth.

PubMed

Poleszczuk, Jan; Macklin, Paul; Enderling, Heiko

2016-01-01

Computational modeling of tumor growth has become an invaluable tool to simulate complex cell-cell interactions and emerging population-level dynamics. Agent-based models are commonly used to describe the behavior and interaction of individual cells in different environments. Behavioral rules can be informed and calibrated by in vitro assays, and emerging population-level dynamics may be validated with both in vitro and in vivo experiments. Here, we describe the design and implementation of a lattice-based agent-based model of cancer stem cell driven tumor growth.

In vitro flow assessment: from PC-MRI to computational fluid dynamics including fluid-structure interaction

NASA Astrophysics Data System (ADS)

Kratzke, Jonas; Rengier, Fabian; Weis, Christian; Beller, Carsten J.; Heuveline, Vincent

2016-04-01

Initiation and development of cardiovascular diseases can be highly correlated to specific biomechanical parameters. To examine and assess biomechanical parameters, numerical simulation of cardiovascular dynamics has the potential to complement and enhance medical measurement and imaging techniques. As such, computational fluid dynamics (CFD) have shown to be suitable to evaluate blood velocity and pressure in scenarios, where vessel wall deformation plays a minor role. However, there is a need for further validation studies and the inclusion of vessel wall elasticity for morphologies being subject to large displacement. In this work, we consider a fluid-structure interaction (FSI) model including the full elasticity equation to take the deformability of aortic wall soft tissue into account. We present a numerical framework, in which either a CFD study can be performed for less deformable aortic segments or an FSI simulation for regions of large displacement such as the aortic root and arch. Both of the methods are validated by means of an aortic phantom experiment. The computational results are in good agreement with 2D phase-contrast magnetic resonance imaging (PC-MRI) velocity measurements as well as catheter-based pressure measurements. The FSI simulation shows a characteristic vessel compliance effect on the flow field induced by the elasticity of the vessel wall, which the CFD model is not capable of. The in vitro validated FSI simulation framework can enable the computation of complementary biomechanical parameters such as the stress distribution within the vessel wall.

Nrf2-related gene expression and exposure to traffic-related air pollution in elderly subjects with cardiovascular disease: An exploratory panel study

PubMed Central

Wittkopp, Sharine; Staimer, Norbert; Tjoa, Thomas; Stinchcombe, Timothy; Daher, Nancy; Schauer, James J.; Shafer, Martin M.; Sioutas, Constantinos; Gillen, Daniel L.; Delfino, Ralph J.

2015-01-01

Gene expression changes are linked to air pollutant exposures in in vitro and animal experiments. However, limited data are available on how these outcomes relate to ambient air pollutant exposures in humans. We performed an exploratory analysis testing whether gene expression levels were associated with air pollution exposures in a Los Angeles area cohort of elderly subjects with coronary artery disease. Candidate genes (35) were selected from published studies of gene expression-pollutant associations. Expression levels were measured weekly in 43 subjects (≤12 weeks) using quantitative PCR. Exposures included gaseous pollutants O3, nitrogen oxides (NOx), and CO; particulate matter (PM) pollutants elemental and black carbon (EC, BC); and size-fractionated PM mass. We measured organic compounds from PM filter extracts, including polycyclic aromatic hydrocarbons (PAHs), and determined the in vitro oxidative potential of particle extracts. Associations between exposures and gene expression levels were analyzed using mixed-effects regression models. We found positive associations of traffic-related pollutants (EC, BC, primary organic carbon, PM0.25-2.5 PAH and/or PM0.25 PAH, and NOx) with NFE2L2, Nrf2-mediated genes (HMOX1, NQO1, and SOD2), CYP1B1, IL1B, and SELP. Findings suggest that NFE2L2 gene expression links associations of traffic-related air pollution with phase I and II enzyme genes at the promoter transcription level. PMID:25564368

Nrf2-related gene expression and exposure to traffic-related air pollution in elderly subjects with cardiovascular disease: An exploratory panel study.

PubMed

Wittkopp, Sharine; Staimer, Norbert; Tjoa, Thomas; Stinchcombe, Timothy; Daher, Nancy; Schauer, James J; Shafer, Martin M; Sioutas, Constantinos; Gillen, Daniel L; Delfino, Ralph J

2016-01-01

Gene expression changes are linked to air pollutant exposures in in vitro and animal experiments. However, limited data are available on how these outcomes relate to ambient air pollutant exposures in humans. We performed an exploratory analysis testing whether gene expression levels were associated with air pollution exposures in a Los Angeles area cohort of elderly subjects with coronary artery disease. Candidate genes (35) were selected from published studies of gene expression-pollutant associations. Expression levels were measured weekly in 43 subjects (≤ 12 weeks) using quantitative PCR. Exposures included gaseous pollutants O3, nitrogen oxides (NOx), and CO; particulate matter (PM) pollutants elemental and black carbon (EC, BC); and size-fractionated PM mass. We measured organic compounds from PM filter extracts, including polycyclic aromatic hydrocarbons (PAHs), and determined the in vitro oxidative potential of particle extracts. Associations between exposures and gene expression levels were analyzed using mixed-effects regression models. We found positive associations of traffic-related pollutants (EC, BC, primary organic carbon, PM 0.25-2.5 PAH and/or PM 0.25 PAH, and NOx) with NFE2L2, Nrf2-mediated genes (HMOX1, NQO1, and SOD2), CYP1B1, IL1B, and SELP. Findings suggest that NFE2L2 gene expression links associations of traffic-related air pollution with phase I and II enzyme genes at the promoter transcription level.

Phosphorus Amendment Efficacy for In Situ Remediation of ...

EPA Pesticide Factsheets

A validated method is needed to measure reductions of in vitro bioaccessible (IVBA) Pb in urban soil remediated with amendments. This study evaluated the effect of in vitro extraction solution pH and glycine buffer on bioaccesible Pb in P-treated soils. Two Pb-contaminated soils (790-1300 mg Pb kg-1), one from a garden and one from a city lot in Cleveland, OH, were incubated in a bench scale experiment for 1 yr. Six phosphate amendments, including bone meal, fish bone, poultry litter, monoammonium phosphate, diammonium phosphate, and triple superphosphate, were added to containers at two application rates. Lead IVBA was assessed using USEPA Method 1340 and three modified versions of this method. Modifications included using solutions with pH 1.5 and 2.5 as well as using solutions with and without 0.4 mol L-1 glycine. Soil amendments were effective in reducing IVBA Pb in these soils as measured by pH 1.5 with glycine buffer. The greatest reductions in IVBA Pb, from 5 to 26%, were found using pH 2.5 extractions. Lead mineral results showed several soil amendments promoted Pb phosphate formation, an indicator of remediation success. A significant negative linear relationship between reduction in IVBA Pb and Pb-phosphate formation was found only for pH 2.5 without glycine extraction solution. A modified USEPA Method 1340 without glycine and using pH 2.5 has the potential to predict P soil treatment efficacy and reductions in bioavailable Pb. Developing mana

Withangulatin I, a new cytotoxic withanolide from Physalis angulata.

PubMed

Lee, Shwu-Woan; Pan, Min-Hsiung; Chen, Chiu-Ming; Chen, Zong-Tsi

2008-02-01

A new withanolide, withangulatin I (2), was isolated from the whole plant of Physalis angulata. Its structure was established as (20S,22R)-15alpha-acetoxy-5beta,6beta-epoxy-14alpha-hydroxy-1,4-dioxo-witha-2,16,24-trienolide on the basis of chemical and spectroscopic methods including 2D-NMR and circular dichroism (CD) experiments. Withangulatin A (1) and withangulatin I (2) were tested for their cytotoxic activities against two human cancer cell lines, colorectal carcinoma COLO 205 and gastric carcinoma AGS, in vitro. Compounds 1 and 2 exhibited inhibitory activities against these two human cancer cells with IC(50) values of 16.6 and 1.8 and 53.6 and 65.4 muM, respectively.

The twin children of Auschwitz-Birkenau: conference on Nazi medicine.

PubMed

Segal, Nancy L

2013-06-01

The twin children who survived the Holocaust and the horrific medical experiments conducted by Nazi doctors are sometimes overlooked in the relevant literature. This topic and more were discussed as part of an annual conference hosted by students from Yeshiva University's Medical Ethics Society in October 2012. A selective summary of this meeting is followed by summaries of recent twin studies concerning genetic influences on twinning, in vitro fertilization versus spontaneous twin pregnancies, gender identity disorder, and royal support for twin registries. Several human interest stories are also worth noting. They include identical twin school principals, twin loss at Sandy Hook Elementary School, timely twin documentaries, new twin and twin-like reunions, and the passing of two prominent twins.

[Preliminary investigation of the in vitro inhibitory effect of antibiotics on algae of the genus Prototheca].

PubMed

Casal, M; Gutierrez, J

1981-07-10

We investigated the possible inhibitory action 'in vitro' by antibiotic containing discs against microscopic alga that are potential human pathogens of the genus Prototheca especially P. wickerhamii. The results indicated that inhibitors in vitro included amikacin, colistin, dibecacin, framicetin, gentamycin, kanamycin, lividomycin, neomycin, polymyxin, paromomycin, ribostamycin, sisomycin and tobramycin. We emphasize the interest in the effectiveness at these drugs in a case of human protothecosis.

The in vitro isolated whole guinea pig brain as a model to study epileptiform activity patterns.

PubMed

de Curtis, Marco; Librizzi, Laura; Uva, Laura

2016-02-15

Research on ictogenesis is based on the study of activity between seizures and during seizures in animal models of epilepsy (chronic condition) or in in vitro slices obtained from naïve non-epileptic brains after treatment with pro-convulsive drugs, manipulations of the extracellular medium and specific stimulation protocols. The in vitro isolated guinea pig brain retains the functional connectivity between brain structures and maintains interactions between neuronal, glial and vascular compartments. It is a close-to-in vivo preparation that offers experimental advantages not achieved with the use of other experimental models. Neurophysiological and imaging techniques can be utilized in this preparation to study brain activity during and between seizures induced by pharmacological or functional manipulations. Cellular and network determinants of interictal and ictal discharges that reproduce abnormal patterns observed in human focal epilepsies and the associated changes in extracellular ion and blood-brain permeability can be identified and analyzed in the isolated guinea pig brain. Ictal and interictal patterns recorded in in vitro slices may show substantial differences from seizure activity recorded in vivo due to slicing procedure itself. The isolated guinea pig brain maintained in vitro by arterial perfusion combines the typical facilitated access of in vitro preparations, that are difficult to approach during in vivo experiments, with the preservation of larger neuronal networks. The in vitro whole isolated guinea pig brain preparation offers an unique experimental model to study systemic and neurovascular changes during ictogenesis. Published by Elsevier B.V.

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

PubMed

Siril, E A; Joseph, Nisha

2013-01-01

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 μM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 μM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

In vivo and in vitro measurements of cerebral aneurysm hemodynamics

NASA Astrophysics Data System (ADS)

Amili, Omid; Toloui, Mostafa; van de Moortele, Pierre-Francois; Jagadeesan, Bharathi; Coletti, Filippo

2017-11-01

The hemodynamics of cerebral aneurysms is thought to play a critical role in their formation, growth, and potential rupture. Our understanding in this area, however, comes mostly from in vitro experiments and numerical simulations, which have limited realism. In vivo measurements of the intracranial blood flow can be obtained by Magnetic Resonance Imaging (MRI), but they typically suffer from limited accuracy and inadequate resolution. Here we present a direct comparison between in vivo and in vitro measurements of the flow inside an internal carotid artery aneurysm. For both, we use 4D (i.e. volumetric and time-resolved) MRI velocimetry performed in a 7 Tesla magnet at sub-millimeter resolution. The in vitro measurements are carried out in a 3D printed aneurysm replica scaled up by a factor three, effectively increasing the spatial resolution. The patient-specific inflow waveform and the corresponding Reynolds and Womersley numbers are matched in a flow loop that mimics the impedance of the vascular bed. Direct comparison of the velocity fields allows assessing the robustness of the in vivo measurements, while highlighting the insight achievable in vitro. The data also represents a comprehensive test case for numerical simulations.