Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

PubMed

Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Ribosome display: next-generation display technologies for production of antibodies in vitro.

PubMed

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Micropropagation of frost-resistant Eucalyptus

Treesearch

Michel Boulay

1983-01-01

A method for the in vitro propagation of frost resistant eucalyptus is presented. It was used for the propagation of 2-30 years old trees. This method is presently used for the fast production of mother trees from selected trees.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Detection of Hydroxyapatite in Calcified Cardiovascular Tissues

PubMed Central

Lee, Jae Sam; Morrisett, Joel D.; Tung, Ching-Hsuan

2012-01-01

Objective The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. Methods A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. Results Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. Conclusion Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases. PMID:22877867

Vibrational monitor of early demineralization in tooth enamel after in vitro exposure to phosphoridic liquid

NASA Astrophysics Data System (ADS)

Pezzotti, Giuseppe; Adachi, Tetsuya; Gasparutti, Isabella; Vincini, Giulio; Zhu, Wenliang; Boffelli, Marco; Rondinella, Alfredo; Marin, Elia; Ichioka, Hiroaki; Yamamoto, Toshiro; Marunaka, Yoshinori; Kanamura, Narisato

2017-02-01

The Raman spectroscopic method has been applied to quantitatively assess the in vitro degree of demineralization in healthy human teeth. Based on previous evaluations of Raman selection rules (empowered by an orientation distribution function (ODF) statistical algorithm) and on a newly proposed analysis of phonon density of states (PDOS) for selected vibrational modes of the hexagonal structure of hydroxyapatite, a molecular-scale evaluation of the demineralization process upon in vitro exposure to a highly acidic beverage (i.e., CocaCola™ Classic, pH = 2.5) could be obtained. The Raman method proved quite sensitive and spectroscopic features could be directly related to an increase in off-stoichiometry of the enamel surface structure since the very early stage of the demineralization process (i.e., when yet invisible to other conventional analytical techniques). The proposed Raman spectroscopic algorithm might possess some generality for caries risk assessment, allowing a prompt non-contact diagnostic practice in dentistry.

In vitro assessment of biodurability: acellular systems.

PubMed Central

de Meringo, A; Morscheidt, C; Thélohan, S; Tiesler, H

1994-01-01

The assessment of biodurability of man-made vitreous fibers is essential to the limitation of health hazards associated with human exposure to environments in which respirable fibers are present. In vitro acellular systems provide effective test methods of measuring fiber solubility provided care is taken to select the most suitable solvent and test conditions for the specific fiber type and dimension. PMID:7882955

Comparative assessment of three in vitro exposure methods for combustion toxicity.

PubMed

Lestari, Fatma; Markovic, Boban; Green, Anthony R; Chattopadhyay, Gautam; Hayes, Amanda J

2006-01-01

A comparative assessment of three approaches for the use of human cells in vitro to investigate combustion toxicity was conducted. These included one indirect and two direct (passive and dynamic) exposure methods. The indirect method used an impinger system in which culture medium was used to trap the toxicants, whilst the direct exposure involved the use of a Horizontal Harvard Navicyte Chamber at the air/liquid interface. The cytotoxic effects of thermal decomposition products were assessed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega) on a selection of human cells including: HepG2, A549 and skin fibroblasts. A small scale laboratory fire test using a vertical tube furnace was designed for the generation of combustion products. Polymethyl methacrylate (PMMA) was selected as a model polymer to study the cytotoxic effects of combustion products. NOAEC (no observable adverse effect concentration), IC10 (10% inhibitory concentration), IC50 (50% inhibitory concentration) and TLC (total lethal concentration) values were determined from dose response curves. Assessment using the NRU (neutral red uptake) and ATP (adenosine triphosphate) assays on human lung derived cells (A549) was also undertaken. Comparison between in vitro cytotoxicity results against published toxicity data for PMMA combustion and predicted LC50 (50% lethal concentration) values calculated from identified compounds using GCMS (gas chromatography mass spectrometry) was determined. The results suggested that the indirect exposure method did not appear to simulate closely exposure via inhalation, whilst exposure at the air/liquid interface by using the dynamic method proved to be a more representative method of human inhalation. This exposure method may be a potential system for in vitro cytotoxicity testing in combustion toxicity. Copyright 2005 John Wiley & Sons, Ltd.

Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro

PubMed Central

2013-01-01

Background Good quality spermatozoa are important to achieve fertilization, viable embryos and offspring. Single Layer Centrifugation (SLC) through a colloid (Androcoll-P) selects good quality spermatozoa. However, it has not been established previously whether porcine spermatozoa selected by this method maintain their fertility. Methods The semen was prepared either by SLC or by standard centrifugation (control) and used for in vitro fertilization (IVF) at oocyte:spermatozoa ratios of 1:50; 1:100 and 1:300 (or 4 x 103, 8 x 103 and 24 x 103 spermatozoa/ml) to evaluate their subsequent ability to generate blastocysts. In addition, sperm motility was assessed by computer assisted sperm motility analysis. Results Total and progressive motility were significantly higher in sperm samples prepared by SLC compared to uncentrifuged samples. Sperm binding ability, polyspermy, cleavage and blastocyst rates were affected by the oocyte:sperm ratio, but not by sperm treatment. Conclusion The use of SLC does not adversely affect the in vitro fertilizing and embryo-generating ability of the selected spermatozoa compared to their unselected counterparts, but further modifications in the IVF conditions would be needed to improve the monospermy in IVF systems. Since SLC did not appear to have a negative effect on sperm fertilizing ability, and may in fact select for spermatozoa with a greater potential for fertilization, an in vivo trial to determine the usefulness of this sperm preparation technique prior to artificial insemination is warranted. PMID:23497680

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 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.

[Screening specific recognition motif of RNA-binding proteins by SELEX in combination with next-generation sequencing technique].

PubMed

Zhang, Lu; Xu, Jinhao; Ma, Jinbiao

2016-07-25

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.

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.

Screening of Lactobacillus isolates from gastrointestinal tract of guinea fowl for probiotic qualities using in vitro tests to select species-specific probiotic candidates.

PubMed

Vineetha, P G; Tomar, S; Saxena, V K; Susan, C; Sandeep, S; Adil, K; Mukesh, K

2016-08-01

A total of 32 Lactobacillus isolates, 8 each from the crop (LGFCP1-LGFCP8), proventriculus (LGFP9-LGFP16), ileum (LGFI17-LGFI24) and caeca (LGFCM25-LGFCM32) were isolated from 25 adult guinea fowl (Pearl variety), 22-28 weeks of age, and characterised morphologically, physiologically, biochemically and by molecular methods. Isolates were screened for their probiotic quality using range of in vitro tests: aggregation test, cell surface hydrophobicity, resistance to bile salts and acidic conditions, enzymatic tests and coaggregation and antagonistic test. Based on in vitro test results and a novel scoring method, the two best isolates were selected and partial 16S rRNA sequencing was done. BLAST (Basic Local Alignment Search Tool) analysis of sequence of isolate LGFCP4 showed 99% genetic identity with Lactobacillus plantarum and LGFP16 with Lactobacillus reuteri. The study shows that these two microbial agents may be suitable as potential probiotic candidates in guinea fowl, as well as in a feed supplement for other poultry species.

Quality by design approach for understanding the critical quality attributes of cyclosporine ophthalmic emulsion.

PubMed

Rahman, Ziyaur; Xu, Xiaoming; Katragadda, Usha; Krishnaiah, Yellela S R; Yu, Lawrence; Khan, Mansoor A

2014-03-03

Restasis is an ophthalmic cyclosporine emulsion used for the treatment of dry eye syndrome. There are no generic products for this product, probably because of the limitations on establishing in vivo bioequivalence methods and lack of alternative in vitro bioequivalence testing methods. The present investigation was carried out to understand and identify the appropriate in vitro methods that can discriminate the effect of formulation and process variables on critical quality attributes (CQA) of cyclosporine microemulsion formulations having the same qualitative (Q1) and quantitative (Q2) composition as that of Restasis. Quality by design (QbD) approach was used to understand the effect of formulation and process variables on critical quality attributes (CQA) of cyclosporine microemulsion. The formulation variables chosen were mixing order method, phase volume ratio, and pH adjustment method, while the process variables were temperature of primary and raw emulsion formation, microfluidizer pressure, and number of pressure cycles. The responses selected were particle size, turbidity, zeta potential, viscosity, osmolality, surface tension, contact angle, pH, and drug diffusion. The selected independent variables showed statistically significant (p < 0.05) effect on droplet size, zeta potential, viscosity, turbidity, and osmolality. However, the surface tension, contact angle, pH, and drug diffusion were not significantly affected by independent variables. In summary, in vitro methods can detect formulation and manufacturing changes and would thus be important for quality control or sameness of cyclosporine ophthalmic products.

Development of small bisquaternary cholinesterase inhibitors as drugs for pre-treatment of nerve agent poisonings

PubMed Central

Kuca, Kamil; Karasova, Jana Zdarova; Soukup, Ondrej; Kassa, Jiri; Novotna, Eva; Sepsova, Vendula; Horova, Anna; Pejchal, Jaroslav; Hrabinova, Martina; Vodakova, Eva; Jun, Daniel; Nepovimova, Eugenie; Valis, Martin; Musilek, Kamil

2018-01-01

Background Intoxication by nerve agents could be prevented by using small acetylcholinesterase inhibitors (eg, pyridostigmine) for potentially exposed personnel. However, the serious side effects of currently used drugs led to research of novel potent molecules for prophylaxis of organophosphorus intoxication. Methods The molecular design, molecular docking, chemical synthesis, in vitro methods (enzyme inhibition, cytotoxicity, and nicotinic receptors modulation), and in vivo methods (acute toxicity and prophylactic effect) were used to study bispyridinium, bisquinolinium, bisisoquinolinium, and pyridinium-quinolinium/isoquinolinium molecules presented in this study. Results The studied molecules showed non-competitive inhibitory ability towards human acetylcholinesterase in vitro that was further confirmed by molecular modelling studies. Several compounds were selected for further studies. First, their cytotoxicity, nicotinic receptors modulation, and acute toxicity (lethal dose for 50% of laboratory animals [LD50]; mice and rats) were tested to evaluate their safety with promising results. Furthermore, their blood levels were measured to select the appropriate time for prophylactic administration. Finally, the protective ratio of selected compounds against soman-induced toxicity was determined when selected compounds were found similarly potent or only slightly better to standard pyridostigmine. Conclusion The presented small bisquaternary molecules did not show overall benefit in prophylaxis of soman-induced in vivo toxicity. PMID:29563775

Detection of hydroxyapatite in calcified cardiovascular tissues.

PubMed

Lee, Jae Sam; Morrisett, Joel D; Tung, Ching-Hsuan

2012-10-01

The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

[Selection of a melanine concentrating hormone receptor-1 (MCHR1) antagonists' focused library and its biological screening with AequoScreen].

PubMed

Flachner, Beáta; Hajdú, István; Dobi, Krisztina; Lorincz, Zsolt; Cseh, Sándor; Dormán, György

2013-01-01

Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.

Production, Preservation, and Transfer of South American Camelid Embryos

PubMed Central

Trasorras, Virginia L.; Carretero, María Ignacia; Neild, Deborah M.; Chaves, Maria Graciela; Giuliano, Susana M.; Miragaya, Marcelo H.

2017-01-01

The current review summarizes progress in the field of in vitro and in vivo production of South American Camelid embryos. Both methods require ovarian superstimulation (with FSH and eCG) to obtain multiple ovulations (in vivo embryo production) or to induce follicle growth for oocyte collection (in vitro embryo production). Moreover, superstimulation entails prior administration of hormones that inhibit follicular growth (progesterone, progestagens, and estrogens). Cumulus-oocyte complexes obtained must mature in vivo (buserelin administration) or in vitro to then be subjected to in vitro fertilization or intracytoplasmic sperm injection. All these techniques also require morphologically normal, motile spermatozoa to achieve fertilization. Methods used to decrease semen viscosity and to select the best spermatozoa (Percoll®; Androcoll-ETM) are described. Additionally, nuclear transfer or cloning has been applied in llamas. Up to now, embryo deep-freezing and vitrification have progressed slowly but are at the height of development. Embryos that are obtained by any of these techniques, either in vivo or in vitro, need to be transferred to synchronized recipient females. The best results are achieved after transfer to the left uterine horn with an ipsilateral ovulation. No live offspring have been obtained after the transfer of cryopreserved embryos. Applying reproductive biotechnologies, such as those described, will permit the expansion of genetically selected animals in the population and also that of wild camelid species, vicunas, and guanacos, whose embryos could then be transferred to the uterus of domestic species. PMID:29181380

Phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein DAS181

PubMed Central

Triana-Baltzer, Gallen B.; Sanders, Rebecca L.; Hedlund, Maria; Jensen, Kellie A.; Aschenbrenner, Laura M.; Larson, Jeffrey L.; Fang, Fang

2011-01-01

Background Influenza viruses (IFVs) frequently achieve resistance to antiviral drugs, necessitating the development of compounds with novel mechanisms of action. DAS181 (Fludase®), a sialidase fusion protein, may have a reduced potential for generating drug resistance due to its novel host-targeting mechanism of action. Methods IFV strains B/Maryland/1/59 and A/Victoria/3/75 (H3N2) were subjected to >30 passages under increasing selective pressure with DAS181. The DAS181-selected IFV isolates were characterized in vitro and in mice. Results Despite extensive passaging, DAS181-selected viruses exhibited a very low level of resistance to DAS181, which ranged between 3- and 18-fold increase in EC50. DAS181-selected viruses displayed an attenuated phenotype in vitro, as exhibited by slower growth, smaller plaque size and increased particle to pfu ratios relative to wild-type virus. Further, the DAS181 resistance phenotype was unstable and was substantially reversed over time upon DAS181 withdrawal. In mice, the DAS181-selected viruses exhibited no greater virulence than their wild-type counterparts. Genotypic and phenotypic analysis of DAS181-selected viruses revealed mutations in the haemagglutinin (HA) and neuraminidase (NA) molecules and also changes in HA and NA function. Conclusions Results indicate that resistance to DAS181 is minimal and unstable. The DAS181-selected IFV isolates exhibit reduced fitness in vitro, likely due to altered HA and NA functions. PMID:21097900

Colorimetric method for the detection of melamine using in-situ formed silver nanoparticles via tannic acid

NASA Astrophysics Data System (ADS)

Alam, Md. Fazle; Laskar, Amaj Ahmed; Ahmed, Shahbaz; Shaida, Mohd. Azfar; Younus, Hina

2017-08-01

Melamine toxicity has recently attracted worldwide attention as it causes renal failure and the death of humans and animals. Therefore, developing a simple, fast and sensitive method for the routine detection of melamine is the need of the hour. Herein, we have developed a selective colorimetric method for the detection of melamine in milk samples based upon in-situ formation of silver nanoparticles (AgNPs) via tannic acid. The AgNPs thus formed were characterized by UV-Visible spectrophotometer, transmission electron microscope (TEM), zetasizer and dynamic light scattering (DLS). The AgNPs were used to detect melamine under in vitro condition and in raw milk spiked with melamine. Under optimal conditions, melamine could be selectively detected in vitro within the concentration range of 0.05-1.4 μM with a limit of detection (LOD) of 0.01 μM, which is lower than the strictest melamine safety requirement of 1 ppm. In spiked raw milk, the recovery percentage range was 99.5-106.5% for liquid milk and 98.5-105.5% for powdered milk. The present method shows extreme selectivity with no significant interference with other substances like urea, glucose, glycine, ascorbic acid etc. This assay method does not utilize organic cosolvents, enzymatic reactions, light sensitive dye molecules and sophisticated instrumentation, thereby overcoming some of the limitations of the other conventional methods.

Formulation and Evaluation of Mouth Dissolving Tablets of Cinnarizine

PubMed Central

Patel, B. P.; Patel, J. K.; Rajput, G. C.; Thakor, R. S.

2010-01-01

The purpose of this research was to develop mouth dissolve tablets of cinnarizine by effervescent, superdisintegrant addition and sublimation methods. All the three formulations were evaluated for disintegration time, hardness and friability, among these superdisintegrant addition method showed lowest disintegration time; hence it was selected for further studies. Further nine batches (B1-B9) were prepared by using crospovidone, croscarmellose sodium and L-HPC in different concentrations such as 5, 7.5 and 10%. All the formulations were evaluated for weight variation, hardness, friability, drug content, in vitro disintegration time, wetting time, in vitro dissolution. Formulation with 10% L-HPC showed the less disintegration time (25.3 s) and less wetting time (29.1 s). In vitro dissolution studies showed total drug release at the end of 6 min. PMID:21218071

Generation and development of RNA ligase ribozymes with modular architecture through "design and selection".

PubMed

Fujita, Yuki; Ishikawa, Junya; Furuta, Hiroyuki; Ikawa, Yoshiya

2010-08-26

In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed "design and selection," new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection.

Novel selection methods for DNA-encoded chemical libraries

PubMed Central

Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

2015-01-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation.

PubMed

De Baere, S; Eeckhaut, V; Steppe, M; De Maesschalck, C; De Backer, P; Van Immerseel, F; Croubels, S

2013-06-01

A rapid and sensitive HPLC-UV method for the quantitative determination of four short-chain fatty acids (SCFAs) and lactic acid (LA) produced during in vitro fermentation is presented. Extraction of SCFAs from supernatants of bacterial cultures is aggravated due to their polarity and volatility. Detection can only be performed at a short, non-selective UV wavelength (210nm), due to the lack of any significant chromophore. Therefore special attention was paid to the optimization of the sample preparation procedure and the HPLC-UV conditions. The final extraction procedure consisted of a liquid-liquid back extraction using diethylether. Prior to HPLC-UV analysis the samples were acidified (pH<2) in order to improve retention of the SCFA's and LA on the Hypersil Gold aQ column. Matrix-matched calibration graphs were prepared for all analytes of interest (range 0.5-50mM) and correlation and goodness-of-fit coefficients were between 0.9951-0.9993 and 3.88-8.27%, respectively. Limits of detection and quantification ranged from 0.13 to 0.33mM and 0.5 to 1.0mM, respectively. The results for the within-day and between-day precision and accuracy fell within the ranges specified. The reported validated method has been successfully used for the in vitro screening of supernatants of bacterial cultures for the presence of butyric acid, aiming to select for butyric acid-producing bacteria. In addition, the method has been used to determine the production pattern of selected fatty acids by bacterial species isolated from human feces and chicken caeca. Copyright © 2013 Elsevier B.V. All rights reserved.

A comparison of in vitro cytotoxicity assays in medical device regulatory studies.

PubMed

Liu, Xuemei; Rodeheaver, Denise P; White, Jeffrey C; Wright, Ann M; Walker, Lisa M; Zhang, Fan; Shannon, Stephen

2018-06-06

Medical device biocompatibility testing is used to evaluate the risk of adverse effects on tissues from exposure to leachates/extracts. A battery of tests is typically recommended in accordance with regulatory standards to determine if the device is biocompatible. In vitro cytotoxicity, a key element of the standards, is a required endpoint for all types of medical devices. Each validated cytotoxicity method has different methodology and acceptance criteria that could influence the selection of a specific test. In addition, some guidances are more specific than others as to the recommended test methods. For example, the International Organization for Standardization (ISO 1 ) cites preference for quantitative methods (e.g., tetrazolium (MTT/XTT), neutral red (NR), or colony formation assays (CFA)) over qualitative methods (e.g., elution, agar overlay/diffusion, or direct), while a recent ISO standard for contact lens/lens care solutions specifically requires a qualitative direct test. Qualitative methods are described in United States Pharmacopeia (USP) while quantitative CFAs are listed in Japan guidance. The aim of this review is to compare the methodologies such as test article preparation, test conditions, and criteria for six cytotoxicity methods recommended in regulatory standards in order to inform decisions on which method(s) to select during the medical device safety evaluation. Copyright © 2018. Published by Elsevier Inc.

In vitro Induction and Generation of Tetraploid Plants of Sophora tonkinensis Gapnep

PubMed Central

Wei, Kun Hua; Xu, Jian Ping; Li, Lin Xuan; Cai, Jin Yuan; Miao, Jian Hua; Li, Min Hui

2018-01-01

Background: Sophora tonkinensis Gapnep. is an important medical plant in China. Early researches of S. tonkinensis were focused on rapid propagation and quality analysis of in vitro tissue culture plantlet, and still no research focuses on the plant breeding of and there were no excellent varieties for artificial cultivation of S. tonkinensis. Objective: To set up a method to generate and select the best varieties of S. tonkinensis by polyploid breeding after induction by colchicine treatment. Materials and Methods: The adventitious buds were submerged in different concentrations of aqueous colchicine solution for different lengths of time to induce polyploidy in the plants, and the induced buds were identified by root-tip chromosome determination and leaf characteristics comparison. The contents of matrine and oxymatrine of radix ex rhizoma in 13 selected tetraploid lines were collected after 90 days in vitro rooting culture and were evaluated to provide evidence of good qualities of tetraploid S. tonkinensis. Results: The results showed that the highest percentage of tetraploid induction was 23.33% and occurred in the 0.2% (w/v) colchicine treatment for 30 h. Fifty lines of tetraploid plants were obtained and 12 of the 13 selected tetraploid lines exhibited higher productivity of total contents of matrine and oxymatrine when compared to controls. Conclusion: The data demonstrate that polyploidy induction can be beneficial for improving the medicinal value of S. tonkinensis. SUMMARY Colchicine has a good in vitro induction effect on the tetraploid plants of Sophora tonkinensisThe leaf indices and stomatal apparatus of tetraploid plants were slightly larger than diploid plantsThe total content of matrine and oxymatrine of some tetraploid lines for 90-day-old in vitro rooting culture was higher than the diploid. Abbreviations used: MS medium: Murashige and Skoog medium; BAP: 6-benzylaminopurine; NAA: A-naphthaleneacetic acid; IAA: Indole-3-acetic acid; KT: Kinetin; IBA: Indole-3-butyric acid; ABT: Rooting power. PMID:29720823

Asymbiotic in vitro seed propagation of Dendrobium.

PubMed

Teixeira da Silva, Jaime A; Tsavkelova, Elena A; Ng, Tzi Bun; Parthibhan, S; Dobránszki, Judit; Cardoso, Jean Carlos; Rao, M V; Zeng, Songjun

2015-10-01

The ability to germinate orchids from seeds in vitro presents a useful and viable method for the propagation of valuable germplasm, maintaining the genetic heterogeneity inherent in seeds. Given the ornamental and medicinal importance of many species within the genus Dendrobium, this review explores in vitro techniques for their asymbiotic seed germination. The influence of abiotic factors (such as temperature and light), methods of sterilization, composition of basal media, and supplementation with organic additives and plant growth regulators are discussed in context to achieve successful seed germination, protocorm formation, and further seedling growth and development. This review provides both a basis for the selection of optimal conditions, and a platform for the discovery of better ones, that would allow the development of new protocols and the exploration of new hypotheses for germination and conservation of Dendrobium seeds and seedlings.

Enhancement of in vitro Guayule propagation

NASA Technical Reports Server (NTRS)

Dastoor, M. N.; Schubert, W. W.; Petersen, G. R. (Inventor)

1982-01-01

A method for stimulating in vitro propagation of Guayule from a nutrient medium containing Guayule tissue by adding a substituted trialkyl amine bioinducing agent to the nutrient medium is described. Selective or differentiated propagation of shoots or callus is obtained by varying the amounts of substituted trialky amine present in the nutrient medium. The luxuriant growth provided may be processed for its poly isoprene content or may be transferred to a rooting medium for production of whole plants as identical clones of the original tissue. The method also provides for the production of large numbers of Guayule plants having identical desirable properties such as high polyisoprene levels.

Development and in vitro/in vivo Evaluation of a Silastic Intravaginal Ring for Mifepristone Delivery.

PubMed

Duan, X; Ning, M

2015-01-01

Preparation and in vitro/in vivo evaluation of mifepristone intravaginal ring formulations were investigated. In the present study, it is reported that a mifepristone intravaginal ring of reservoir design comprising of a mifepristone silicone elastomer core enclosed in a silicone layer. During the preparation of intravaginal ring solid dispersion method was employed which improved the release rate of drug from the intravaginal ring. In vitro release studies performed under sink conditions and the released drug amounts were estimated using UV spectrometry at 310 nm. In addition, the in vivo release profile of in-house devices was evaluated in female New Zealand white rabbits. The rabbit plasma samples were processed and analyzed using a validated HPLC-MS method. Norgestrel was used as internal standard, and plasma samples contained mifepristone and internal standard were deproteinized, and then subjected to HPLC-MS analysis under condition of electrospray ionization in the selected ion monitoring mode. The drug release from intravaginal ring made in house was constant for 21 days in rabbits, which suggested the mifepristone intravaginal ring release system would be useful in clinical practice in the future. The result indicated the in vitro/in vivo correlation is perfect, which explained in vitro release analysis method developed was feasible.

Novel selection methods for DNA-encoded chemical libraries.

PubMed

Chan, Alix I; McGregor, Lynn M; Liu, David R

2015-06-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Vitro culture of axe-head glochidia in pink heelsplitter Potamilus alatus and mechanism of its high host specialists

PubMed Central

Ma, Xue Yan; Zheng, Bing Qing; Xu, Pao; Xu, Liang; Hua, Dan; Yuan, Xin Hua; Gu, Ruo Bo

2018-01-01

The basal media M199 or MEM was utilized in the classical method of vitro culture of glochidia where 1–5% CO2 was required to maintain stable physiological pH for completion of non-parasitic metamorphosis. The classical method encounters a great challenge to those glochidia which undergo development of visceral tissue but significantly increase in size during metamorphosis. The improved in vitro culture techniques and classical methods were firstly compared for non-parasitic metamorphosis and development of glochidia in pink heelsplitter. Based on the improved method, the optimal vitro culture media was further selected from 14 plasmas or sera, realizing the non-parasitic metamorphosis of axe-head glochidia for the first time. The results showed that addition of different plasma (serum) had significant effect on glochidial metamorphosis in pink heelsplitter. Only glochidia in the skewband grunt and red drum groups could complete metamorphosis, the metamorphosis rate in skewband grunt was 93.3±3.1% at 24±0.5°C, significantly higher than in marine and desalinated red drum. Heat-inactivated treatment on the plasma of yellow catfish and Barbus capito had significant effect on glochidia survival and shell growth. The metamorphosis rate also varied among different gravid period, and generally decreased with gravid time. Further comparison of free amino acid and fatty acid indicated that the taurine of high concentration was the only amino acid that might promote the rapid growth of glochidial shell, and the lack of adequate DPA and DHA might be an important reason leading to the abnormal foot and visceral development. Combined with our results of artificial selection of host fish, we tentatively established the mechanism of its host specialists in pink heelsplitter for the first time. This is the first report on non-parasite metamorphosis of axe-head glochidia based on our improved vitro culture method, which should provide important reference to fundamental theory research of glochidia metamorphosis and also benefit for better understand of mechanism of host specialists and generalists of Unionidae species. PMID:29447194

Susceptibility of selected ornamental plants to Phytophthora ramorum

Treesearch

K. Kaminski; S. Wagner; S. Werres

2008-01-01

Within the European project ?Risk Analysis for Phytophthora ramorum? (RAPRA), susceptibility of economically and ecologically important plants in Europe towards P. ramorum was tested via in vitro inoculation methods. In these studies different species and/or cultivars of Buxus, Calluna...

An In Vitro Translation, Selection, and Amplification System for Peptide Nucleic Acids

PubMed Central

Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.

2009-01-01

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory. PMID:20081830

In vitro antiplasmodial and cytotoxic activities of sesquiterpene lactones from Vernonia fimbrillifera Less. (Asteraceae).

PubMed

Bordignon, Annélise; Frédérich, Michel; Ledoux, Allison; Campos, Pierre-Eric; Clerc, Patricia; Hermann, Thomas; Quetin-Leclercq, Joëlle; Cieckiewicz, Ewa

2018-06-01

Due to the in vitro antiplasmodial activity of leaf extracts from Vernonia fimbrillifera Less. (Asteraceae), a bioactivity-guided fractionation was carried out. Three sesquiterpene lactones were isolated, namely 8-(4'-hydroxymethacrylate)-dehydromelitensin (1), onopordopicrin (2) and 8α-[4'-hydroxymethacryloyloxy]-4-epi-sonchucarpolide (3). Their structures were elucidated by spectroscopic methods (1D and 2D NMR and MS analyses) and by comparison with published data. The isolated compounds exhibited antiplasmodial activity with IC 50 values ≤ 5 μg/mL. Cytotoxicity of the compounds against a human cancer cell line (HeLa) and a mouse lung epithelial cell line (MLE12) was assessed to determine selectivity. Compound 3 displayed promising selective antiplasmodial activity (SI > 10).

Replacement of RNA hairpins by in vitro selected tetranucleotides.

PubMed Central

Dichtl, B; Pan, T; DiRenzo, A B; Uhlenbeck, O C

1993-01-01

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA. Images PMID:7680121

Population dynamics and in vitro antibody pressure of porcine parvovirus indicate a decrease in variability.

PubMed

Streck, André Felipe; Homeier, Timo; Foerster, Tessa; Truyen, Uwe

2013-09-01

To estimate the impact of porcine parvovirus (PPV) vaccines on the emergence of new phenotypes, the population dynamic history of the virus was calculated using the Bayesian Markov chain Monte Carlo method with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed with consecutive passages of the 'Challenge' strain (a virulent field strain) and NADL2 strain (a vaccine strain) in a PK-15 cell line supplemented with polyclonal antibodies raised against the vaccine strain. A decrease in genetic diversity was observed in the presence of antibodies in vitro or after vaccination (as estimated by the in silico model). We hypothesized that the antibodies induced a selective pressure that may reduce the incidence of neutral selection, which should play a major role in the emergence of new mutations. In this scenario, vaccine failures and non-vaccinated populations (e.g. wild boars) may have an important impact in the emergence of new phenotypes.

[Study on sustained release preparations of Epimedium component].

PubMed

Yan, Hong-mei; Ding, Dong-mei; Zhang, Zhen-hai; Sun, E; Song, Jie; Jia, Xiao-bin

2015-04-01

The formulation for sustained release tablet of Epinedium component was selected and the evaluation equation of in vitro release was established. The liquidity of component was improved with the help of colloidal silica aided by spray drying, which would be the main drug in the sustained release tablets. Dissolution was selected as an evaluation index to investigate skeletal material type, fillers, impact porogen, lubricants and other materials on the quality of sustained release tablet. The sustained release tablets were prepared by dry compression. Formulation of sustained release preparations was main drug 35%, HPMC K(4M) 20% and HPMC K(15M) 10% as skeleton material, MCC 31% as filler, PEG6000 2% as porogen and magnesium stearate 2% as lubricant. The sustained release tablets released up to 80% in 8 h. The zero order equation, primary equation and Higuchi equation could simulate the release characteristics of sustained release tablets in vitro, the correlation coefficients r were larger than 0.96. The primary equation was most similar in vitro release characteristics and its correlation coefficient r was 0.9950. The preparation method is simple and the results of formulation selection are reliable. It can be used to guide the production of Epimedium component sustained release preparations.

Resorption behavior of a nanostructured bone substitute: in vitro investigation and clinical application.

PubMed

Reichert, Christoph; Götz, Werner; Reimann, Susanne; Keilig, Ludger; Hagner, Martin; Bourauel, Christoph; Jäger, Andreas

2013-03-01

To develop an in vitro assay for quantitative analysis of the degradation to which a bone substitute is exposed by osteoclasts. The aim of establishing this method was to improve the predictability of carrying out tooth movements via bone substitutes and to provide a basis for verification in exemplary clinical cases. After populating a bone substitute (NanoBone®; ArtOss, Germany) with osteoclastic cells, inductively-coupled mass spectrometry was used to evaluate changing calcium levels in the culture medium as a marker of resorption activity. It was observed that calcium levels increased substantially in the culture medium with the cells populating the bone substitute. This in vitro assay is a valid method that can assist clinicians in selecting the appropriate materials for certain patients. While tooth movements occurring through this material were successful, uncertainty about the approach will remain as long-term results are not available.

Design Optimization and In Vitro-In Vivo Evaluation of Orally Dissolving Strips of Clobazam

PubMed Central

Bala, Rajni; Khanna, Sushil; Pawar, Pravin

2014-01-01

Clobazam orally dissolving strips were prepared by solvent casting method. A full 32 factorial design was applied for optimization using different concentration of film forming polymer and disintegrating agent as independent variable and disintegration time, % cumulative drug release, and tensile strength as dependent variable. In addition the prepared films were also evaluated for surface pH, folding endurance, and content uniformity. The optimized film formulation showing the maximum in vitro drug release, satisfactory in vitro disintegration time, and tensile strength was selected for bioavailability study and compared with a reference marketed product (frisium5 tablets) in rabbits. Formulation (F6) was selected by the Design-expert software which exhibited DT (24 sec), TS (2.85 N/cm2), and in vitro drug release (96.6%). Statistical evaluation revealed no significant difference between the bioavailability parameters of the test film (F6) and the reference product. The mean ratio values (test/reference) of C max (95.87%), t max (71.42%), AUC0−t (98.125%), and AUC0−∞ (99.213%) indicated that the two formulae exhibited comparable plasma level-time profiles. PMID:25328709

[Everted intestinal sac method for quick finding absorption ingredients of Wuzhuyu decoction].

PubMed

Gong, Muxin; Wang, Yaxun; Song, Yafang; Wang, Zhimin; Zhang, Qiwei; Wang, Weihao; Zhu, Jingjing

2010-06-01

To establish a method for quick finding the absorption ingredients of Wuzhuyu decoction in order to select the index to control its quality. The absorption of three concentration of Wuzhuyu decotion was investigated with the in vitro-everted intestinal sac model. The intestinal bag fluid of jejunum and ileum were collected in different time and the eight ingredients, which were evodiamine (Ev), rutaecarpine (Ru), limonin (Li), ginsenoside-Rb1, -Rg1, -Re (Rb1, Rg1, Re), isorhamnetin-3-O-beta-D-glucosyl(6''-->1'")-alpha-L-rhamnoside (Irs)and 6-gingerol (6-Gi), were detected by HPLC as the represent constituents in samples. Eight ingredients except Ru in samples could be detected, but Ev could not be detected in high concentration samples. The ratios between absorption ingredients were different from in Wuzhuyu decotion. The in vitro-everted intestinal sac canc absorb the ingredients of Wuzhuyu decotion selectivity. Compare with the ileum, the jejunum can provide the more absorption information and faster, the best test time is 60-90 min.

Eukaryotic ribosome display with in situ DNA recovery.

PubMed

He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Automated analysis of clonal cancer cells by intravital imaging

PubMed Central

Coffey, Sarah Earley; Giedt, Randy J; Weissleder, Ralph

2013-01-01

Longitudinal analyses of single cell lineages over prolonged periods have been challenging particularly in processes characterized by high cell turn-over such as inflammation, proliferation, or cancer. RGB marking has emerged as an elegant approach for enabling such investigations. However, methods for automated image analysis continue to be lacking. Here, to address this, we created a number of different multicolored poly- and monoclonal cancer cell lines for in vitro and in vivo use. To classify these cells in large scale data sets, we subsequently developed and tested an automated algorithm based on hue selection. Our results showed that this method allows accurate analyses at a fraction of the computational time required by more complex color classification methods. Moreover, the methodology should be broadly applicable to both in vitro and in vivo analyses. PMID:24349895

In vitro motility evaluation of aggregated cancer cells by means of automatic image processing.

PubMed

De Hauwer, C; Darro, F; Camby, I; Kiss, R; Van Ham, P; Decaesteker, C

1999-05-01

Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

miRNA studies in in vitro and in vivo activated hepatic stellate cells

PubMed Central

Maubach, Gunter; Lim, Michelle Chin Chia; Chen, Jinmiao; Yang, Henry; Zhuo, Lang

2011-01-01

AIM: To understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation. METHODS: We used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated. RESULTS: We found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs. Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type I transcription. CONCLUSION: Our results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA. PMID:21734783

A review study on the effect of Iranian herbal medicines against in vitro replication of herpes simplex virus

PubMed Central

Moradi, Mohammad-Taghi; Rafieian-Kopaei, Mahmoud; Karimi, Ali

2016-01-01

Objective: There are a number of published data indicating in vitro anti-HSV activity of some of Iranian herbal extracts with no systematic review to discuss these results. Therefore, this article was aimed to review and discuss the methods carried out and the phytochemistry and bioactivity of the extracts used and also conclusions provided in these publications. Materials and Methods: Published articles both in English (from Medline, Science Direct, EMBASE, Scopus, Pro Quest, Google scholar, Cochrane Library) and in Persian (from SID, Iran Medex and Magiran) databases, from 1966 to October 2014 were incorporated in this review. The in vitro studies that lacked CC50, IC50, were excluded. Results: Only 42 published reports were found to examine Iranian herbs against HSV replication in vitro. Seventeen out of 42 studies in which 23 kinds of medicinal plants were subjected to crude extraction were included. The review of data showed that some of the herbal extracts including Hyssopus officinalis methanolic extract, Melissa officinalis aqueous extract, Quercus persica L. hydroalcoholic extract and Securigeras ecuridaca methanolic extract with selective index (SI) of 234, 877, >778 and 250, respectively were highly effective against HSV in vitro. Conclusion: More comprehensive studies using more advanced methods are needed to be done to achieve promising anti-HSV agents from the bioactive compounds isolated from these herbs. PMID:27761420

Rapidly-disintegrating sublingual tablets of epinephrine: role of non-medicinal ingredients in formulation development.

PubMed

Rachid, Ousama; Rawas-Qalaji, Mutasem; Simons, F Estelle R; Simons, Keith J

2012-11-01

Epinephrine is the drug of choice in the management of anaphylaxis. For first-aid treatment in the community, epinephrine autoinjectors (E-autos) are commonly prescribed, but are underutilized. In our laboratory, we developed a series of first-generation rapidly-disintegrating sublingual tablets (RDSTs) containing 40mg of epinephrine. One RDST had similar bioavailability to epinephrine 0.3mg from an auto-injector, as confirmed in a validated rabbit model, while other formulations containing different non-medicinal ingredients (NMIs) and with similar in vitro characteristics demonstrated much lower bioavailability. Subsequently, we evaluated the effect of changing the grade and proportion of NMIs, specifically mannitol and microcrystalline cellulose (MCC), on the in vitro characteristics of second- and third-generation RDSTs. Weight variation, content uniformity, breaking force, and friability were tested using official USP methods. Novel validated methods that simulate ambient conditions of the sublingual cavity were developed to test disintegration time, wetting time, and dissolution. Using these methods, it was possible to measure the effects of making small changes in NMIs on the in vitro characteristics of the formulations. The RDST formulation that resulted in the best in vitro characteristics contained the optimum proportion of mannitol and a specific ratio of coarse and fine particle grades of MCC. Appropriate comparative testing resulted in the selection of the RDST with the optimum in vitro characteristics. Copyright © 2012 Elsevier B.V. All rights reserved.

An In Vitro Comparison of Propex II Apex Locator to Standard Radiographic Method

PubMed Central

Chakravarthy Pishipati, Kalyan Vinayak

2013-01-01

Introduction The aim of this in vitro study was to compare the accuracy of radiography in assessing working length to Propex II apex locator. Materials and Methods Thirty single canal extracted human teeth with patent apical foramen were selected. Access cavities were prepared. Anatomic length (AL) was determined by inserting a K-file into the root canal until the file tip was just visible at the most coronal aspect of the apical foramen; subsequently 0.5 mm was deducted from this measured length. Working length by radiographic method (RL) was determined using Ingle’s method. Propex II apex locator was used to determine the electronic working length (EL). From these calculated lengths, AL was deducted to obtain D-value. D-value in the range of +/-0.5 mm was considered to be acceptable. Results The percentage accuracy of RL and Propex II apex locator was 76.6% and 86.6%, respectively. Paired t-test revealed significant difference between the RL and Propex II apex locator (P<0.05). Conclusion: Under these in vitro conditions, Propex II apex locator has determined working length more accurately than radiographic method. PMID:23922572

Evaluation of an in vitro cell assay to select attenuated bacterial mutants of Aeromonas hydrophila and Edwardsiella tarda to channel catfish

USDA-ARS?s Scientific Manuscript database

To evaluate the feasibility of using an in vitro cell assay to select attenuated bacterial mutants. Using catfish gill cells G1B, the feasibility of using an in vitro assay instead of in vivo virulence assay using live fish to select attenuated bacterial mutants was evaluated in this study. Pearson ...

Radio-methyl vorozole and methods for making and using the same

DOEpatents

Kim, Sung Won; Biegon, Anat; Fowler, Joanna S.

2014-08-12

Radiotracer vorozole compounds for in vivo and in vitro assaying, studying and imaging cytochrome P450 aromatase enzymes in humans, animals, and tissues and methods for making and using the same are provided. [N-radio-methyl] vorozole substantially separated from an N-3 radio-methyl isomer of vorozole is provided. Separation is accomplished through use of chromatography resins providing multiple mechanisms of selectivity.

Radio-methyl vorozole and methods for making and using the same

DOEpatents

Kim, Sung Won; Biegon, Anat; Fowler, Joanna S.

2014-08-05

Radiotracer vorozole compounds for in vivo and in vitro assaying, studying and imaging cytochrome P450 aromatase enzymes in humans, animals, and tissues and methods for making and using the same are provided. [N-radio-methyl] vorozole substantially separated from an N-3 radio-methyl isomer of vorozole is provided. Separation is accomplished through use of chromatography resins providing multiple mechanisms of selectivity.

[Multiresistant Pseudomonas spp. in vitro susceptibility to a combination of two antibiotics].

PubMed

Pliego-Castañeda, Q F B Amanda; Yánez-Viguri, Jorge Antonio; López-Valle, Tiburcio

2005-01-01

In vitro antibiotic combination testing would guide therapy selection in patients severely affected by multi-drug resistant Pseudomonas. In vitro, a two-antibiotic combination susceptible against multi-drug resistant Pseudomonas isolated at the Laboratorio Clínico of the Hospital de Oncología, Centro Médico Nacional Siglo XXI in Mexico City were analyzed to determine which antibiotic combination showed the best bactericidal activity. During 10 months, 30 multi-drug resistant Pseudomonas strains were tested. An automated method was used, including a diluting solution with a well-known concentration of a second antibiotic. Quality controls recommended by the NCCLS were used. Pseudomonas aeruginosa ATCC 27853; Escherichia coli ATCC 25922; and Escherichia coli ATCC 35218. Combinations were betalactamics-aminoglycosides; carbapenemis-amikacin; fluoroquinolones-cefepime; and ciprofloxacin-ampicillin. Ampicillin-ciprofloxacin combination was bactericidal against 100% of the isolates. Cefazolin, cefixime and ticarcillin with amikacin: <50%; aztreonam, cefoxilin, cefuroxime, cefotaxime, ceftazidime and piperacillin with amikacin: 50-60%; cefepime with gentamicin: 76%; cefepime with amikacin: 86%; imipenem and meropenem with amikacin: 70% and 76%; cefepime with ciprofloxacin: 83%; cefepime with levofloxacin: 73%. In vitro antibiotic combination susceptibilities against multi-drug resistant bacteria would be the only way to guide clinicians to select the best therapy in severe infections. We found that the ampicillin-ciprofloxacin combination showed the best in vitro effect against multi-drug resistant Pseudomonas.

Comparison of normal and abnormal fertilization of in vitro-matured human oocyte according to insemination method.

PubMed

Park, Ju Hee; Jee, Byung Chul; Kim, Seok Hyun

2016-04-01

Our purpose was to compare the normal fertilization rate, multi-pronuclei (PN) formation rate, and embryonic development of in vitro-matured oocytes between conventional insemination and intracytoplasmic sperm injection (ICSI). A total of 213 stimulated in vitro fertilization (IVF) cycles were selected, in which at least one immature oocyte was obtained (from 2010 to 2014). Immature oocytes were assigned to germinal vesicle (GV)-stage or metaphase I (MI)-stage oocyte groups. Cycles with obligatory ICSI due to male-factor infertility were excluded. Cycles were divided into two groups according to fertilization method: there were 97 cycles with conventional insemination and 116 cycles with ICSI. After in vitro maturation of 324 GV-stage oocytes and 341 MI-stage oocytes, the fertilization rate, multi-PN formation rate, and embryonic development were compared according to the fertilization method. The normal fertilization rate was similar in the conventional insemination and the ICSI both in GV-derived and MI-derived oocytes. Both fertilization methods resulted in a similar multi-PN formation rate in GV-derived oocytes; however, in MI-derived oocytes, the multi-PN formation rate was zero with ICSI and this was significantly lower than that with conventional insemination (9.6%, P = 0.001). In non-male-factor infertility, ICSI should be considered when MI oocytes are matured. © 2016 Japan Society of Obstetrics and Gynecology.

Circulating tumor cell isolation: the assets of filtration methods with polycarbonate track-etched filters

PubMed Central

Dolfus, Claire; Piton, Nicolas; Toure, Emmanuel

2015-01-01

Circulating tumor cells (CTCs) arise from primary or secondary tumors and enter the bloodstream by active or passive intravasation. Given the low number of CTCs, enrichment is necessary for detection. Filtration methods are based on selection of CTCs by size using a filter with 6.5 to 8 µm pores. After coloration, collected CTCs are evaluated according to morphological criteria. Immunophenotyping and fluorescence in situ hybridization techniques may be used. Selected CTCs can also be cultivated in vitro to provide more material. Analysis of genomic mutations is difficult because it requires adapted techniques due to limited DNA materials. Filtration-selected CTCs have shown prognostic value in many studies but multicentric validating trials are mandatory to strengthen this assessment. Other clinical applications are promising such as follow-up, therapy response prediction and diagnosis. Microfluidic emerging systems could optimize filtration-selected CTCs by increasing selection accuracy. PMID:26543334

Effect of processing on the in vitro and in vivo protein quality of red and green lentils (Lens culinaris).

PubMed

Nosworthy, Matthew G; Medina, Gerardo; Franczyk, Adam J; Neufeld, Jason; Appah, Paulyn; Utioh, Alphonsus; Frohlich, Peter; House, James D

2018-02-01

In order to determine the effect of extrusion, baking and cooking on the protein quality of red and green lentils, a rodent bioassay was conducted and compared to an in vitro method of protein quality determination. On average, the Protein Digestibility-Corrected Amino Acid Score of red lentils (55.0) was higher than that of green lentils (50.8). Extruded lentil flour had higher scores (63.01 red, 57.09 green) than either cooked (57.40 red, 52.92 green) or baked (53.84 red, 47.14 green) flours. The average Digestible Indispensable Amino Acid Score of red lentils (0.54) was higher than green lentils (0.49). The Protein Efficiency Ratio of the extruded lentil flours (1.30 red, 1.34 green) was higher than that of the baked flour (0.98 red, 1.09 green). A correlation was found between in vivo and in vitro methods of determining protein digestibility (R 2 =0.8934). This work could influence selection of processing method during product development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Labeled ALPHA4BETA2 ligands and methods therefor

DOEpatents

Mukherjee, Jogeshwar; Pichika, Ramaiah; Potkin, Steven; Leslie, Frances; Chattopadhyay, Sankha

2013-02-19

Contemplated compositions and methods are employed to bind in vitro and in vivo to an .alpha.4.beta.2 nicotinic acetylcholine receptor in a highly selective manner. Where such compounds are labeled, compositions and methods employing such compounds can be used for PET and SPECT analysis. Alternatively, and/or additionally contemplated compounds can be used as antagonists, partial agonists or agonists in the treatment of diseases or conditions associated with .alpha.4.beta..beta.2 dysfunction.

Next-Generation Sequencing of Antibody Display Repertoires

PubMed Central

Rouet, Romain; Jackson, Katherine J. L.; Langley, David B.; Christ, Daniel

2018-01-01

In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation. PMID:29472918

CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals.

PubMed

Adriaens, E; Alépée, N; Kandarova, H; Drzewieckac, A; Gruszka, K; Guest, R; Willoughby, J A; Verstraelen, S; Van Rompay, A R

2017-10-01

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reprint of "CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals".

PubMed

Adriaens, E; Alépée, N; Kandarova, H; Drzewieckac, A; Gruszka, K; Guest, R; Willoughby, J A; Verstraelen, S; Van Rompay, A R

2018-06-01

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals. Copyright © 2018. Published by Elsevier Ltd.

Informing Selection of Nanomaterial Concentrations for ToxCast In Vitro Testing using the Multi-Path Particle Dosimetry Model

EPA Science Inventory

Currently, little justification is provided for nanomaterial testing concentrations in in vitro assays. The in vitro concentrations typically used may be higher than those experienced in exposed humans. Selection of concentration levels for hazard evaluation based on real-world ...

Factors Affecting Leaf Selection by Foregut-fermenting Proboscis Monkeys: New Insight from in vitro Digestibility and Toughness of Leaves

PubMed Central

Matsuda, Ikki; Clauss, Marcus; Tuuga, Augustine; Sugau, John; Hanya, Goro; Yumoto, Takakazu; Bernard, Henry; Hummel, Jürgen

2017-01-01

Free-living animals must make dietary choices in terms of chemical and physical properties, depending on their digestive physiology and availability of food resources. Here we comprehensively evaluated the dietary choices of proboscis monkeys (Nasalis larvatus) consuming young leaves. We analysed the data for leaf toughness and digestibility measured by an in vitro gas production method, in addition to previously reported data on nutrient composition. Leaf toughness, in general, negatively correlated with the crude protein content, one of the most important nutritional factors affecting food selection by leaf-eating primates. This result suggests that leaf toughness assessed by oral sensation might be a proximate cue for its protein content. We confirmed the importance of the leaf chemical properties in terms of preference shown by N. larvatus; leaves with high protein content and low neutral detergent fibre levels were preferred to those of the common plant species. We also found that these preferred leaves were less tough and more digestible than the alternatives. Our in vitro results also suggested that N. larvatus were little affected by secondary plant compounds. However, the spatial distribution pattern of plant species was the strongest factor explaining the selection of the preferred leaf species. PMID:28211530

Method For Determining And Modifying Protein/Peptide Solubilty

DOEpatents

Waldo, Geoffrey S.

2005-03-15

A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

Deducing the Kinetics of Protein Synthesis In Vivo from the Transition Rates Measured In Vitro

PubMed Central

Rudorf, Sophia; Thommen, Michael; Rodnina, Marina V.; Lipowsky, Reinhard

2014-01-01

The molecular machinery of life relies on complex multistep processes that involve numerous individual transitions, such as molecular association and dissociation steps, chemical reactions, and mechanical movements. The corresponding transition rates can be typically measured in vitro but not in vivo. Here, we develop a general method to deduce the in-vivo rates from their in-vitro values. The method has two basic components. First, we introduce the kinetic distance, a new concept by which we can quantitatively compare the kinetics of a multistep process in different environments. The kinetic distance depends logarithmically on the transition rates and can be interpreted in terms of the underlying free energy barriers. Second, we minimize the kinetic distance between the in-vitro and the in-vivo process, imposing the constraint that the deduced rates reproduce a known global property such as the overall in-vivo speed. In order to demonstrate the predictive power of our method, we apply it to protein synthesis by ribosomes, a key process of gene expression. We describe the latter process by a codon-specific Markov model with three reaction pathways, corresponding to the initial binding of cognate, near-cognate, and non-cognate tRNA, for which we determine all individual transition rates in vitro. We then predict the in-vivo rates by the constrained minimization procedure and validate these rates by three independent sets of in-vivo data, obtained for codon-dependent translation speeds, codon-specific translation dynamics, and missense error frequencies. In all cases, we find good agreement between theory and experiment without adjusting any fit parameter. The deduced in-vivo rates lead to smaller error frequencies than the known in-vitro rates, primarily by an improved initial selection of tRNA. The method introduced here is relatively simple from a computational point of view and can be applied to any biomolecular process, for which we have detailed information about the in-vitro kinetics. PMID:25358034

In vitro selection of catalytic RNAs

NASA Technical Reports Server (NTRS)

Chapman, K. B.; Szostak, J. W.

1994-01-01

In vitro selection techniques are poised to allow a rapid expansion of the study of catalysis by RNA enzymes (ribozymes). This truly molecular version of genetics has already been applied to the study of the structures of known ribozymes and to the tailoring of their catalytic activity to meet specific requirements of substrate specificity or reaction conditions. During the past year, in vitro selection has been successfully used to isolate novel RNA catalysts from random sequence pools.

Development of a HPLC method for determination of four UV filters in sunscreen and its application to skin penetration studies.

PubMed

Souza, Carla; Maia Campos, Patrícia M B G

2017-12-01

This study describes the development, validation and application of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the in vitro skin penetration profile of four UV filters on porcine skin. Experiments were carried out on a gel-cream formulation containing the following UV filters: diethylamino hydroxybenzoyl hexyl benzoate (DHHB), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT) and ethylhexyl triazone (EHT). The HPLC method demonstrated suitable selectivity, linearity (10.0-50.0 μg/mL), precision, accuracy and recovery from porcine skin and sunscreen formulation. The in vitro skin penetration profile was evaluated using Franz vertical diffusion cells for 24 h after application on porcine ear skin. None of the UV filters penetrated the porcine skin. Most of them stayed on the skin surface (>90%) and only BEMT, EHT and DHHB reached the dermis plus epidermis layer. These results are in agreement with previous results in the literature. Therefore, the analytical method was useful to evaluate the in vitro skin penetration of the UV filters and may help the development of safer and effective sunscreen products. Copyright © 2017 John Wiley & Sons, Ltd.

Construction of human antibody gene libraries and selection of antibodies by phage display.

PubMed

Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

2014-01-01

Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

PubMed

Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T

2017-03-17

Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus).

PubMed

Martín-Coello, J; González, R; Crespo, C; Gomendio, M; Roldan, E R S

2008-10-01

Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm-oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5 IU pregnant mare's serum gonadotrophin (PMSG) and 5 IU human chorionic gonadotrophin (hCG) were injected 48 h apart, and oocytes collected 14 h post-hCG, good responses were obtained in Mus musculus (18+/-1.3 oocytes/female; mean+/-S.E.M.) and Mus spretus (12+/-0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19+/-1.2) and M. spretus (12+/-1.1) but not in M. spicilegus (5+/-0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48 h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23+/-0.9; M. spretus, 17+/-1.1; M. spicilegus, 10+/-0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83-87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

PubMed

Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

2014-12-10

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

PubMed

Tanigawa, Shunsuke; Taguchi, Atsuhiro; Sharma, Nirmala; Perantoni, Alan O; Nishinakamura, Ryuichi

2016-04-26

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Antimalarial evaluation of selected medicinal plant extracts used in Iranian traditional medicine

PubMed Central

Haddad, Mohammad Hossein Feiz; Mahbodfar, Hamidreza; Zamani, Zahra; Ramazani, Ali

2017-01-01

Objective(s): In an attempt to discover new natural active extracts against malaria parasites, the present study evaluated the antiplasmodial properties of selected plants based on Iranian traditional medicine. Materials and Methods: Ten plant species found in Iran were selected and collected based on the available literature about the Iranian traditional medicine. The methanolic extracts of these plants were investigated for in vitro antimalarial properties against chloroquine-sensitive (3D7) and multi-drug resistant (K1) strains of Plasmodium falciparum. Their in vivo activity against Plasmodium berghei infection in mice was also determined. Cytotoxicity tests were carried out using the Raji cells line using the MTT assay. The extracts were phytochemically screened for their active constituents. Results: According to the IC50 and selectivity index (SI) values, of the 10 selected plant species, Citrullus colocynthis, Physalis alkekengi, and Solanum nigrum displayed potent in vitro antimalarial activity against both 3D7 and K1 strains with no toxicity (IC50= 2.01-18.67 µg/ml and SI=3.55 to 19.25). Comparisons between treated and untreated control mice showed that the mentioned plant species reduced parasitemia by 65.08%, 57.97%, and 60.68%, respectively. The existence of antiplasmodial compounds was detected in these plant extracts. Conclusion: This was the first study to highlight the in vitro and in vivo antiplasmodial effects of C. colocynthis, P. alkekengi, and S. nigrum in Iran. Future studies can use these findings to design further biological tests to identify the active constituents of the mentioned plant species and clarify their mechanism of action. PMID:28804611

In vitro test systems supporting the development of improved pest control methods: a case study with chemical mixtures and bivalve biofoulers.

PubMed

Silva, Carlos; Nunes, Bruno; Nogueira, António Ja; Gonçalves, Fernando; Pereira, Joana L

2016-11-01

Using the bivalve macrofouler Corbicula fluminea, the suitability of in vitro testing as a stepping stone towards the improvement of control methods based on chemical mixtures was addressed in this study. In vitro cholinesterase (ChE) activity inhibition following single exposure of C. fluminea tissue to four model chemicals (the organophosphates dimethoate and dichlorvos, copper and sodium dodecyl phosphate [SDS]) was first assessed. Consequently, mixtures of dimethoate with copper and dichlorvos with SDS were tested and modelled; mixtures with ChE revealed synergistic interactions for both chemical pairs. These synergic combinations were subsequently validated in vivo and the increased control potential of these selected combinations was verified, with gains of up to 50% in C. fluminea mortality relative to corresponding single chemical treatments. Such consistency supports the suitability of using time- and cost-effective surrogate testing platforms to assist the development of biofouling control strategies incorporating mixtures.

In Vitro Antioxidant Activity of Selected 4-Hydroxy-chromene-2-one Derivatives—SAR, QSAR and DFT Studies

PubMed Central

Mladenović, Milan; Mihailović, Mirjana; Bogojević, Desanka; Matić, Sanja; Nićiforović, Neda; Mihailović, Vladimir; Vuković, Nenad; Sukdolak, Slobodan; Solujić, Slavica

2011-01-01

The series of fifteen synthesized 4-hydroxycoumarin derivatives was subjected to antioxidant activity evaluation in vitro, through total antioxidant capacity, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), hydroxyl radical, lipid peroxide scavenging and chelating activity. The highest activity was detected during the radicals scavenging, with 2b, 6b, 2c, and 4c noticed as the most active. The antioxidant activity was further quantified by the quantitative structure-activity relationships (QSAR) studies. For this purpose, the structures were optimized using Paramethric Method 6 (PM6) semi-empirical and Density Functional Theory (DFT) B3LYP methods. Bond dissociation enthalpies of coumarin 4-OH, Natural Bond Orbital (NBO) gained hybridization of the oxygen, acidity of the hydrogen atom and various molecular descriptors obtained, were correlated with biological activity, after which we designed 20 new antioxidant structures, using the most favorable structural motifs, with much improved predicted activity in vitro. PMID:21686153

Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies.

PubMed

Peippo, Jaana; Viitala, Sirja; Virta, Jouni; Räty, Mervi; Tammiranta, Niina; Lamminen, Terttu; Aro, Johanna; Myllymäki, Hannu; Vilkki, Johanna

2007-11-01

We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy. (c) 2007 Wiley-Liss, Inc.

Level A in vitro-in vivo correlation: Application to establish a dissolution test for artemether and lumefantrine tablets.

PubMed

Rivelli, Graziella Gomes; Ricoy, Letícia Brandão Magalhães; César, Isabela Costa; Fernandes, Christian; Pianetti, Gérson Antônio

2018-06-05

Malaria is the most incident parasite infection worldwide. Artemisinin based combination therapy (ACT) has been proposed as a promising treatment for malaria, and artemether + lumefantrine (20 + 120 mg) is the recommended association in endemic areas. Despite its widespread use, there is still scarce information about dissolution of artemether and lumefantrine, reflecting in the absence of a specific method in pharmacopoeias and international compendia. Because the of their low solubility, both artemether and lumefantrine are candidates for in vitro-in vivo correlation (IVIVC) studies. Previous equilibrium solubility studies have been carried out for both drugs using the shake-flask method and dissolution profiles. Experiments were conducted with a range of parameters such as medium composition, pH and surfactants. In vivo data obtained in a previous pharmacokinetic study was used to select the optimum conditions for dissolution test, based on IVIVC. For drug quantitation, a selective method by high performance liquid chromatography was optimized and validated. For this dosage form, the best dissolution conditions found for artemether were: paddles, 900 mL of dissolution medium containing phosphate buffer pH 6.8 with 1.0% sodium lauryl sulfate and rotation speed of 100 rpm. The same was obtained for lumefantrine, except the dissolution medium, which was pH 1.2 with 1.0% polysorbate 80. After obtaining the curve of in vitro dissolved fraction versus in vivo absorbed fraction, the calculated coefficient of determination (R squared) was close to 1.00 for both drugs, indicating a level A correlation. Therefore, a novel method for assessing dissolution of arthemeter and lumefantrine tablets was established and validated. Copyright © 2018 Elsevier B.V. All rights reserved.

Informing Selection of Nanomaterial Concentrations for ToxCast In Vitro Testing based on Occupational Exposure Potential

EPA Science Inventory

Little justification is generally provided for selection of in vitro assay testing concentrations for engineered nanomaterials (ENMs). Selection of concentration levels for hazard evaluation based on real-world exposure scenarios is desirable. We reviewed published ENM concentr...

Nitric oxide selective electrodes.

PubMed

Davies, Ian R; Zhang, Xueji

2008-01-01

Since nitric oxide (NO) was identified as the endothelial-derived relaxing factor in the late 1980s, many approaches have attempted to provide an adequate means for measuring physiological levels of NO. Although several techniques have been successful in achieving this aim, the electrochemical method has proved the only technique that can reliably measure physiological levels of NO in vitro, in vivo, and in real time. We describe here the development of electrochemical sensors for NO, including the fabrication of sensors, the detection principle, calibration, detection limits, selectivity, and response time. Furthermore, we look at the many experimental applications where NO selective electrodes have been successfully used.

In situ electrochemical assessment of cytotoxicity of chlorophenols in MCF-7 and HeLa cells.

PubMed

Qin, Hongwei; Liu, Jiguang; Zhang, Zeshi; Li, Jinlian; Gao, Guanggang; Yang, Yuxin; Yuan, Xing; Wu, Dongmei

2014-10-01

An in situ electrochemical method was used to assess the cytotoxicity of chlorophenols using human breast cancer (MCF-7) and cervical carcinoma (HeLa) cells as models. On treatment with different chlorophenols, the electrochemical responses of the selected cells, resulting from the oxidation of guanine and xanthine in the cytoplasm, indicated the cell viability. In addition, the in situ in vitro electrochemical method was further compared with the traditional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Although similar cytotoxicity data were obtained from both methods, the effective concentrations of chlorophenols that inhibited 50% cell growth (EC50 values) from the electrochemical method were only slightly lower than those from the MTT assay. These results indicate that the in situ in vitro electrochemical method paves a simple, rapid, strongly responsive, and label-free way to the cytotoxicity assessment of different chlorophenol pollutants. Copyright © 2014 Elsevier Inc. All rights reserved.

Microbiological Horticultural Internship Final Abstract

NASA Technical Reports Server (NTRS)

Palmer, Shane R.; Spencer, Lashelle (Editor)

2017-01-01

GMO dwarf plum (Prunus domestica) is being evaluated as a candidate food crop for long duration space flight missions. A project was undertaken to develop a protocol for transferring selected genetic lines of GMO plum (previously maintained in pots and propagated by cuttings at NASA's Kennedy Space Center in Florida) into in vitro tissue culture. In vitro culture may reduce the space, materials, and labor required to maintain the current lines of GMO plum and better preserve them for future study. Fresh plant material from three selected GMO plum lines (NASA-5, NASA-10, and NASA-11) and a non-modified control line (Control-5) were processed aseptically into in vitro culture on four separate occasions. The impact of multiple treatments on the successful growth of GMO plum tissue in vitro were tested: Parent explant tissue type (leaf petioles, stem nodes containing buds and internodes without buds), tissue sterilization method [soaking in 10 bleach only (5 min for petioles or 10 min for nodesinternodes), or soaking in 70 EtOH (30 sec) followed by 10 bleach (5 min for petioles and 10 min for nodesinternodes)], and media type [three Murashige and Skoog-based medias (SGM, SRM, and SRM+2,4-D) and one recipe containing woody plant media (WPM)]. 22.2 of the plates containing tissue sterilized with bleach alone developed microbial contamination after two weeks, while only 11.8 of plates containing tissue sterilized sequentially with EtOH and bleach developed contamination. Node bud tissue from all four genetic lines of plum produced leafy plantlets on SGM and SRM media after 4-6 weeks. The most numerous and well-developed plantlets were present on SGM. Upon reaching suitable size, plantlets were transferred to larger media containers for further growth. Some node bud growth occurred on SRM+2,4-D and WPM 2.5 weeks after plating, however as of yet no pieces on SRM+2,4-D have adequate development for transferring. Tissue pieces from NASA-5 plated on WPM are developing leaves and will be ready for transferring soon. Petioles and internode tissue lacking bud meristem failed to produce any plantlets on any plates, however petioles developed large masses of undifferentiated callus tissue on SRM+2,4-D media. These callused pieces were then transferred to SRM+TDZ media, which resulted in even larger callus growth but no differentiation. All four selected plum lines were successfully transitioned into in vitro culture. Nodes from NASA-5 and NASA-10 lines produced the most numerous and well-developed leafy plantlets in vitro, while those from NASA-11 and Control-5 were generally smaller, slower growing and less numerous. The best method overall was to use young stem node tissue with buds, surface sterilize the pieces sequentially with 70 EtOH and 10 bleach, and then plate them onto SGM media. Future areas of study will include introducing additional genetic lines of GMO plum into in vitro culture, attempting to induce shoot growth in petiole callus tissue, testing methods (such as cold storage) that extend the time interval between transferring explants into new media, and testing viability of plantlets transferred from in vitro culture back to traditional pot culture.

Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method.

PubMed

Kitamura, Koichiro; Komatsu, Masayuki; Biyani, Madhu; Futakami, Masae; Kawakubo, Tomoyo; Yamamoto, Kenji; Nishigaki, Koichi

2012-12-01

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Error baseline rates of five sample preparation methods used to characterize RNA virus populations.

PubMed

Kugelman, Jeffrey R; Wiley, Michael R; Nagle, Elyse R; Reyes, Daniel; Pfeffer, Brad P; Kuhn, Jens H; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

2017-01-01

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequence-independent single-primer amplification (SISPA) as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5) of all compared methods.

Error baseline rates of five sample preparation methods used to characterize RNA virus populations

PubMed Central

Kugelman, Jeffrey R.; Wiley, Michael R.; Nagle, Elyse R.; Reyes, Daniel; Pfeffer, Brad P.; Kuhn, Jens H.; Sanchez-Lockhart, Mariano; Palacios, Gustavo F.

2017-01-01

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic “no amplification” method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a “targeted” amplification method, sequence-independent single-primer amplification (SISPA) as a “random” amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced “no amplification” method, and Illumina TruSeq RNA Access as a “targeted” enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4−5) of all compared methods. PMID:28182717

Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

NASA Technical Reports Server (NTRS)

Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

Inhibitory effects of Agaricus blazei extracts on human myeloid leukemia cells.

PubMed

Kim, Chi-Fai; Jiang, Jing-Jing; Leung, Kwok-Nam; Fung, Kwok-Pui; Lau, Clara Bik-San

2009-03-18

Agaricus blazei has been used as an adjuvant in cancer chemotherapy and is found to inhibit the growth of various types of tumor cells. Our study has adopted a systematic and bioassay-guided approach to optimize the extraction of Agaricus blazei for anti-leukemic bioactive components. The tumor-selective growth inhibitory activity of the extracts on leukemic cell lines was evaluated in vitro and in vivo using tumor-bearing nude mice. Agaricus blazei extracts were prepared using different methods. MTT and tritiated thymidine incorporation assays were used to evaluate the in vitro anti-leukemic effects. The most potent extract was further investigated using NB-4 cells-bearing nude mice and mechanistic studies using DNA fragmentation assay and cell death detection ELISA. The JAB80E70 extract showed the most potent tumor-selective growth inhibitory activity against human leukemia NB-4 and K-562 cells. This is the first report of anti-leukemic activity of JAB80E70 in athymic nude mice bearing NB-4 cells. Using DNA fragmentation assays and cell death detection ELISA, JAB80E70 was found to induce apoptosis in NB-4 cells. However, the polysaccharide enriched fractions failed to show significant cytotoxicity on NB-4 cells in vitro. The JAB80E70 extract exhibited potent anti-leukemic effect in vitro and in vivo. The effect can be attributed, at least in part, to the induction of apoptosis. Besides, polysaccharides in Agaricus blazei may not possess direct anti-leukemic activity in vitro.

Evaluation of accuracy of shade selection using two spectrophotometer systems: Vita Easyshade and Degudent Shadepilot

PubMed Central

Kalantari, Mohammad Hassan; Ghoraishian, Seyed Ahmad; Mohaghegh, Mina

2017-01-01

Objective: The aim of this in vitro study was to evaluate the accuracy of shade matching using two spectrophotometric devices. Materials and Methods: Thirteen patients who require a full coverage restoration for one of their maxillary central incisors were selected while the adjacent central incisor was intact. 3 same frameworks were constructed for each tooth using computer-aided design and computer-aided manufacturing technology. Shade matching was performed using Vita Easyshade spectrophotometer, Shadepilot spectrophotometer, and Vitapan classical shade guide for the first, second, and third crown subsequently. After application, firing, and glazing of the porcelain, the color was evaluated and scored by five inspectors. Results: Both spectrophotometric systems showed significantly better results than visual method (P < 0.05) while there were no significant differences between Vita Easyshade and Shadepilot spectrophotometers (P < 0.05). Conclusion: Spectrophotometers are a good substitute for visual color selection methods. PMID:28729792

Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.

PubMed

Prasad, Bhagwat; Unadkat, Jashvant D

2014-07-01

Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.

Circular Samples as Objects for Magnetic Resonance Imaging - Mathematical Simulation, Experimental Results

NASA Astrophysics Data System (ADS)

Frollo, Ivan; Krafčík, Andrej; Andris, Peter; Přibil, Jiří; Dermek, Tomáš

2015-12-01

Circular samples are the frequent objects of "in-vitro" investigation using imaging method based on magnetic resonance principles. The goal of our investigation is imaging of thin planar layers without using the slide selection procedure, thus only 2D imaging or imaging of selected layers of samples in circular vessels, eppendorf tubes,.. compulsorily using procedure "slide selection". In spite of that the standard imaging methods was used, some specificity arise when mathematical modeling of these procedure is introduced. In the paper several mathematical models were presented that were compared with real experimental results. Circular magnetic samples were placed into the homogenous magnetic field of a low field imager based on nuclear magnetic resonance. For experimental verification an MRI 0.178 Tesla ESAOTE Opera imager was used.

YSL-12, a novel microtubule-destabilizing agent, exerts potent anti-tumor activity against colon cancer in vitro and in vivo.

PubMed

Cai, De; Qiu, Zhiqing; Yao, Weimin; Liu, Yuyu; Huang, Haixiang; Liao, Sihai; Luo, Qun; Xie, Liming; Lin, Zhixiu

2016-06-01

Microtubules play a central role in various fundamental cell functions and thus become an attractive target for cancer therapy. A novel compound YSL-12 is a combretastatin A-4 (CA-4) analogue with more stability. We investigated its anti-tumor activity and mechanisms in vitro and in vivo for the first time. Cytotoxicity was evaluated by MTT method. In vitro microtubule polymerization assay was performed using a fluorescence-based method by multifunction fluorescence microplate reader. Intracellular microtubule network was detected by immunofluorescence method. Cell cycle analysis and apoptosis were measured by flow cytometry. Metabolic stability was recorded by liquid chromatography-ultraviolet detection and liquid chromatography-mass spectrometry. In vivo anti-tumor activity was assessed using HT-29 colon carcinoma xenografts established in BALB/c nude mice. YSL-12 displayed nanomolar-level cytotoxicity against various human cancer cell lines. A high selectivity toward normal cells and potent activity toward drug-resistant cells were also observed. YSL-12 was identified as tubulin polymerization inhibitor evidenced by effectively inhibits tubulin polymerization and heavily disrupted microtubule networks in living HT-29 cells. YSL-12 displayed potent disruption effect of pre-established tumor vasculature in vitro. In addition, YSL-12 treatment also caused cell cycle arrest in the G2/M phase and induced cell apoptosis in a dose-dependent manner. In vitro metabolic stability study revealed YSL-12 displayed considerable better stability than CA-4 in liver microsomes. In vivo, YSL-12 delayed tumor growth with 69.4 % growth inhibition. YSL-12 is a promising microtubule inhibitor that has great potential for the treatment of colon carcinoma in vitro and in vivo and worth being a candidate for further development of cancer therapy.

Development of achiral and chiral 2D HPLC methods for analysis of albendazole metabolites in microsomal fractions using multivariate analysis for the in vitro metabolism.

PubMed

Belaz, Kátia Roberta A; Pereira-Filho, Edenir Rodrigues; Oliveira, Regina V

2013-08-01

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral-chiral and chiral-chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0×0.46cmI.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0×0.46cmI.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Systematic wavelength selection for improved multivariate spectral analysis

DOEpatents

Thomas, Edward V.; Robinson, Mark R.; Haaland, David M.

1995-01-01

Methods and apparatus for determining in a biological material one or more unknown values of at least one known characteristic (e.g. the concentration of an analyte such as glucose in blood or the concentration of one or more blood gas parameters) with a model based on a set of samples with known values of the known characteristics and a multivariate algorithm using several wavelength subsets. The method includes selecting multiple wavelength subsets, from the electromagnetic spectral region appropriate for determining the known characteristic, for use by an algorithm wherein the selection of wavelength subsets improves the model's fitness of the determination for the unknown values of the known characteristic. The selection process utilizes multivariate search methods that select both predictive and synergistic wavelengths within the range of wavelengths utilized. The fitness of the wavelength subsets is determined by the fitness function F=.function.(cost, performance). The method includes the steps of: (1) using one or more applications of a genetic algorithm to produce one or more count spectra, with multiple count spectra then combined to produce a combined count spectrum; (2) smoothing the count spectrum; (3) selecting a threshold count from a count spectrum to select these wavelength subsets which optimize the fitness function; and (4) eliminating a portion of the selected wavelength subsets. The determination of the unknown values can be made: (1) noninvasively and in vivo; (2) invasively and in vivo; or (3) in vitro.

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

[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.

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

PubMed Central

Appel, David I.; Brinda, Bryan; Markowitz, John S.; Newcorn, Jeffrey H.; Zhu, Hao-Jie

2012-01-01

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography- tandem mass spectrometry (LC-MS/MS) was developed. This assay represents the first LC-MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3-atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/ml and 10 nM for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3 ng/ml to 900 ng/ml and 10 nM to 10 μM for human plasma and cellular samples, respectively (r2 > 0.999). The intra- and inter-day assay accuracy and precision were evaluated using quality control samples at 3 different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect, and recovery were also successfully demonstrated. The present assay is superior to previously published LC-MS and LC-MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. PMID:22275222

Systematic Evaluation of In Vitro and In Vivo Adventitious Virus Assays for the Detection of Viral Contamination of Cell Banks and Biological Products1

PubMed Central

Gombold, James; Karakasidis, Stephen; Niksa, Paula; Podczasy, John; Neumann, Kitti; Richardson, James; Sane, Nandini; Johnson-Leva, Renita; Randolph, Valerie; Sadoff, Jerald; Minor, Phillip; Schmidt, Alexander; Duncan, Paul; Sheets, Rebecca L.

2015-01-01

Viral vaccines and the cell substrates used to manufacture them are subjected to tests for adventitious agents, including viruses, which might contaminant them. Some of the compendial methods (in vivo and in vitro in cell culture) were established in the mid-20th century. These methods have not been subjected to current assay validation, as new methods would need to be. This study was undertaken to provide insight into the breadth (selectivity) and sensitivity (limit of detection) of the routine methods, two such validation parameters. Sixteen viral stocks were prepared and characterized. These stocks were tested in serial dilutions by the routine methods to establish which viruses were detected by which methods and above what limit of detection. Sixteen out of sixteen viruses were detected in vitro, though one (bovine viral diarrhea virus) required special conditions to detect and another (rubella virus) was detected with low sensitivity. Many were detected at levels below 1 TCID50 or PFU (titers were established on the production cell line in most cases). In contrast, in vivo, only 6/11 viruses were detected, and 4 of these were detected only at amounts one or more logs above 1 TCID50 or PFU. Only influenza virus and vesicular stomatitis virus were detected at lower amounts in vivo than in vitro. Given the call to reduce, refine, or replace (3 R's) the use of animals in product safety testing and the emergence of new technologies for the detection of viruses, a re-examination of the current adventitious virus testing strategies seems warranted. Suggested pathways forward are offered. PMID:24681273

Stability indicating HPLC-DAD method for analysis of Ketorolac binary and ternary mixtures in eye drops: Quantitative analysis in rabbit aqueous humor.

PubMed

El Yazbi, Fawzy A; Hassan, Ekram M; Khamis, Essam F; Ragab, Marwa A A; Hamdy, Mohamed M A

2017-11-15

Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture (mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were analyzed using a validated HPLC-DAD method. The developed method was suitable for the in vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The analysis in dosage form (eye drops) was a stability indicating one at which drugs were separated from possible degradation products arising from different stress conditions (in vitro analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard and the method was validated according to FDA regulation for analysis in biological fluids. Agilent 5 HC-C18(2) 150×4.6mm was used as stationary phase with a gradient eluting solvent of 20mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64min for PHE, GUF, KTC and CPM, respectively. The method was sensitive and selective to analyze simultaneously the three drugs in presence of possible forced degradation products and dosage form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous humor interferences (analysis in biological fluid), at a single wavelength (261nm). No extraction procedure was required for analysis in aqueous humor. The simplicity of the method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in vitro (in pharmaceutical formulations). Copyright © 2017 Elsevier B.V. All rights reserved.

Selectivity Mechanism of ATP-Competitive Inhibitors for PKB and PKA.

PubMed

Wu, Ke; Pang, Jingzhi; Song, Dong; Zhu, Ying; Wu, Congwen; Shao, Tianqu; Chen, Haifeng

2015-07-01

Protein kinase B (PKB) acts as a central node on the PI3K kinase pathway. Constitutive activation and overexpression of PKB have been identified to involve in various cancers. However, protein kinase A (PKA) sharing high homology with PKB is essential for metabolic regulation. Therefore, specific targeting on PKB is crucial strategy in drug design and development for antitumor. Here, we had revealed the selectivity mechanism for PKB inhibitors with molecular dynamics simulation and 3D-QSAR methods. Selective inhibitors of PKB could form more hydrogen bonds and hydrophobic contacts with PKB than those with PKA. This could explain that selective inhibitor M128 is more potent to PKB than to PKA. Then, 3D-QSAR models were constructed for these selective inhibitors and evaluated by test set compounds. 3D-QSAR model comparison of PKB inhibitors and PKA inhibitors reveals possible methods to improve the selectivity of inhibitors. These models can be used to design new chemical entities and make quantitative prediction of the specific selective inhibitors before resorting to in vitro and in vivo experiment. © 2014 John Wiley & Sons A/S.

Selective and self-guided micro-ablation of tissue with plasmonic nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Koneva, Irina I.; Oginsky, Alexander O.; La Francesca, Saverio; Lapotko, Dmitri O.

2010-01-01

Background The accuracy, selectivity and safety of surgical and laser methods for tissue elimination are often limited at microscale. Materials and methods We developed a novel agent, the plasmonic nanobubble (PNB), for optically guided selective elimination of the target tissue with micrometer precision. PNBs were tested in vitro in the two different models of superficial tumors and vascular plaques. Results PNBs were selectively generated around gold nanoparticles (delivered to the target tissues) with short laser pulses. Monolayers of cancerous cells and atherosclerotic plaque tissue were eliminated with PNBs with micrometer accuracy and without thermal and mechanical damage to collateral normal tissues. The effect of the PNB was dynamically controlled through the fluence of laser pulses (532 nm, duration 0.5 and 10 ns) and was guided through the optical scattering by PNB. Conclusions plasmonic nanobubbles were shown to provide precise, tunable, selective and guided ablation of tissue at a microcscopic level and could be employed as a new generation of surgical tools. PMID:21176913

Arisugacins A and B, novel and selective acetylcholinesterase inhibitors from Penicillium sp. FO-4259. I. Screening, taxonomy, fermentation, isolation and biological activity.

PubMed

Kuno, F; Otoguro, K; Shiomi, K; Iwai, Y; Omura, S

1996-08-01

An in vitro screening method for selective acetylcholinesterase (AChE) inhibitors was established. Inhibitory activity of AChE and butyrylcholinesterase (BuChE) was measured and the culture broths of microorganisms that showed selective inhibition against AChE were characterized. By using this method, a strain producing the novel and selective inhibitors of AChE, arisugacins A and B, was picked out among over seven thousand microorganisms tested. Arisugacins were obtained as white powders from the culture broth together with three known compounds, territrems B and C and cyclopenin that also showed selective inhibition against AChE. Arisugacins and territrems are members of the meroterpenoid compounds. They showed potent inhibitory activities against AChE with IC50 values in range of 1.0 approximately 25.8 nM. Furthermore, they showed greater than 2,000-fold more potent inhibition against AChE than BuChE.

Compositions and methods related to serotonin 5-HT1A receptors

DOEpatents

Mukherjee, Jogeshwar; Saigal, Neil

2010-06-08

Contemplated substituted arylpiperazinyl compounds, and most preferably ¹⁸F-Mefway, exhibit desirable in vitro and in vivo binding characteristics to the 5-HT1A receptor. Among other advantageous parameters, contemplated compounds retain high binding affinity, display optimal lipophilicity, and are radiolabeled efficiently with ¹⁸F-fluorine in a single step. Still further, contemplated compounds exhibit high target to non-target ratios in receptor-rich regions both in vitro and in vivo, and selected compounds can be effectively and sensitively displaced by serotonin, thus providing a quantitative tool for measuring 5-HT1A receptors and serotonin concentration changes in the living brain.

Compositions and methods related to serotonin 5-HT1A receptors

DOEpatents

Mukherjee, Jogeshwar [Irvine, CA; Saigal, Neil [Fresno, CA; Saigal, legal representative, Harsh

2012-09-25

Contemplated substituted arylpiperazinyl compounds, and most preferably .sup.18F-Mefway, exhibit desirable in vitro and in vivo binding characteristics to the 5-HT1A receptor. Among other advantageous parameters, contemplated compounds retain high binding affinity, display optimal lipophilicity, and are radiolabeled efficiently with .sup.18F-fluorine in a single step. Still further, contemplated compounds exhibit high target to non-target ratios in receptor-rich regions both in vitro and in vivo, and selected compounds can be effectively and sensitively displaced by serotonin, thus providing a quantitative tool for measuring 5-HT1A receptors and serotonin concentration changes in the living brain.

Compositions and methods related to serotonin 5-HT1A receptors

DOEpatents

Mukherjee, Jogeshwar; Saigal, Neil; Saigal, legal representative, Harsh

2012-09-25

Contemplated substituted arylpiperazinyl compounds, and most preferably ¹⁸F-Mefway, exhibit desirable in vitro and in vivo binding characteristics to the 5-HT1A receptor. Among other advantageous parameters, contemplated compounds retain high binding affinity, display optimal lipophilicity, and are radiolabeled efficiently with ¹⁸F-fluorine in a single step. Still further, contemplated compounds exhibit high target to non-target ratios in receptor-rich regions both in vitro and in vivo, and selected compounds can be effectively and sensitively displaced by serotonin, thus providing a quantitative tool for measuring 5-HT1A receptors and serotonin concentration changes in the living brain.

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.

Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

NASA Astrophysics Data System (ADS)

Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

1999-01-01

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

A novel in vitro whole plant system for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum.

PubMed

Srivastava, Shivani; Cahill, David M; Conlan, Xavier A; Adholeya, Alok

2014-10-15

Plants are an important source for medicinal compounds. Chemical screening and selection is critical for identification of compounds of interest. Ocimum basilicum (Basil) is a rich source of polyphenolics and exhibits high diversity, therefore bioprospecting of a suitable cultivar is a necessity. This study reports on the development of a true to type novel "in vitro system" and its comparison with a conventional system for screening and selection of cultivars for high total phenolics, individual polyphenolics, and antioxidant content. We have shown for the first time using online acidic potassium permanganate chemiluminescence that extracts from Ocimum basilicum showed antioxidant potential. The current study identified the cultivar specific composition of polyphenolics and their antioxidant properties. Further, a distinct relationship between plant morphotype and polyphenolic content was also found. Of the 15 cultivars examined, "Holy Green", "Red Rubin", and "Basil Genovese" were identified as high polyphenolic producing cultivars while "Subja" was determined to be a low producer. The "in vitro system" enabled differentiation of the cultivars in their morphology, polyphenolic content, and antioxidant activity and is a cheap and efficient method for bioprospecting studies.

In Vitro Reconstitution of Metabolic Pathways: Insights into Nature’s Chemical Logic

PubMed Central

Lowry, Brian; Walsh, Christopher T.

2015-01-01

In vitro analysis of metabolic pathways is becoming a powerful method to gain a deeper understanding of Nature’s core biochemical transformations. With astounding advancements in biotechnology, purification of a metabolic pathway’s constitutive enzymatic components is becoming a tractable problem, and such in vitro studies allow scientists to capture the finer details of enzymatic reaction mechanisms, kinetics, and the identity of organic product molecules. In this review, we present eleven metabolic pathways that have been the subject of in vitro reconstitution studies in the literature in recent years. In addition, we have selected and analyzed subset of four case studies within these eleven examples that exemplify remarkable organic chemistry occurring within biology. These examples serves as tangible reminders that Nature’s biochemical routes obey the fundamental principles of organic chemistry, and the chemical mechanisms are reminiscent of those featured in traditional synthetic organic routes. The illustrations of biosynthetic chemistry depicted in this review may inspire the development of biomimetic chemistries via abiotic chemical techniques. PMID:26207083

In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts.

PubMed

Iki, Taichiro; Ishikawa, Masayuki; Yoshikawa, Manabu

2017-01-01

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

Measurement of metabolizable energy in poultry feeds by an in vitro system.

PubMed

Valdes, E V; Leeson, S

1992-09-01

A two-stage in vitro system (IVDE) for estimating AMEn in poultry feeds was investigated. For 71 diets ranging from 2.2 to 3.4 kcal/g, the average AMEn was 2.889 kcal/g and the mean IVDE value was 3.005 kcal/g. From the 71 diets, 30 (42.2%) showed differences between AMEn and IVDE of less than .100 kcal/g and represented diets across the AMEn range of values. The statistical analysis of the data showed a standard error of the estimate (SEE) of .152 kcal/g for the 71 diets assayed. No clear differences in accuracy of AMEn among the diets, as related to the composition and proportion of ingredients, were observed. Thus, the IVDE method gave different AMEn for diets of similar composition. The application of the IVDE system to selected ingredients showed that the AMEn of corn was underestimated by the method. However the AMEn of roasted, extruded soybeans and oats was estimated accurately by the IVDE method. Other ingredients were greatly overestimated by the in vitro technique (soybean meal, corn gluten meal, and barley). The results of applying the IVDE method for estimating AMEn showed the limitations of this technique with regard to the universality of its application. Although the method was successful in estimating AMEn values of diets and ingredients, for many samples the IVDE technique did not give acceptable results.

Diagnosis of coccidioidomycosis by culture: safety considerations, traditional methods, and susceptibility testing.

PubMed

Sutton, Deanna A

2007-09-01

The recovery of Coccidioides spp. by culture and confirmation utilizing the AccuProbe nucleic acid hybridization method by GenProbe remain the definitive diagnostic method. Biosafety considerations from specimen collection through culture confirmation in the mycology laboratory are critical, as acquisition of coccidioidomycosis by laboratory workers is well documented. The designation of Coccidioides spp. as select agents of potential bioterrorism has mandated strict regulation of their transport and inventory. The genus appears generally susceptible, in vitro, although no defined breakpoints exist. Susceptibility testing may assist in documenting treatment failures.

In vitro and in vivo antimalarial and cytotoxic activity of five plants used in congolese traditional medicine.

PubMed

Lusakibanza, M; Mesia, G; Tona, G; Karemere, S; Lukuka, A; Tits, M; Angenot, L; Frédérich, M

2010-06-16

The in vitro antiplasmodial activity and cytotoxicity of methanolic and dichloromethane extracts from five Congolese plants were evaluated. The plants were selected following an ethnobotanical survey conducted in D.R. Congo and focusing on plants used traditionally to treat malaria. The in vivo antimalarial activity of aqueous and methanolic extracts active in vitro was also determined in mice infected by Plasmodium berghei berghei. The growth inhibition of Plasmodium falciparum strains was evaluated using the measurement of lactate dehydrogenase activity. The extracts (aqueous, CH(3)OH, EtOH and CH(2)Cl(2)) were prepared by maceration and tested in vitro against the 3D7 (chloroquine sensitive) and W2 (chloroquine resistant) strains of Plasmodium falciparum and against the human normal fetal lung fibroblasts WI-38 to determine the selectivity index. Some extracts were also used at the dose of 300 mg/kg to evaluate their activity in mice infected since 4 days by Plasmodium berghei. Two plants presented a very high activity (IC(50)<3 microg/ml). These plants were Strychnos icaja roots bark (MeOH and CH(2)Cl(2)) and Physalis angulata leaves (MeOH and CH(2)Cl(2)). One plant (Anisopappus chinensis whole plant, MeOH and CH(2)Cl(2)) presented a high activity (IC50<15 microg/ml). The extracts of Anisopappus chinensis and Physalis angulata showed also a good inhibition of parasitemia in vivo. Flavonoids, phenolic acids and terpenes were identified in these plants by a general phytochemical screening method. Three plants showed a very interesting antiplasmodial activity (Anisopappus chinensis, Physalis angulata and Strychnos icaja) and one of them showed a good selectivity index (>10, Anisopappus chinensis). Anisopappus chinensis and Physalis angulata were also active in vivo. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Analysis of arsenical metabolites in biological samples.

PubMed

Hernandez-Zavala, Araceli; Drobna, Zuzana; Styblo, Miroslav; Thomas, David J

2009-11-01

Quantitation of iAs and its methylated metabolites in biological samples provides dosimetric information needed to understand dose-response relations. Here, methods are described for separation of inorganic and mono-, di-, and trimethylated arsenicals by thin layer chromatography. This method has been extensively used to track the metabolism of the radionuclide [(73)As] in a variety of in vitro assay systems. In addition, a hydride generation-cryotrapping-gas chromatography-atomic absorption spectrometric method is described for the quantitation of arsenicals in biological samples. This method uses pH-selective hydride generation to differentiate among arsenicals containing trivalent or pentavalent arsenic.

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt.

PubMed

Berg, G; Kurze, S; Buchner, A; Wellington, E M; Smalla, K

2000-12-01

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.

Towards Personalized Medicine Mediated by in Vitro Virus-Based Interactome Approaches

PubMed Central

Ohashi, Hiroyuki; Miyamoto-Sato, Etsuko

2014-01-01

We have developed a simple in vitro virus (IVV) selection system based on cell-free co-translation, using a highly stable and efficient mRNA display method. The IVV system is applicable to the high-throughput and comprehensive analysis of proteins and protein–ligand interactions. Huge amounts of genomic sequence data have been generated over the last decade. The accumulated genetic alterations and the interactome networks identified within cells represent a universal feature of a disease, and knowledge of these aspects can help to determine the optimal therapy for the disease. The concept of the “integrome” has been developed as a means of integrating large amounts of data. We have developed an interactome analysis method aimed at providing individually-targeted health care. We also consider future prospects for this system. PMID:24756093

A review of microdialysis coupled to microchip electrophoresis for monitoring biological events

PubMed Central

Saylor, Rachel A.; Lunte, Susan M.

2015-01-01

Microdialysis is a powerful sampling technique that enables monitoring of dynamic processes in vitro and in vivo. The combination of microdialysis with chromatographic or electrophoretic methods yields along with selective detection methods yields a “separation-based sensor” capable of monitoring multiple analytes in near real time. Analysis of microdialysis samples requires techniques that are fast (<1 min), have low volume requirements (nL–pL), and, ideally, can be employed on-line. Microchip electrophoresis fulfills these requirements and also permits the possibility of integrating sample preparation and manipulation with detection strategies directly on-chip. Microdialysis coupled to microchip electrophoresis has been employed for monitoring biological events in vivo and in vitro. This review discusses technical considerations for coupling microdialysis sampling and microchip electrophoresis, including various interface designs, and current applications in the field. PMID:25637011

Development and stability of semisolid preparations based on a supercritical CO2 Arnica extract.

PubMed

Bilia, Anna Rita; Bergonzi, Maria Camilla; Mazzi, Giovanni; Vincieri, Franco Francesco

2006-05-03

Conventional herbal drug preparations (HDP) based on Arnica montana L. have a low content of the active principles, sesquiterpene lactones, which show poor stability and low physical compatibility in semisolid formulations. Recently, an innovative supercritical carbon dioxide (CO2) extract with high sesquiterpene content has been marketed. Development of six semisolid preparations (cetomacrogol, polysorbate 60, polawax, anphyphil, natrosol and sepigel) based on this innovative CO2 extract is discussed. Stability of these preparations was investigated according to ICH guidelines. The evaluation of in vitro release of active constituents was performed using the cell method reported in the European Pharmacopoeia. Preliminary data on in vivo permeation of three selected formulations is demonstrated using the "skin stripping" test, according to the FDA, in healthy subjects. Analysis of sesquiterpene lactones within the extract and in vitro and in vivo studies was performed by RP-HPLC-DAD-MS method. The cetomacrogol showed the best release profile in the in vitro test, while in the in vivo test the best preparation resulted polysorbate 60 and polawax.

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

PubMed

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Informing Selection of Nanomaterial Concentrations for ToxCast In Vitro Testing using the Multiple-Path Particle Dosimetry Model - 3rd Annual International Conference on the Environmental Implications of NanoTechnology (ICEIN) & EPA Nano Grantees Meeting (2011)

EPA Science Inventory

Currently, little justification is provided for nanomaterial testing concentrations in in vitro assays. The in vitro concentrations typically used may be higher than those experienced by exposed humans. Selection of concentration levels for hazard evaluation based on real-world e...

Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

NASA Technical Reports Server (NTRS)

Harada, Kazuo; Orgel, Leslie E.

1993-01-01

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

Rationalizing the selection of oral lipid based drug delivery systems by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water soluble drugs.

PubMed

Dahan, Arik; Hoffman, Amnon

2008-07-02

As a consequence of modern drug discovery techniques, there has been a consistent increase in the number of new pharmacologically active lipophilic compounds that are poorly water soluble. A great challenge facing the pharmaceutical scientist is making these molecules into orally administered medications with sufficient bioavailability. One of the most popular approaches to improve the oral bioavailability of these molecules is the utilization of a lipid based drug delivery system. Unfortunately, current development strategies in the area of lipid based delivery systems are mostly empirical. Hence, there is a need for a simplified in vitro method to guide the selection of a suitable lipidic vehicle composition and to rationalize the delivery system design. To address this need, a dynamic in vitro lipolysis model, which provides a very good simulation of the in vivo lipid digestion process, has been developed over the past few years. This model has been extensively used for in vitro assessment of different lipid based delivery systems, leading to enhanced understanding of the suitability of different lipids and surfactants as a delivery system for a given poorly water soluble drug candidate. A key goal in the development of the dynamic in vitro lipolysis model has been correlating the in vitro data of various drug-lipidic delivery system combinations to the resultant in vivo drug profile. In this paper, we discuss and review the need for this model, its underlying theory, practice and limitations, and the available data accumulated in the literature. Overall, the dynamic in vitro lipolysis model seems to provide highly useful initial guidelines in the development process of oral lipid based drug delivery systems for poorly water soluble drugs, and it predicts phenomena that occur in the pre-enterocyte stages of the intestinal absorption cascade.

Does time-lapse imaging have favorable results for embryo incubation and selection compared with conventional methods in clinical in vitro fertilization? A meta-analysis and systematic review of randomized controlled trials.

PubMed

Chen, Minghao; Wei, Shiyou; Hu, Junyan; Yuan, Jing; Liu, Fenghua

2017-01-01

The present study aimed to undertake a review of available evidence assessing whether time-lapse imaging (TLI) has favorable outcomes for embryo incubation and selection compared with conventional methods in clinical in vitro fertilization (IVF). Using PubMed, EMBASE, Cochrane library and ClinicalTrial.gov up to February 2017 to search for randomized controlled trials (RCTs) comparing TLI versus conventional methods. Both studies randomized women and oocytes were included. For studies randomized women, the primary outcomes were live birth and ongoing pregnancy, the secondary outcomes were clinical pregnancy and miscarriage; for studies randomized oocytes, the primary outcome was blastocyst rate, the secondary outcome was good quality embryo on Day 2/3. Subgroup analysis was conducted based on different incubation and embryo selection between groups. Ten RCTs were included, four randomized oocytes and six randomized women. For oocyte-based review, the pool-analysis observed no significant difference between TLI group and control group for blastocyst rate [relative risk (RR) 1.08, 95% CI 0.94-1.25, I2 = 0%, two studies, including 1154 embryos]. The quality of evidence was moderate for all outcomes in oocyte-based review. For woman-based review, only one study provided live birth rate (RR 1,23, 95% CI 1.06-1.44,I2 N/A, one study, including 842 women), the pooled result showed no significant difference in ongoing pregnancy rate (RR 1.04, 95% CI 0.80-1.36, I2 = 59%, four studies, including 1403 women) between two groups. The quality of the evidence was low or very low for all outcomes in woman-based review. Currently there is insufficient evidence to support that TLI is superior to conventional methods for human embryo incubation and selection. In consideration of the limitations and flaws of included studies, more well designed RCTs are still in need to comprehensively evaluate the effectiveness of clinical TLI use.

Methods in Molecular Biology: Germline Stem Cells | Center for Cancer Research

Cancer.gov

The protocols in Germline Stem Cells are intended to present selected genetic, molecular, and cellular techniques used in germline stem cell research. The book is divided into two parts. Part I covers germline stem cell identification and regulation in model organisms. Part II covers current techniques used in in vitro culture and applications of germline stem cells.

Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

PubMed Central

Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

2016-01-01

Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation and invasion, arrested the cell cycle, and induced apoptosis in vitro. Conclusion The asymmetric mono-carbonyl analog of curcumin Da0324 exhibited significantly improved antigastric cancer activity. Da0324 may be a promising NF-κB inhibitor for the selective targeting of cancer cells. However, further studies are needed in animals to validate these findings for the therapeutic use of Da0324. PMID:27042000

Impact of exogenous ascorbic acid on biochemical activities of rice callus treated with salt stress

NASA Astrophysics Data System (ADS)

Alhasnawi, Arshad Naji; Zain, Che Radziah Che Mohd; Kadhimi, Ahsan A.; Isahak, Anizan; Mohamad, Azhar; Ashraf, Mehdi Farshad; Doni, Febri; Yusoff, Wan Mohtar Wan

2016-11-01

The application of in vitro systems can lead to new methods of crop amelioration. This method has been widely utilized for breeding tenacities, particularly for stress tolerance selection. Salinity causes oxidative stress in callus by enhancing the production of Reactive Oxygen Species (ROS), resulting in an efficient antioxidant system. The exogenous application of ascorbic acid (AsA) is an important requirement for tolerance. The present study aimed to examine in vitro selection strategy for callus induction in rice mature embryo culture on MS culture medium and to produce salt-tolerant callus under sodium chloride (NaCl) and AsA conditions in callus rice variety, MR269. This study also highlights changes in the activities of proline and antioxidants peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) of callus under NaCl stress to understand their possible role in salt tolerance. However, various levels of exogenously applied AsA under saline conditions improved callus, and the antioxidant enzyme activities of AsA are related to resistance to oxidative stress. Our results provide strong support for the hypothesis that AsA-dependent antioxidant enzymes play a significant role in the salinity tolerance of callus rice.

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

PubMed Central

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

PubMed

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.

PubMed

Eigenmann, Daniela Elisabeth; Jähne, Evelyn Andrea; Smieško, Martin; Hamburger, Matthias; Oufir, Mouhssin

2016-03-01

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.

Correlation Between An In-vitro Method And An In-vivo Method In Assessing Bioavailable Arsenic In Two Pesticide-Amended Soils

NASA Astrophysics Data System (ADS)

Quazi, S.; Sarkar, D.; Sylvia, V.; Datta, R.

2006-05-01

Health risk assessment of Arsenic (As) enriched soil requires the estimation of bioavailable fraction of total metal. Research has been conducted to gain a better understanding of the relationship between metal availability and risk assessment. Some baseline risk assessments developed for contaminated sites have used the conservative assumption that all (i.e. 100%) of the As present in soils and wastes is bioavailable, due to tremendous cost associated with in-vivo bioavailability studies. This potentially overestimates the actual health risk, elevating the expenses associated with site cleanup. Health risk from direct exposure to soil-As via the hand-to-mouth exposure route is restricted only to those fractions of As in the soil that are available to the human gastrointestinal system. A reasonable approach is to develop in-vitro methods that simulate the complex and dynamic human gastrointestinal system and correlate well with the results of in-vivo method. Thus this study aims in addressing the potential of one such in-vitro method developed by our research group in assessing the bioavailability of soil-As. Two soils with drastically different chemical characteristics in regards to As reactivity (Immokalee-low As retention capacity; Millhopper-high As retention capacity) spiked with a pesticide (sodium arsenate) were used. Soils were amended at two rates representing concentrations typically found at Superfund sites: 675 and 1500 mg/kg of As. In-vitro bioavailability experiments were performed in order to obtain an estimate of the amount of As likely to be available in the human gastrointestinal system as well as the fraction potentially absorbed onto the intestinal linings. Following the in-vitro study selective in-vivo bioavailability studies using As-contaminated soils were conducted on male and female mice to validate the in-vitro results via comparison with the in-vivo data. Soils were administered orally to the BALB/c mice immediately after spiking. Treatments comprised of a soil group (As in soil), a positive control group (only As) and a negative control group (no soil, no As). Blood samples were collected at different time periods to determine As concentrations. Correlation between the in-vitro and in-vivo data was determined. Information obtained from this study will serve as the first step towards the future development of a semi-quantitative model for predicting bioavailable As. This in turn will result in designing appropriate, cost-effective remedial strategies for As contaminated sites. Keywords: Bioavailability, In-vitro, In-vivo, Arsenic, Soil, Risk Assessment

In vivo and in vitro testing for selenium and selenium compounds bioavailability assessment in foodstuff.

PubMed

Moreda-Piñeiro, Jorge; Moreda-Piñeiro, Antonio; Bermejo-Barrera, Pilar

2017-03-04

The assessment of selenium and selenium species bioavailability in foodstuff is of special concern on the context of human nutrition. In vivo (human and animal), and in vitro tests are important approaches for estimating the bioavailability of toxic and essential compounds to humans. An overview on in vivo and in vitro bioavailability assays for releasing selenium and selenium species in foodstuffs is summarized. Se and Se species content in a foodstuff critically influence Se bioavailability and bioactivity to humans and animals. Se bioavailability is affected by foodstuff-matrix major composition and minor components. Foodstuffs processing and/or treatments could enhancement or decrease Se bioavailability. Experimental conditions such as the selection of healthy status of examined people (in in vivo humans approaches), the selection of animal model (in vivo animals approaches), or the selection of GI conditions (in in vitro tests) could determines the results. Thus, international standardized protocol for in vivo and in vitro approaches assessment is mandatory.

Use of in vitro methods to rank surfactants for irritation potential in support of new product development.

PubMed

Casterton, P L; Potts, L F; Klein, B D

1994-08-01

11 surfactant raw materials with potential applications in light-duty liquid cleaning products were evaluated in vitro using a human skin analogue (ATS SKIN(2) Model ZK1100) for predicting cytotoxicity (MTT reduction) and inflammation [prostaglandin E(2) (PGE(2)) release]. Two of the 11 raw materials, both in the same compound family, were selected to be individually combined with each of the other nine in a 90:10 (raw:selected raw) mixture. Selection criteria were based on desired performance characteristics and low irritation potential as suggested from the individual surfactant assay data. To determine whether irritation potential was mitigated, MTT and PGE(2) scores were again determined for each of the 18 combinations with the resulting data being compared with the untreated raw material data. A plot of the data indicated that one of two selected materials may have an 'anti-irritant' effect. For raw materials with intrinsic MTT scores of less than 50 mug/ml and with the original data corrected for possible dilution effects, a statistical comparison between individual raw materials and the two sets of combinations was done using a one-sample analysis. Both cytotoxicity (MTT) and inflammation (PGE(2)) were significantly decreased by the milder of the two selected raw materials. By factoring the data into future new product decisions, this methodology has become a useful and practical tool for Amway product development.

Analysis of the shapes of hemocytes of Callista brevisiphonata in vitro (Bivalvia, Veneridae).

PubMed

Karetin, Yu A; Pushchin, I I

2015-08-01

Fractal formalism in conjunction with linear methods of image analysis is suitable for the comparative analysis of such "irregular" shapes (from the point of view of classical Euclidean geometry) as flattened amoeboid cells of invertebrates in vitro. Cell morphology of in vitro spreading hemocytes from the bivalve mollusc Callista brevisiphonata was analyzed using correlation, factor and cluster analysis. Four significantly different cell types were identified on the basis of 36 linear and nonlinear parameters. The analysis confirmed the adequacy of the selected methodology for numerical description of the shape and the adequacy of classification of nonlinear shapes of spread hemocytes belonging to the same species. Investigation has practical significance for the description of the morphology of cultured cells, since cell shape is a result of summation of a number of extracellular and intracellular factors. © 2015 International Society for Advancement of Cytometry.

Bioaccessibility of metals in alloys: Evaluation of three surrogate biofluids

PubMed Central

Hillwalker, Wendy E.; Anderson, Kim A.

2014-01-01

Bioaccessibility in vitro tests measure the solubility of materials in surrogate biofluids. However, the lack of uniform methods and the effects of variable test parameters on material solubility limit interpretation. One aim of this study was to measure and compare bioaccessibility of selected economically important alloys and metals in surrogate physiologically based biofluids representing oral, inhalation and dermal exposures. A second aim was to experimentally test different biofluid formulations and residence times in vitro. A third aim was evaluation of dissolution behavior of alloys with in vitro lung and dermal biofluid surrogates. This study evaluated the bioaccessibility of sixteen elements in six alloys and 3 elemental/metal powders. We found that the alloys/metals, the chemical properties of the surrogate fluid, and residence time all had major impacts on metal solubility. The large variability of bioaccessibility indicates the relevancy of assessing alloys as toxicologically distinct relative to individual metals. PMID:24212234

In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection.

PubMed

K C, Tara Bahadur; Tada, Seiichi; Zhu, Liping; Uzawa, Takanori; Minagawa, Noriko; Luo, Shyh-Chyang; Zhao, Haichao; Yu, Hsiao-Hua; Aigaki, Toshiro; Ito, Yoshihiro

2018-05-17

An electrosensitive peptide probe has been developed from an in vitro selection technique using biorthogonal tRNA prepared with an electroreactive non-natural amino acid, 3,4-ethylenedioxythiophene-conjugated aminophenylalanine. The selected probe quantitatively detected the influenza virus based on a signal "turn-on" mechanism. The developed strategy could be used to develop electrochemical biosensors toward a variety of targets.

Rational selection of structurally diverse natural product scaffolds with favorable ADME properties for drug discovery.

PubMed

Samiulla, D S; Vaidyanathan, V V; Arun, P C; Balan, G; Blaze, M; Bondre, S; Chandrasekhar, G; Gadakh, A; Kumar, R; Kharvi, G; Kim, H O; Kumar, S; Malikayil, J A; Moger, M; Mone, M K; Nagarjuna, P; Ogbu, C; Pendhalkar, D; Rao, A V S Raja; Rao, G Venkateshwar; Sarma, V K; Shaik, S; Sharma, G V R; Singh, S; Sreedhar, C; Sonawane, R; Timmanna, U; Hardy, L W

2005-01-01

Natural product analogs are significant sources for therapeutic agents. To capitalize efficiently on the effective features of naturally occurring substances, a natural product-based library production platform has been devised at Aurigene for drug lead discovery. This approach combines the attractive biological and physicochemical properties of natural product scaffolds, provided by eons of natural selection, with the chemical diversity available from parallel synthetic methods. Virtual property analysis, using computational methods described here, guides the selection of a set of natural product scaffolds that are both structurally diverse and likely to have favorable pharmacokinetic properties. The experimental characterization of several in vitro ADME properties of twenty of these scaffolds, and of a small set of designed congeners based upon one scaffold, is also described. These data confirm that most of the scaffolds and the designed library members have properties favorable to their utilization for creating libraries of lead-like molecules.

Evaluation of accuracy of shade selection using two spectrophotometer systems: Vita Easyshade and Degudent Shadepilot.

PubMed

Kalantari, Mohammad Hassan; Ghoraishian, Seyed Ahmad; Mohaghegh, Mina

2017-01-01

The aim of this in vitro study was to evaluate the accuracy of shade matching using two spectrophotometric devices. Thirteen patients who require a full coverage restoration for one of their maxillary central incisors were selected while the adjacent central incisor was intact. 3 same frameworks were constructed for each tooth using computer-aided design and computer-aided manufacturing technology. Shade matching was performed using Vita Easyshade spectrophotometer, Shadepilot spectrophotometer, and Vitapan classical shade guide for the first, second, and third crown subsequently. After application, firing, and glazing of the porcelain, the color was evaluated and scored by five inspectors. Both spectrophotometric systems showed significantly better results than visual method ( P < 0.05) while there were no significant differences between Vita Easyshade and Shadepilot spectrophotometers ( P < 0.05). Spectrophotometers are a good substitute for visual color selection methods.

In vitro-in vivo sequence studies as a method of selecting the most efficacious alcohol-based solution for hygienic hand disinfection.

PubMed

Herruzo, R; Vizcaino, M J; Herruzo, I

2010-05-01

The use of alcohol-based hand rubs serves to reduce hospital-acquired infections. Many products of this type are now on offer and it is essential to know how to rank their efficacy. A sequence of tests is proposed here to compare any given new alcohol-based solution against the reference solution (60% 2-isopropyl-alcohol) with 30 s of contact time: (i) in vitro (with pig skin as carrier) testing of >30 species of microorganism; (ii) in vitro assessment of residual efficacy (after 30 min of drying); (iii) in vivo study of transient microbiota (modification of the EN 1500 standard procedure) using four ATCC strains; (iv) in vivo study of resident hand microbiota. After performing the in vitro evaluation of seven alcohol-based hand rubs, the two most efficacious (chlorhexidine-quac-alcohol and mecetronium- alcohol) were chosen and studied, comparatively with the reference solution (60% isopropyl alcohol), in vitro (for chemical sustainability on the skin) and in vivo (against transient and resident microbiota). Chlorhexidine-quac-alcohol proved to be significantly superior to mecetronium-alcohol or the reference solution in all tests, except against resident microbiota for which the improvement was not statistically significant.

Development and Characterization of a Differentiated Thyroid Cancer Cell Line Resistant to VEGFR-Targeted Kinase Inhibitors

PubMed Central

Isham, Crescent R.; Netzel, Brian C.; Bossou, Ayoko R.; Milosevic, Dragana; Cradic, Kendall W.; Grebe, Stefan K.

2014-01-01

Background: Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differentiated thyroid cancer. Unfortunately, drug resistance uniformly develops, limiting their therapeutic efficacies and thereby constituting a major clinical problem. Approach and Methods: To study acquired drug resistance and elucidate underlying mechanisms in this setting, BHP2–7 human differentiated thyroid cancer cells were subjected to prolonged continuous in vitro selection with 18 μM pazopanib, a clinically relevant concentration; acquisition of pazopanib resistance was serially assessed, with the resulting resistant cells thereafter subcloned and characterized to assess potential mechanisms of acquired pazopanib resistance. Results: Stable 2- to 4-fold in vitro pazopanib resistance emerged in response to pazopanib selection associated with similar in vitro growth characteristics but with markedly more aggressive in vivo xenograft growth. Selected cells were cross-resistant to sunitinib and to a lesser extent sorafenib but not to MAPK kinase (MEK1/2) inhibition by GSK1120212. Genotyping demonstrated acquisition of a novel activating KRAS codon 13 GGC to GTT (glycine to valine) mutation, consistent with the observed resistance to upstream vascular endothelial growth factor receptor inhibition yet sensitivity to downstream MAPK kinase (MEK1/2) inhibition. Conclusions: Selection of thyroid cancer cells with clinically utilized therapeutics can lead to acquired drug resistance and altered in vivo xenograft behavior that can recapitulate analogous drug resistance observed in patients. This approach has the potential to lead to insights into acquired treatment-related drug resistance in thyroid cancers that can be subjected to subsequent validation in serially collected patient samples and that has the potential to yield preemptive and responsive approaches to dealing with this important clinical problem. PMID:24628546

Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin

PubMed Central

Prueksaritanont, Thomayant; Chu, Xiaoyan; Evers, Raymond; Klopfer, Stephanie O; Caro, Luzelena; Kothare, Prajakti A; Dempsey, Cynthia; Rasmussen, Scott; Houle, Robert; Chan, Grace; Cai, Xiaoxin; Valesky, Robert; Fraser, Iain P; Stoch, S Aubrey

2014-01-01

Aims Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion-transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. Methods The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. Results Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration–time curve were 5.7- and 7.6-fold for pitavastatin and 4.4- and 3.3-fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. Conclusions The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose. PMID:24617605

The consequences of physical post-treatments (microwave and electron-beam) on food/packaging interactions: A physicochemical and toxicological approach.

PubMed

Riquet, A M; Breysse, C; Dahbi, L; Loriot, C; Severin, I; Chagnon, M C

2016-05-15

The safety of microwave and electron-beam treatments has been demonstrated, in regards to the formation of reaction products that could endanger human health. An integrated approach was used combining the potential toxicity of all the substances likely to migrate to their chemical characterizations. This approach was applied to polypropylene (PP) films prepared with a selection of additives. Components were identified by liquid and gas chromatography using a mass selective detector system. Their potential toxicity was assessed using three in vitro short-term bioassays and their migrations were carried out using a standards-based approach. After the electron-beam treatment some additives decomposed and there was a significant increase in the polyolefin oligomeric saturated hydrocarbons concentration. PP prepared with Irgafos 168 led to a significantly strong cytotoxic effect and PP prepared with Irganox 1076 induced a dose-dependant estrogenic effect in vitro. Migration values were low and below the detection limit of the analytical method applied. Copyright © 2015 Elsevier Ltd. All rights reserved.

The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck).

PubMed

Boscariol, R L; Almeida, W A B; Derbyshire, M T V C; Mourão Filho, F A A; Mendes, B M J

2003-09-01

A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.

Electrochemical Cathodic Polarization, a Simplified Method That Can Modified and Increase the Biological Activity of Titanium Surfaces: A Systematic Review

PubMed Central

2016-01-01

Background The cathodic polarization seems to be an electrochemical method capable of modifying and coat biomolecules on titanium surfaces, improving the surface activity and promoting better biological responses. Objective The aim of the systematic review is to assess the scientific literature to evaluate the cellular response produced by treatment of titanium surfaces by applying the cathodic polarization technique. Data, Sources, and Selection The literature search was performed in several databases including PubMed, Web of Science, Scopus, Science Direct, Scielo and EBSCO Host, until June 2016, with no limits used. Eligibility criteria were used and quality assessment was performed following slightly modified ARRIVE and SYRCLE guidelines for cellular studies and animal research. Results Thirteen studies accomplished the inclusion criteria and were considered in the review. The quality of reporting studies in animal models was low and for the in vitro studies it was high. The in vitro and in vivo results reported that the use of cathodic polarization promoted hydride surfaces, effective deposition, and adhesion of the coated biomolecules. In the experimental groups that used the electrochemical method, cellular viability, proliferation, adhesion, differentiation, or bone growth were better or comparable with the control groups. Conclusions The use of the cathodic polarization method to modify titanium surfaces seems to be an interesting method that could produce active layers and consequently enhance cellular response, in vitro and in vivo animal model studies. PMID:27441840

Does time-lapse imaging have favorable results for embryo incubation and selection compared with conventional methods in clinical in vitro fertilization? A meta-analysis and systematic review of randomized controlled trials

PubMed Central

Yuan, Jing; Liu, Fenghua

2017-01-01

Objective The present study aimed to undertake a review of available evidence assessing whether time-lapse imaging (TLI) has favorable outcomes for embryo incubation and selection compared with conventional methods in clinical in vitro fertilization (IVF). Methods Using PubMed, EMBASE, Cochrane library and ClinicalTrial.gov up to February 2017 to search for randomized controlled trials (RCTs) comparing TLI versus conventional methods. Both studies randomized women and oocytes were included. For studies randomized women, the primary outcomes were live birth and ongoing pregnancy, the secondary outcomes were clinical pregnancy and miscarriage; for studies randomized oocytes, the primary outcome was blastocyst rate, the secondary outcome was good quality embryo on Day 2/3. Subgroup analysis was conducted based on different incubation and embryo selection between groups. Results Ten RCTs were included, four randomized oocytes and six randomized women. For oocyte-based review, the pool-analysis observed no significant difference between TLI group and control group for blastocyst rate [relative risk (RR) 1.08, 95% CI 0.94–1.25, I2 = 0%, two studies, including 1154 embryos]. The quality of evidence was moderate for all outcomes in oocyte-based review. For woman-based review, only one study provided live birth rate (RR 1,23, 95% CI 1.06–1.44,I2 N/A, one study, including 842 women), the pooled result showed no significant difference in ongoing pregnancy rate (RR 1.04, 95% CI 0.80–1.36, I2 = 59%, four studies, including 1403 women) between two groups. The quality of the evidence was low or very low for all outcomes in woman-based review. Conclusions Currently there is insufficient evidence to support that TLI is superior to conventional methods for human embryo incubation and selection. In consideration of the limitations and flaws of included studies, more well designed RCTs are still in need to comprehensively evaluate the effectiveness of clinical TLI use. PMID:28570713

Probing antioxidant activity of 2'-hydroxychalcones: crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation.

PubMed

Rossi, Miriam; Caruso, Francesco; Crespi, Erica J; Pedersen, Jens Z; Nakano, Gail; Duong, Michelle; McKee, Celia; Lee, Sharon; Jiwrajka, Manasi; Caldwell, Charles; Baffour, Francis; Karlin, Dylan Alex; Lidoff, Genevieve; Leone, Stefano; Balducci, Valentina; Miler, Jaroslav; Incerpi, Sandra

2013-10-01

In order to better understand the antioxidant behavior of a series of polyphenolic 2'-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV-vis spectroscopic method indicate that a hydroxyl group in position 5' induces the highest antioxidant activity. Consequently, 2,2',5'-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2',5'-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa(-)/fa(-)) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2',5'-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons

PubMed Central

Santos, Daniel; Gonzalez-Perez, Francisco; Navarro, Xavier

2016-01-01

Although peripheral axons can regenerate after nerve transection and repair, functional recovery is usually poor due to inaccurate reinnervation. Neurotrophic factors promote directional guidance to regenerating axons and their selective application may help to improve functional recovery. Hence, we have characterized in organotypic cultures of spinal cord and dorsal root ganglia the effect of GDNF, FGF-2, NGF, NT-3, and BDNF at different concentrations on motor and sensory neurite outgrowth. In vitro results show that GDNF and FGF-2 enhanced both motor and sensory neurite outgrowth, NGF and NT-3 were the most selective to enhance sensory neurite outgrowth, and high doses of BDNF selectively enhanced motor neurite outgrowth. Then, NGF, NT-3, and BDNF (as the most selective factors) were delivered in a collagen matrix within a silicone tube to repair the severed sciatic nerve of rats. Quantification of Fluorogold retrolabeled neurons showed that NGF and NT-3 did not show preferential effect on sensory regeneration whereas BDNF preferentially promoted motor axons regeneration. Therefore, the selective effects of NGF and NT-3 shown in vitro are lost when they are applied in vivo, but a high dose of BDNF is able to selectively enhance motor neuron regeneration both in vitro and in vivo. PMID:27867665

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

Micro-optical fiber probe for use in an intravascular Raman endoscope.

PubMed

Komachi, Yuichi; Sato, Hidetoshi; Aizawa, Katsuo; Tashiro, Hideo

2005-08-01

We believe that we have developed the narrowest optical-fiber Raman probe ever reported, 600 microm in total diameter, that can be inserted into coronary arteries. The selection of suitable optical fibers, filters, and a processing method is discussed. Custom-made filters attached to the front end of a probe eliminate the background Raman signals of the optical fiber itself. The experimental evaluation of various optical fibers is carried out for the selection of suitable fibers. Measurement of the Raman spectra of an atherosclerotic lesion of a rabbit artery in vitro demonstrates the excellent performance of the micro-Raman probe.

A novel strategy for selection of allosteric ribozymes yields RiboReporter™ sensors for caffeine and aspartame

PubMed Central

Ferguson, Alicia; Boomer, Ryan M.; Kurz, Markus; Keene, Sara C.; Diener, John L.; Keefe, Anthony D.; Wilson, Charles; Cload, Sharon T.

2004-01-01

We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter™ sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids. PMID:15026535

Formulation, in vitro and in vivo evaluation of lidocaine HCl ocular inserts for topical ocular anesthesia.

PubMed

Shukr, Marwa

2014-07-01

Topical anesthesia is a safe and cost-effective method considered as the first-choice in many procedures. The objective of the present study was to develop ocular inserts as a new form of lidocaine HCl to give a sufficient level of anesthetic. Ocuserts were prepared using HPMC and PVA in different ratios with lidocaine HCl alone and lidocaine HCl β-cyclodextrins complex. Drug polymer interactions were studied by Fourier transform infrared spectroscopic studies. The prepared ocular inserts were characterized by means of ocusert thickness, weight variation, folding endurance, surface pH, moisture absorption, drug content and in-vitro drug release. Stability study was conducted on selected formulations, and in vivo evaluation of lidocaine HCl was also carried out. The results revealed that F7 formulations containing drug β-cyclodextrins with 4 % HPMC and 2 % PVA were found to have good physical characteristics and appropriate flexibility. In addition to the highest initial and cumulative percentage of drug released in vitro. The selected F7 ocuserts retained their characteristics during the stability study. The results of in vivo study showed that the addition of β-cyclodextrins in F7 significantly increase the drug content in the aqueous humor when compared with F3 ocuserts containing lidocaine HCl alone.

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

Methacrylate micro/nano particles prepared by spray drying: a preliminary in vitro/in vivo study.

PubMed

Muñoz Ortega, Begoña; Sallam, Marwa Ahmed; Marín Boscá, M Teresa

2016-09-01

Delivery systems controlling drug release only in the colon holds great promises since they improve utilization of drug and decrease the dosing times comparison with conventional forms. The aim of the present study was to prepare polymeric microparticles on the basis of Ciprofloxacin via oral route for the treatment of inflammatory bowel disease. Ciprofloxacin was selected because of its extensive coverage for intestinal flora, relatively favorable side-effect profile and preliminary data suggesting its efficacy in the treatment of active Crohn's Disease. Microparticles were prepared using different acrylic compounds, namely Eudragit® RL (PO) and RS (PO) and a mixture of both. Spray-drying was used as a preparation method of Ciprofloxacin/Eudragit® microparticles using a Mini Spray Dryer B-290 (Büchi, Postfach, Switzerland). In vitro dissolution studies were performed to choose the best formulation and selected microparticles were characterized by size and morphology by environmental scanning electron microscopy. Yield and encapsulation efficiency were calculated and in vivo/ex vivo experiments were investigated both of which suggest that selected microparticles can be used for colon targeting of drugs increasing residence time of the drug in the affected area.

CON4EI: SkinEthic™ Human Corneal Epithelium Eye Irritation Test (SkinEthic™ HCE EIT) for hazard identification and labelling of eye irritating chemicals.

PubMed

Van Rompay, A R; Alépée, N; Nardelli, L; Hollanders, K; Leblanc, V; Drzewiecka, A; Gruszka, K; Guest, R; Kandarova, H; Willoughby, J A; Verstraelen, S; Adriaens, E

2018-06-01

Assessment of ocular irritancy is an international regulatory requirement and a necessary step in the safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as a stand-alone assay. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed with the goal of assessing the reliability of eight in vitro/alternative test methods as well as establishing an optimal tiered-testing strategy. One of the in vitro assays selected was the validated SkinEthic™ Human Corneal Epithelium Eye Irritation Test method (SkinEthic™ HCE EIT). The SkinEthic™ HCE EIT has already demonstrated its capacity to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage (No Category). The goal of this study was to evaluate the performance of the SkinEthic™ HCE EIT test method in terms of the important in vivo drivers of classification. For the performance with respect to the drivers all in vivo Cat 1 and No Cat chemicals were 100% correctly identified. For Cat 2 chemicals the liquids and the solids had a sensitivity of 100% and 85.7%, respectively. For the SkinEthic™ HCE EIT test method, 100% concordance in predictions (No Cat versus No prediction can be made) between the two participating laboratories was obtained. The accuracy of the SkinEthic™ HCE EIT was 97.5% with 100% sensitivity and 96.9% specificity. The SkinEthic™ HCE EIT confirms its excellent results of the validation studies. Copyright © 2017. Published by Elsevier Ltd.

Selection of endophytic fungi from comfrey (Symphytum officinale L.) for in vitro biological control of the phytopathogen Sclerotinia sclerotiorum (Lib.)

PubMed Central

Rocha, Rafaeli; da Luz, Daniela Eleutério; Engels, Cibelle; Pileggi, Sônia Alvim Veiga; de Souza Jaccoud Filho, David; Matiello, Rodrigo Rodrigues; Pileggi, Marcos

2009-01-01

Biological control consists of using one organism to attack another that may cause economic damage to crops. Integrated Pest Management (IPM) is a very common strategy. The white mold produced by Sclerotinia sclerotiorum (Lib.) causes considerable damage to bean crops. This fungus is a soil inhabitant, the symptoms of which are characterized by water-soaked lesions covered by a white cottony fungal growth on the soil surface and/or the host plant. Possible biological control agents taken from plants are being investigated as phytopathogen inhibitors. These are endophytic microorganisms that inhabit the intercellular spaces of vegetal tissues and are often responsible for antimicrobial production. The objective of the present study was to select endophytic fungi isolated from comfrey (Symphytum officinale L.) leaves with in vitro antagonist potential against the phytopathogenic fungus S. sclerotiorum. Twelve isolates of endophytic fungi and a pathogenic strain of S. sclerotiorum were used in the challenge method. With the aid of this method, four endophytes with the best antagonistic activity against S. sclerotiorum were selected. Pathogen growth inhibition zones were considered indicative of antibiosis. The percentages of pathogenic mycelia growth were measured both with and without the antagonist, resulting in growth reductions of 46.7% to 50.0% for S. sclerotiorum. These analyses were performed by evaluating the endophytic/pathogenic mycelia growth in mm/day over an eight-day period of antagonistic tests. PMID:24031320

The synergy of honokiol and fluconazole against clinical isolates of azole-resistant Candida albicans.

PubMed

Jin, J; Guo, N; Zhang, J; Ding, Y; Tang, X; Liang, J; Li, L; Deng, X; Yu, L

2010-09-01

To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole-resistant (azole-R) clinical isolates of Candida albicans. A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole-R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC-resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by Delta E models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time-killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Synergistic activity of HNK and FLC against clinical isolates of FLC-resistant C. albicans was observed in vitro and in vivo. This report might provide a potential therapeutic method to overcome the problem of drug-resistance in C. albicans.

Recent trends in SELEX technique and its application to food safety monitoring

PubMed Central

Mei, Zhanlong; Yao, Li; Wang, Xin; Zheng, Lei; Liu, Jian; Liu, Guodong; Peng, Chifang; Chen, Wei

2014-01-01

The method referred to as “systemic evolution of ligands by exponential enrichment” (SELEX) was introduced in 1990 and ever since has become an important tool for the identification and screening of aptamers. Such nucleic acids can recognize and bind to their corresponding targets (analytes) with high selectivity and affinity, and aptamers therefore have become attractive alternatives to traditional antibodies not the least because they are much more stable. Meanwhile, they have found numerous applications in different fields including food quality and safety monitoring. This review first gives an introduction into the selection process and to the evolution of SELEX, then covers applications of aptamers in the surveillance of food safety (with subsections on absorptiometric, electrochemical, fluorescent and other methods), and then gives conclusions and perspectives. The SELEX method excels by its features of in vitro, high throughput and ease of operation. This review contains 86 references. PMID:25419005

Biological method to quantify progressive stages of decay in five commercial woods by Coriolus versicolor.

PubMed

Olfat, A M; Karimi, A N; Parsapajouh, D

2007-04-01

Biologic agar-block method was developed that allowed wood samples to be evaluated and monitored in terms of colonization and development of the decay by Basidiomycetes fungi (Coriolus versicolor) and to be directly classified based on mean mass loss. In this research, the in vitro decay of five commercial woods by Coriolus versicolor was studied by the agar-block method. The selected wood samples were Abies alba, Populus alba, Fagus orientalis, Platanus orientalis and Ulmus glabra. The results demonstrated the strong resistance of Ulmus glabra and the lowest resistance in Fagus orientalis. The mass losses (%) were 16.8 and 42.4%, respectively. There were also a high correlation between the mass loss and apparent damage. Therefore biological evaluation of wood regarding biodegradation and the selection of wood types for various application respects will be of high priority.

Identification of Potential Plasma Biomarkers for Nonalcoholic Fatty Liver Disease by Integrating Transcriptomics and Proteomics in Laying Hens.

PubMed

Tsai, Meng-Tsz; Chen, Yu-Jen; Chen, Ching-Yi; Tsai, Mong-Hsun; Han, Chia-Li; Chen, Yu-Ju; Mersmann, Harry J; Ding, Shih-Torng

2017-03-01

Background: Prevalent worldwide obesity is associated with increased incidence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. The identification of noninvasive biomarkers for NAFLD is of recent interest. Because primary de novo lipogenesis occurs in chicken liver as in human liver, adult chickens with age-associated steatosis resembling human NAFLD is an appealing animal model. Objective: The objective of this study was to screen potential biomarkers in the chicken model for NAFLD by transcriptomic and proteomic analysis. Methods: Hy-Line W-36 laying hens were fed standard feed from 25 to 45 wk of age to induce fatty liver. They were killed every 4 wk, and liver and plasma were collected at each time point to assess fatty liver development and for transcriptomic and proteomic analysis. Next, selected biomarkers were confirmed in additional experiments by providing supplements of the hepatoprotective nutrients betaine [300, 600, or 900 parts per million (ppm) in vivo; 2 mM in vitro] or docosahexaenoic acid (DHA; 1% in vivo; 100 μM in vitro) to 30-wk-old Hy-Line W-36 laying hens for 4 mo and to Hy-Line W-36 chicken primary hepatocytes with oleic acid-induced steatosis. Liver or hepatocyte lipid contents and the expression of biomarkers were then examined. Results: Plasma acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL), and glutathione S -transferase (GST) concentrations are well-established biomarkers for NAFLD. Selected biomarkers had significant positive associations with hepatic lipid deposition ( P < 0.001). Betaine (900 ppm in vivo; 2 mM in vitro) and DHA (1% in vivo; 100 μM in vitro) supplementation both resulted in lower steatosis accompanied by the reduced expression of selected biomarkers in vivo and in vitro ( P < 0.05). Conclusion: This study used adult laying hens to identify biomarkers for NAFLD and indicated that AACS, DPP4, GLUL, and GST could be considered to be potential diagnostic indicators for NAFLD in the future. © 2017 American Society for Nutrition.

Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker

PubMed Central

Ray, Partha; Rialon-Guevara, Kristy L.; Veras, Emanuela; Sullenger, Bruce A.; White, Rebekah R.

2012-01-01

Most cases of pancreatic cancer are not diagnosed until they are no longer curable with surgery. Therefore, it is critical to develop a sensitive, preferably noninvasive, method for detecting the disease at an earlier stage. In order to identify biomarkers for pancreatic cancer, we devised an in vitro positive/negative selection strategy to identify RNA ligands (aptamers) that could detect structural differences between the secretomes of pancreatic cancer and non-cancerous cells. Using this molecular recognition approach, we identified an aptamer (M9-5) that differentially bound conditioned media from cancerous and non-cancerous human pancreatic cell lines. This aptamer further discriminated between the sera of pancreatic cancer patients and healthy volunteers with high sensitivity and specificity. We utilized biochemical purification methods and mass-spectrometric analysis to identify the M9-5 target as cyclophilin B (CypB). This molecular recognition–based strategy simultaneously identified CypB as a serum biomarker and generated a new reagent to recognize it in body fluids. Moreover, this approach should be generalizable to other diseases and complementary to traditional approaches that focus on differences in expression level between samples. Finally, we suggest that the aptamer we identified has the potential to serve as a tool for the early detection of pancreatic cancer. PMID:22484812

Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker.

PubMed

Ray, Partha; Rialon-Guevara, Kristy L; Veras, Emanuela; Sullenger, Bruce A; White, Rebekah R

2012-05-01

Most cases of pancreatic cancer are not diagnosed until they are no longer curable with surgery. Therefore, it is critical to develop a sensitive, preferably noninvasive, method for detecting the disease at an earlier stage. In order to identify biomarkers for pancreatic cancer, we devised an in vitro positive/negative selection strategy to identify RNA ligands (aptamers) that could detect structural differences between the secretomes of pancreatic cancer and non-cancerous cells. Using this molecular recognition approach, we identified an aptamer (M9-5) that differentially bound conditioned media from cancerous and non-cancerous human pancreatic cell lines. This aptamer further discriminated between the sera of pancreatic cancer patients and healthy volunteers with high sensitivity and specificity. We utilized biochemical purification methods and mass-spectrometric analysis to identify the M9-5 target as cyclophilin B (CypB). This molecular recognition-based strategy simultaneously identified CypB as a serum biomarker and generated a new reagent to recognize it in body fluids. Moreover, this approach should be generalizable to other diseases and complementary to traditional approaches that focus on differences in expression level between samples. Finally, we suggest that the aptamer we identified has the potential to serve as a tool for the early detection of pancreatic cancer.

Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

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

Cline, Gary W., E-mail: gary.cline@yale.edu; Zhao, Xiaojian; Jakowski, Amy B.

2011-09-02

Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity tomore » islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution characteristics suggest several GPCRs as potential targets for PET imaging of pancreatic BCM.« less

In vitro antiplasmodial, antileishmanial and antitrypanosomal activities of selected medicinal plants used in the traditional Arabian Peninsular region

PubMed Central

2012-01-01

Background Worldwide particularly in developing countries, a large proportion of the population is at risk for tropical parasitic diseases. Several medicinal plants are still used traditionally against protozoal infections in Yemen and Saudi Arabia. Thus the present study investigated the in vitro antiprotozoal activity of twenty-five plants collected from the Arabian Peninsula. Methods Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC50 < 10 μg/ml (<5 μg/ml for T. brucei) and selectivity index of >4. Results Antiplasmodial activity was found in the extracts of Chrozophora oblongifolia, Ficus ingens, Lavandula dentata and Plectranthus barbatus. Amastigotes of T. cruzi were affected by Grewia erythraea, L. dentata, Tagetes minuta and Vernonia leopoldii. Activity against T. brucei was obtained in G. erythraea, L. dentata, P. barbatus and T. minuta. No relevant activity was found against L. infantum. High levels of cytotoxicity (MRC-5 IC50 < 10 μg/ml) and hence non-specific activities were noted in Cupressus sempervirens, Kanahia laniflora and Kniphofia sumarae. Conclusion The results endorse that medicinal plants can be promising sources of natural products with antiprotozoal activity potential. The results support to some extent the traditional uses of some plants for the treatment of parasitic protozoal diseases. PMID:22520595

Novel levocetirizine HCl tablets with enhanced palatability: synergistic effect of combining taste modifiers and effervescence technique

PubMed Central

Labib, Gihan S

2015-01-01

Objectives Levocetirizine HCl, a second-generation piperazine derivative and H1-selective antihistaminic agent, possesses few side effects. The first objective of the study was to compare and evaluate the taste-masking effect of different ratios of 2-hydroxypropyl-β-cyclodextrin and mannitol on levocetirizine HCl using an inclusion complex and solid dispersion, respectively. The second objective was to study the possibility of preparing and evaluating effervescent tablets from the best-chosen taste-masked blends for the purpose of their use either as orodispersible tablets or as water-soluble effervescent tablets, according to patients’ will. Materials and methods Prepared taste-masked blends were prepared and subjected to palatability, Fourier-transform infrared spectroscopy, and differential scanning calorimetry studies. Tablets containing different percentages of effervescent mixtures were prepared by direct compression on the selected taste-modified blends. Evaluation tests were conducted, including flowability and compressibility on the precompressed blends and hardness, friability, wetting time, effervescent time, in vitro, in vivo disintegration time, and in vitro dissolution study on the compressed tablets. Formulated tablets were evaluated and compared to marketed orodispersible tablets for mouth feel and palatability. Results All prepared tablets showed convenient physical and palatability properties compared to the selected brand. The in vitro drug-release study revealed fast release of levocetirizine HCl within 5 minutes from all prepared tablets. Conclusion Levocetirizine HCl effervescent tablets are likely to increase patient compliance with drug administration. Moreover, the use of these effervescent tablets in an orodispersible dosage form can improve oral drug bioavailability and act as an attractive pediatric dosage form. PMID:26379426

Pollen selection under acid rain stress

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

Zhang, Y.

To investigate whether acid rain stress induces pollen selection in nature, three different approaches were used, based on the assumption that the response of pollen grains to acid rain is controlled by an acid sensitive gene product. Germination of pollen from homozygous and heterozygous individuals under acid rain stress was examined to detect any differences in rate of germination between populations of homogeneous and heterogeneous pollen grains. In vitro and in vivo bulked segregant analysis using RAPDs was used to search for differences in DNA constitution between the survivors of acid rain stressed and non-acid rain stressed pollen populations inmore » vitro and between the progenies of acid rain stressed and non-acid rain stressed populations during pollination, respectively. No evidence for the pollen selection under acid rain stress was obtained in any of the test systems. Inhibition of protein synthesis using cycloheximide led to significant reduction of tube elongation at 4 hr and had no effect on pollen germination at any time interval tested. Total proteins extracted from control and acid rain stressed pollen grain populations exhibited no differences. The reduction of corn pollen germination in vitro under acid rain stress was mainly due to pollen rupture. The present data indicates the reduction of pollen germination and tube growth under acid rain stress may be a physiological response rather than a genetic response. A simple, nontoxic, and effective method to separate germinated from ungerminated pollen grains has been developed using pollen from corn (Zea mays, L. cv. Pioneer 3747). The separated germinated pollen grains retained viability and continued tube growth when placed in culture medium.« less

Impact of Fluoroquinolone Resistance Mutations on Gonococcal Fitness and In Vivo Selection for Compensatory Mutations

PubMed Central

Kunz, Anjali N.; Begum, Afrin A.; Wu, Hong; D'Ambrozio, Jonathan A.; Robinson, James M.; Shafer, William M.; Bash, Margaret C.; Jerse, Ann E.

2012-01-01

Background. Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA91/95 and parC86 mutations on gonococcal fitness. Methods. Mutant gyrA91/95 and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR−79 mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection. Results. In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA91/95 mutation conferred an in vivo fitness benefit to wild-type and mtrR−79 mutant gonococci. The gyrA91/95, parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA91/95, parC86, mtrR−79 mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (CipR) mutant was selected during infection with the gyrA91/95, parC86, mtrR−79 mutant in which the mtrR−79 mutation was repaired and the gyrA91 mutation was altered. This in vivo–selected mutant grew as well as the wild-type strain in vitro. Conclusions. gyrA91/95 mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain CipR. PMID:22492860

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes.

PubMed

Husna, A U; Azam, A; Qadeer, S; Awan, M A; Nasreen, S; Shahzad, Q; Fouladi-Nashta, A; Khalid, M; Akhter, S

2018-04-01

Routinely, swim-up method is used to separate high-quality sperm; however, long processing time and close cell-to-cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex ™ and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus-oocyte complexes (COCs) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO 2 incubator at 38.5°C and 5% CO 2 . Matured COCs were rinsed twice in fertilization TALP and placed in the pre-warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex ™ , glass wool filtration and swim-up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15-20 min in CO 2 incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co-incubation with sets of 10-15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA, while in vitro fertilizing rates were compared by chi-squared test using SPSS-20. Least significant difference (LSD) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex ™ filtration improved (p < .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim-up (control). In conclusion, cryopreserved Nili-Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo. © 2017 Blackwell Verlag GmbH.

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.

Validation of a new ELISA method for in vitro potency testing of hepatitis A vaccines.

PubMed

Morgeaux, S; Variot, P; Daas, A; Costanzo, A

2013-01-01

The goal of the project was to standardise a new in vitro method in replacement of the existing standard method for the determination of hepatitis A virus antigen content in hepatitis A vaccines (HAV) marketed in Europe. This became necessary due to issues with the method used previously, requiring the use of commercial test kits. The selected candidate method, not based on commercial kits, had already been used for many years by an Official Medicines Control Laboratory (OMCL) for routine testing and batch release of HAV. After a pre-qualification phase (Phase 1) that showed the suitability of the commercially available critical ELISA reagents for the determination of antigen content in marketed HAV present on the European market, an international collaborative study (Phase 2) was carried out in order to fully validate the method. Eleven laboratories took part in the collaborative study. They performed assays with the candidate standard method and, in parallel, for comparison purposes, with their own in-house validated methods where these were available. The study demonstrated that the new assay provides a more reliable and reproducible method when compared to the existing standard method. A good correlation of the candidate standard method with the in vivo immunogenicity assay in mice was shown previously for both potent and sub-potent (stressed) vaccines. Thus, the new standard method validated during the collaborative study may be implemented readily by manufacturers and OMCLs for routine batch release but also for in-process control or consistency testing. The new method was approved in October 2012 by Group of Experts 15 of the European Pharmacopoeia (Ph. Eur.) as the standard method for in vitro potency testing of HAV. The relevant texts will be revised accordingly. Critical reagents such as coating reagent and detection antibodies have been adopted by the Ph. Eur. Commission and are available from the EDQM as Ph. Eur. Biological Reference Reagents (BRRs).

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.

PubMed

Xu, Juanjuan; Fang, Rui; Chen, Li; Chen, Daozhen; Xiao, Jian-Ping; Yang, Weimin; Wang, Honghua; Song, Xiaoqing; Ma, Ting; Bo, Shiping; Shi, Chong; Ren, Jun; Huang, Lei; Cai, Li-Yi; Yao, Bing; Xie, X Sunney; Lu, Sijia

2016-10-18

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Fundamentals of human embryonic growth in vitro and the selection of high-quality embryos for transfer.

PubMed

Boiso, Irene; Veiga, Anna; Edwards, Robert G

2002-01-01

Knowledge of the nature of embryo growth, and the handling and scoring of quality in human embryos are significant aspects for embryologists in IVF clinics. This review describes the formation, growth and maturation of human oocytes, many aspects of fertilization in vitro, embryonic transcription during preimplantation stages, and the formation of polarities, timing controls, role of mitochondria and functions of endocrine and paracrine systems. Modern concepts are fully discussed, together with their significance in the practice of IVF. This knowledge is essential for the correct clinical care of human embryos growing in vitro, especially in view of their uncharacteristic tendency to vary widely in implantation potential. Underlying causes of such variation have not been identified. Stringent tests must be enforced to ensure human embryos develop under optimal conditions, and are scored for quality using the most advanced techniques. Optimal methods of culture are described, including methods such as co-culture introduced to improve embryo quality but less important today. Detailed attention is given to quality as assessed from embryonic characteristics determined by timers, polarities, disturbed embryo growth and anomalous cell cycles. Methods for classification are described. Approaches to single embryo transfers are described, including the use of sequential media to produce high-quality blastocysts. These approaches, and others involved in surgical methods to remove fragments, transfer ooplasm or utilize newer approaches such as preimplantation diagnosis of chromosomal complements in embryos are covered. New outlooks in this field are summarized.

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.

7 CFR 331.3 - PPQ select agents and toxins.

Code of Federal Regulations, 2011 CFR

2011-01-01

... listed in paragraph (b) of this section if the nucleic acids: (i) Can be expressed in vivo or in vitro; or (ii) Are in a vector or recombinant host genome and can be expressed in vivo or in vitro. (3... select agents and toxins. (a) Except as provided in paragraphs (d) and (e) of this section, the...

Torsemide Fast Dissolving Tablets: Development, Optimization Using Box-Bhenken Design and Response Surface Methodology, In Vitro Characterization, and Pharmacokinetic Assessment.

PubMed

El-Shenawy, Ahmed A; Ahmed, Mahmoud M; Mansour, Heba F; Abd El Rasoul, Saleh

2017-08-01

The present study planed to develop new fast dissolving tablets (FDTs) of torsemide. Solid dispersions (SDs) of torsemide and sorbitol (3:1) or polyvinylpyrrolidone (PVP) k25 were prepared. The prepared SDs were evaluated for in-vitro dissolution. Fourier transform infrared spectroscopy and differential scanning calorimetry for SDs revealed no drug/excipient interactions and transformation of torsemide to the amorphous form. Torsemide/sorbitol SD was selected for formulation of torsemide FDTs by direct compression method. Box-Bhenken factorial design was employed to design 15 formulations using croscarmellose sodium and crospovidone at different concentrations. The response surface methodology was used to analyze the effect of changing these concentrations (independent variables) on disintegration time (Y 1 ), percentage friability (Y 2 ), and amount torsemide released at 10 min. The physical mixtures of torsemide and the used excipients were evaluated for angle of repose, Hausner's ratio, and Carr's index. The prepared FDTs tablets were evaluated for wetting and disintegration time, weight variation, drug content, percentage friability, thickness, hardness, and in vitro release. Based on the in-vitro results and factorial design characterization, F10 and F7 were selected for bioavailability studies following administration to Albino New Zealand rabbits. They showed significantly higher C max and (AUC 0-12 ) and shorter T max than those obtained after administration of the corresponding ordinary commercial Torseretic ® tablets. Stability study was conducted for F10 that showed good stability upon storage at 30°C/75% RH and 40°C/75% RH for 3 months.

Activity of delafloxacin against methicillin-resistant Staphylococcus aureus: resistance selection and characterization.

PubMed

Remy, Joan M; Tow-Keogh, Cheryl A; McConnell, Timothy S; Dalton, James M; Devito, Joseph A

2012-12-01

To determine the potential for delafloxacin to select for resistant mutants in methicillin-resistant Staphylococcus aureus (MRSA), including isolates with existing mutations in the quinolone resistance determining region (QRDR). Susceptibility testing by broth microdilution was performed on 30 MRSA clinical isolates. For four of these isolates, the presence or absence of mutations in the QRDR was characterized. Resistance selection was performed on these four isolates by spreading cells on drug-containing agar plates followed by incubation for 48 h. Resistance frequencies and mutant prevention concentrations (MPCs) were calculated for each; PCR amplification and sequencing were performed using standard methods to characterize mutations in the QRDR. Growth rate analysis was performed and relative fitness was determined. Delafloxacin demonstrated potent in vitro activity against this set of MRSA isolates, with MICs of 0.008-1 mg/L and an MIC(50) and MIC(90) of 0.03 and 0.5 mg/L, respectively. Spontaneous delafloxacin resistance frequencies for the MRSA strains were 2 × 10(-9) to <9.5 × 10(-11). Delafloxacin MPCs were one to four times the MIC for any isolate, lower than those of comparator quinolones. Some delafloxacin-selected mutants showed a fitness cost when co-cultured with the parent strain. Delafloxacin demonstrates excellent antibacterial potency and exhibits a low probability for the selection of resistant mutants in MRSA. Although mutants can be selected at low frequencies in vitro from quinolone-resistant isolates, delafloxacin MICs and MPCs remain low and a fitness cost can be observed. Consequently delafloxacin warrants further investigation for the potential treatment of drug-resistant MRSA infections.

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.

In vitro cytotoxicity and differential cellular sensitivity of derivatives of diamino acids. II. N1-methyl, N1-allyl, N1-(2-chloroethyl) and N1-propargyl nitrosoureas.

PubMed

Dulude, H; Salvador, R; Gallant, G

1995-01-01

The in vitro cytotoxicity and differential cellular sensitivity of a series of new N1-methyl, N1-allyl, N1-2-chloroethyl and N1-propargyl nitrosourea derivatives of diamino acids were determined in the National Cancer Institute's primary antitumor drug screen. The compounds tested showed an in vitro anticancer activity similar to commercialized nitrosoureas such as CCNU, BCNU, MeCCNU, chlorozotocin, streptozotocin and PCNU. The alkylating moiety of the nitrosoureas seems to play a role in the general selectivity of our compounds. The N1-methyl and N1-2-chloroethyl nitrosourea derivatives are more selective for central nervous system cell lines, the N1-allyl nitrosourea derivatives are more selective for lung cancer cell lines and the N1-propargyl nitrosoureas are more selective for leukemia cell lines.

Evaluation of trimethoprim-sulfamethoxazole based combination therapy against Stenotrophomonas maltophilia: in vitro effects and clinical efficacy in cancer patients.

PubMed

Araoka, Hideki; Baba, Masaru; Okada, Chikako; Abe, Masahiro; Kimura, Muneyoshi; Yoneyama, Akiko

2017-05-01

The aim of this study was to evaluate the in vitro effects and clinical efficacies of trimethoprim-sulfamethoxazole (SXT) combined with other antimicrobial agents against Stenotrophomonas maltophilia. In vitro analysis was conducted on 89 S. maltophilia strains isolated from blood and the respiratory tract between June 2012 and October 2014. Levofloxacin (LVX), ticarcillin-clavulanic acid (TIM), and minocycline (MIN) were selected for an examination of their effects when individually combined with SXT by the checkerboard method. In addition, 29 S. maltophilia bacteremia cases were reviewed and the clinical efficacies of SXT-based combination therapies were analyzed. SXT+LVX showed synergy in 21, no interactions in 61, and antagonism in 7. SXT+TIM showed synergy in 71, and no interactions in 18. SXT+MIN showed synergy in 10, and no interactions in 79. The review of clinical data indicated that a combination of SXT+fluoroquinolone was not associated with improved prognosis compared with monotherapy. The in vitro data indicated that SXT+TIM had beneficial microbiological effects and was not antagonistic. Our in vitro and clinical data analyses do not support the routine use of SXT+fluoroquinolone combination therapy for S. maltophilia infection. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

PubMed

Bourgogne, Emmanuel; Culot, Benoit; Dell'Aiera, Sylvie; Chanteux, Hugues; Stockis, Armel; Nicolas, Jean-Marie

2018-06-01

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x 2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction. Copyright © 2018 Elsevier B.V. All rights reserved.

Phenotypic and Genotypic Analysis of In Vitro-Selected Artemisinin-Resistant Progeny of Plasmodium falciparum

PubMed Central

Tucker, Matthew S.; Mutka, Tina; Sparks, Kansas; Patel, Janus

2012-01-01

Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340×3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC50s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistance in vitro than the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification of pfmdr1 increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug. PMID:22083467

Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli - an in vitro study

PubMed Central

2010-01-01

Background Probiotic bacteria are suggested to play a role in the maintenance of oral health. Such health promoting bacteria are added to different commercial probiotic products. The aim of the study was to investigate the ability of a selection of lactobacilli strains, used in commercially available probiotic products, to inhibit growth of oral mutans streptococci and C. albicans in vitro. Methods Eight probiotic lactobacilli strains were tested for growth inhibition on three reference strains and two clinical isolates of mutans streptococci as well as two reference strains and three clinical isolates of Candida albicans with an agar overlay method. Results At concentrations ranging from 109 to 105 CFU/ml, all lactobacilli strains inhibited the growth of the mutans streptococci completely with the exception of L. acidophilus La5 that executed only a slight inhibition of some strains at concentrations corresponding to 107 and 105 CFU/ml. At the lowest cell concentration (103 CFU/ml), only L. plantarum 299v and L. plantarum 931 displayed a total growth inhibition while a slight inhibition was seen for all five mutans streptococci strains by L. rhamnosus LB21, L. paracasei F19, L. reuteri PTA 5289 and L. reuteri ATCC 55730. All the tested lactobacilli strains reduced candida growth but the effect was generally weaker than for mutans streptococci. The two L. plantarum strains and L. reuteri ATCC 55730 displayed the strongest inhibition on Candida albicans. No significant differences were observed between the reference strains and the clinical isolates. Conclusion The selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and Candida albicans in vitro. PMID:20598145

Bioprocessing of wheat and paddy straw for their nutritional up-gradation.

PubMed

Sharma, Rakesh Kumar; Arora, Daljit Singh

2014-07-01

Solid-state bioprocessing of agricultural residues seems to be an emerging and effective method for the production of high quality animal feed. Seven strains of white-rot fungi were selected to degrade wheat and paddy straw (PS) under solid-state conditions. Degradation of different components, i.e., hemicellulose, cellulose and lignin was evaluated along with nutritional parameters including; in vitro digestibility, crude protein, amino acids, total phenolic contents (TPC) etc. Effect of nitrogen-rich supplements on degradation of lignocellulosics was evaluated using two best selected fungal strains (Phlebia brevispora and Phlebia floridensis). The best selected conditions were used to upscale the process up to 200 g batches of wheat and PS. Lignin was selectively degraded up to 30 % with a limited loss of 11-12 % in total organic matter. Finally, the degraded agro-residues demonstrated 50-62 % enhancement in their digestibility. Two-threefold enhancement in other nutritional quality (amino acids, TPCs and antioxidant activity) fortifies the process. Thus the method is quite helpful to design an effective solid-state fermentation system to improve the nutritive quality of agricultural residues by simultaneous production of lignocellulolytic enzyme production and antioxidants.

An in vitro study of the effects of low-level laser radiation on human blood

NASA Astrophysics Data System (ADS)

Siposan, Dan G.; Lukacs, Adalbert

2003-12-01

In the last time the study of the effects of LLLR on the blood is considered to be a subject of great importance in elucidating the mechanisms of action between LLLR and biologic tissues. Different methods of therapy by blood irradiation have been developed and used in clinical purposes with benefic effects. This study investigates some in vitro effects of LLLR on some selected rheologic indices of human blood. After establishing whether or not damaging effects could appear due to laser irradiation of the blood, we tried to find a new method for rejuvenating the blood preserved in MacoPharma-type bags. Blood samples were obtained from adult regular donors (volunteers). HeNe laser and laser diodes were used as radiation source, in a wide range of wavelengths, power densities, doses and other parameters of irradiation protocol. In the first series of experiments we established that LLLR does not alter the fresh blood from healthy donors, for doses between 0 and 10 J/cm3 and power densities between 30 and 180 mW/cm3. In the second series of experiments we established that LLLR does have, in some specific conditions, a revitalizing effect on the erythrocytes in preserved blood. We concluded that laser irradiation of the preserved blood, following a selected protocol of irradiation, could be used as a new method to improve the performances of preservation: prolonging the period of storage and blood rejuvenation before transfusion.

Molecular docking and ex vivo and in vitro anticholinesterase activity studies of Salvia sp. and highlighted rosmarinic acid.

PubMed

Demirezer, Lütfiye Ömür; Gürbüz, Perihan; Kelicen Uğur, Emine Pelin; Bodur, Mine; Özenver, Nadire; Uz, Ayse; Güvenalp, Zühal

2015-01-01

To evaluate acetylcholinesterase (AChE) inhibitory activity and antioxidant capacity of the major molecule from Salvia sp., rosmarinic acid, as a drug candidate molecule for treatment of Alzheimer disease (AD). The AChE inhibitory activity of different extracts from Salvia trichoclada, Salvia verticillata, and Salvia fruticosa was determined by the Ellman and isolated guinea pig ileum methods, and the antioxidant capacity was determined with DPPH. The AChE inhibitory activity of the major molecule rosmarinic acid was determined by in silico docking and isolated guinea pig ileum methods. The methanol extract of Salvia trichoclada showed the highest inhibition on AChE. The same extract and rosmarinic acid showed significant contraction responses on isolated guinea pig ileum. All the extracts and rosmarinic acid showed high radical scavenging capacities. Docking results of rosmarinic acid showed high affinity to the selected target, AChE. In this study in vitro and ex vivo studies and in silico docking research of rosmarinic acid were used simultaneously for the first time. Rosmarinic acid showed promising results in all the methods tested.

Covalent antibody display—an in vitro antibody-DNA library selection system

PubMed Central

Reiersen, Herald; Løbersli, Inger; Løset, Geir Å.; Hvattum, Else; Simonsen, Bjørg; Stacy, John E.; McGregor, Duncan; FitzGerald, Kevin; Welschof, Martin; Brekke, Ole H.; Marvik, Ole J.

2005-01-01

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription–translation mixture of Escherichia coli S30 lysate. Complexes of scFv–P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA–protein complexes. PMID:15653626

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

PubMed

Bode, Gerard H; Coué, Gregory; Freese, Christian; Pickl, Karin E; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; van Winden, Ewoud C; Tziveleka, Leto-Aikaterini; Sideratou, Zili; Engbersen, Johan F J; Singh, Smriti; Albrecht, Krystyna; Groll, Jürgen; Möller, Martin; Pötgens, Andy J G; Schmitz, Christoph; Fröhlich, Eleonore; Grandfils, Christian; Sinner, Frank M; Kirkpatrick, C James; Steinbusch, Harry W M; Frank, Hans-Georg; Unger, Ronald E; Martinez-Martinez, Pilar

2017-04-01

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis and in vitro evaluation of bone-seeking superparamagnetic iron oxide nanoparticles as contrast agents for imaging bone metabolic activity.

PubMed

Panahifar, Arash; Mahmoudi, Morteza; Doschak, Michael R

2013-06-12

In this article, we report the synthesis and in vitro evaluation of a new class of nonionizing bone-targeting contrast agents based on bisphosphonate-conjugated superparamagnetic iron oxide nanoparticles (SPIONs), for use in imaging of bone turnover with magnetic resonance imaging (MRI). Similar to bone-targeting (99m)Technetium medronate, our novel contrast agent uses bisphosphonates to impart bone-seeking properties, but replaces the former radioisotope with nonionizing SPIONs which enables their subsequent detection using MRI. Our reported method is relatively simple, quick and cost-effective and results in BP-SPIONs with a final nanoparticle size of 17 nm under electron microscopy technique (i.e., TEM). In-vitro binding studies of our novel bone tracer have shown selective binding affinity (around 65%) for hydroxyapatite, the principal mineral of bone. Bone-targeting SPIONs offer the potential for use as nonionizing MRI contrast agents capable of imaging dynamic bone turnover, for use in the diagnosis and monitoring of metabolic bone diseases and related bone pathology.

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

NASA Technical Reports Server (NTRS)

Tsang, Joyce; Joyce, Gerald F.

1996-01-01

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

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...

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol.

PubMed

Greer, Michael S; Zhou, Ting; Weselake, Randall J

2014-08-01

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-(14)C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.

Comparative activity of several beta-lactam antibiotics against anaerobes determined by two methods.

PubMed

Zabransky, R J; Birk, R J

1987-01-01

The susceptibility of 120 strains of several species of anaerobes to a number of second and third generation beta-lactam antibiotics was determined by the National Committee for Clinical Laboratory Standards reference agar dilution and microdilution methods. The antibiotics tested were cefoperazone, cefotaxime, cefotetan, ceftizoxime, cefoxitin, and imipenem. The MIC50s ranged from 0.125 to 16 micrograms/ml. The MIC90s were lowest with imipenem at 0.5 micrograms/ml, followed by cefoxitin at 32 micrograms/ml; they were highest with cefotetan at 128 micrograms/ml and were 64 micrograms/ml with the others. In vitro drug activity varied with the antibiotic, the organism, the method used, and the breakpoint selected. Rates of resistance varied considerably between the taxonomic groups of organisms tested and also among species within a group. Overall, reproducibility with the agar dilution method ranged from 44% to 85%; testing with ceftizoxime was the least reproducible. Microdilution results agreed within +/- 1 dilution of the agar dilution mode 79% to 95% of the time, with some variation between drugs and organisms tested. Because there were distinct differences in the activity of some drugs against certain species, no antibiotic can substitute for others in in vitro testing.

Larva-mediated chalkbrood resistance-associated single nucleotide polymorphism markers in the honey bee Apis mellifera.

PubMed

Liu, Y; Yan, L; Li, Z; Huang, W-F; Pokhrel, S; Liu, X; Su, S

2016-06-01

Chalkbrood is a disease affecting honey bees that seriously impairs brood growth and productivity of diseased colonies. Although honey bees can develop chalkbrood resistance naturally, the details underlying the mechanisms of resistance are not fully understood, and no easy method is currently available for selecting and breeding resistant bees. Finding the genes involved in the development of resistance and identifying single nucleotide polymorphisms (SNPs) that can be used as molecular markers of resistance is therefore a high priority. We conducted genome resequencing to compare resistant (Res) and susceptible (Sus) larvae that were selected following in vitro chalkbrood inoculation. Twelve genomic libraries, including 14.4 Gb of sequence data, were analysed using SNP-finding algorithms. Unique SNPs derived from chromosomes 2 and 11 were analysed in this study. SNPs from resistant individuals were confirmed by PCR and Sanger sequencing using in vitro reared larvae and resistant colonies. We found strong support for an association between the C allele at SNP C2587245T and chalkbrood resistance. SNP C2587245T may be useful as a genetic marker for the selection of chalkbrood resistance and high royal jelly production honey bee lines, thereby helping to minimize the negative effects of chalkbrood on managed honey bees. © 2016 The Royal Entomological Society.

Selection and characterization, application of a DNA aptamer targeted to Streptococcus pyogenes in cooked chicken.

PubMed

Huang, Yukun; Wang, Xin; Duan, Nuo; Xia, Yu; Wang, Zhouping; Che, Zhenming; Wang, Lijun; Yang, Xiao; Chen, Xianggui

2018-06-15

An aptamer against Streptococcus pyogenes was selected and identified, and a fluorescent method based on the reported aptamer was established to detect S. pyogenes in the cooked chicken. Through a twelve rounds of whole-bacterium SELEX (systematic evolution of ligands by exponential enrichment) selection in vitro, a set of aptamers binding to the whole cell of S. pyogenes were generated, harvesting a low-level dissociation constant (K d ) value of 44 ± 5 nmol L -1 of aptamer S-12. Aptamer-based quantification of S. pyogenes in the cooked chicken sample was implemented in a fluorescence resonance energy transfer-based assay by using graphene oxide, resulting in a limit of detection of 70 cfu mL -1 . The selected aptamer showed affinity and selectivity recognizing S. pyogenes; besides, more biosensors based on the selected aptamer as a molecular recognition element could be developed in the innovative determinations of S. pyogenes. Copyright © 2018 Elsevier Inc. All rights reserved.

Natural Selection in a Bangladeshi Population from the Cholera-Endemic Ganges River Delta

PubMed Central

Karlsson, Elinor K.; Harris, Jason B.; Tabrizi, Shervin; Rahman, Atiqur; Shlyakhter, Ilya; Patterson, Nick; O'Dushlaine, Colm; Schaffner, Stephen F.; Gupta, Sameer; Chowdhury, Fahima; Sheikh, Alaullah; Shin, Ok Sarah; Ellis, Crystal; Becker, Christine E.; Stuart, Lynda M.; Calderwood, Stephen B.; Ryan, Edward T.; Qadri, Firdausi; Sabeti, Pardis C.; LaRocque, Regina C.

2015-01-01

As an ancient disease with high fatality, cholera has likely exerted strong selective pressure on affected human populations. We performed a genome-wide study of natural selection in a population from the Ganges River Delta, the historic geographic epicenter of cholera. We identified 305 candidate selected regions using the Composite of Multiple Signals (CMS) method. The regions were enriched for potassium channel genes involved in cyclic AMP-mediated chloride secretion and for components of the innate immune system involved in NF-κB signaling. We demonstrate that a number of these strongly selected genes are associated with cholera susceptibility in two separate cohorts. We further identify repeated examples of selection and association in an NF-kB / inflammasome-dependent pathway that is activated in vitro by Vibrio cholerae. Our findings shed light on the genetic basis of cholera resistance in a population from the Ganges River Delta and present a promising approach for identifying genetic factors influencing susceptibility to infectious diseases. PMID:23825302

Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

PubMed

Bell, Thomas J; Eberwine, James

2015-01-01

RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

Mannich-Benzimidazole Derivatives as Antioxidant and Anticholinesterase Inhibitors: Synthesis, Biological Evaluations, and Molecular Docking Study.

PubMed

Alpan, Ayşe Selcen; Sarıkaya, Görkem; Çoban, Güneş; Parlar, Sülünay; Armagan, Güliz; Alptüzün, Vildan

2017-07-01

A series of Mannich bases of benzimidazole derivatives having a phenolic group were designed to assess their anticholinesterase and antioxidant activities. The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities were evaluated in vitro by using Ellman's method. According to the activity results, all of the compounds exhibited moderate to good AChE inhibitory activity (except for 2a), with IC 50 values ranging from 0.93 to 10.85 μM, and generally displayed moderate BuChE inhibitory activity. Also, most of the compounds were selective against BuChE. Compound 4b was the most active molecule on the AChE enzyme and also selective. In addition, we investigated the antioxidant effects of the synthesized compounds against FeCl 2 /ascorbic acid-induced oxidative stress in the rat brain in vitro, and the activity results showed that most of the compounds are effective as radical scavengers. Molecular docking studies and molecular dynamics simulations were also carried out. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A rapid method to increase the number of F₁ plants in pea (Pisum sativum) breeding programs.

PubMed

Espósito, M A; Almirón, P; Gatti, I; Cravero, V P; Anido, F S L; Cointry, E L

2012-08-16

In breeding programs, a large number of F₂ individuals are required to perform the selection process properly, but often few such plants are available. In order to obtain more F₂ seeds, it is necessary to multiply the F₁ plants. We developed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting of seeds using 6-benzylaminopurine. To optimize shoot regeneration, basic medium contained Murashige and Skoog (MS) salts with or without B5 Gamborg vitamins and different concentrations of 6-benzylaminopurine (25, 50 and 75 μM) using five genotypes. We found that modified MS (B5 vitamins + 25 μM 6-benzylaminopurine) is suitable for in vitro shoot regeneration of pea. Thirty-eight hybrid combinations were transferred onto selected medium to produce shoots that were used for root induction on MS medium supplemented with α-naphthalene-acetic acid. Elongated shoots were developed from all hybrid genotypes. This procedure can be used in pea breeding programs and will allow working with a large number of plants even when the F₁ plants produce few seeds.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319

Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding.

PubMed

Tenzer, S; Peters, B; Bulik, S; Schoor, O; Lemmel, C; Schatz, M M; Kloetzel, P-M; Rammensee, H-G; Schild, H; Holzhütter, H-G

2005-05-01

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

A flow cytometric method for assessing viability of intraerythrocytic hemoparasites.

PubMed

Wyatt, C R; Goff, W; Davis, W C

1991-06-24

We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.

Polyphenol bioavailability in nuts and seeds by an in vitro dialyzability approach.

PubMed

Herbello-Hermelo, Paloma; Lamas, Juan Pablo; Lores, Marta; Domínguez-González, Raquel; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2018-07-15

An in vitro dialyzability approach has been undertaken to elucidate the bioavailable fraction of the total polyphenols (TPs) of edible nuts and seeds. The TP contents in samples and in dialyzates were assessed by the Folin-Ciocalteu spectrophotometric method. Antioxidant activity was determined in selected samples, using a modified method against Trolox®. TPs and antioxidant activity in nuts/seeds were determined after applying a pressurized liquid extraction sample pre-treatment. High dialyzability ratios were assessed in most nuts/seeds (TP dialyzability percentages within the 25-91% range). The highest TP dialyzability ratios were found in raw Brazil nuts (81 ± 5%), toasted pistachios (88 ± 9%), and fried cashews (89 ± 9%), whereas TPs in pumpkin seeds were found to be very low (TPs were not detected in the dialyzable fraction). TP dialyzability was correlated with the copper content in nuts and seeds. Copyright © 2018 Elsevier Ltd. All rights reserved.

High-yield in vitro recordings from neurons functionally characterized in vivo.

PubMed

Weiler, Simon; Bauer, Joel; Hübener, Mark; Bonhoeffer, Tobias; Rose, Tobias; Scheuss, Volker

2018-06-01

In vivo two-photon calcium imaging provides detailed information about the activity and response properties of individual neurons. However, in vitro methods are often required to study the underlying neuronal connectivity and physiology at the cellular and synaptic levels at high resolution. This protocol provides a fast and reliable workflow for combining the two approaches by characterizing the response properties of individual neurons in mice in vivo using genetically encoded calcium indicators (GECIs), followed by retrieval of the same neurons in brain slices for further analysis in vitro (e.g., circuit mapping). In this approach, a reference frame is provided by fluorescent-bead tracks and sparsely transduced neurons expressing a structural marker in order to re-identify the same neurons. The use of GECIs provides a substantial advancement over previous approaches by allowing for repeated in vivo imaging. This opens the possibility of directly correlating experience-dependent changes in neuronal activity and feature selectivity with changes in neuronal connectivity and physiology. This protocol requires expertise both in in vivo two-photon calcium imaging and in vitro electrophysiology. It takes 3 weeks or more to complete, depending on the time allotted for repeated in vivo imaging of neuronal activity.

Development and current applications of assisted fertilization.

PubMed

Palermo, Gianpiero D; Neri, Queenie V; Monahan, Devin; Kocent, Justin; Rosenwaks, Zev

2012-02-01

Since the very early establishment of in vitro insemination, it became clear that one of the limiting steps is the achievement of fertilization. Among the different assisted fertilization methods, intracytoplasmic sperm injection emerged as the ultimate technique to allow fertilization with ejaculated, epididymal, and testicular spermatozoa. This work describes the early steps that brought forth the development of intracytoplasmic sperm injection and its role in assisted reproductive techniques. The current methods to select the preferential male gamete will be elucidated and the concerns related to the offspring of severe male factor couples will be discussed. Copyright © 2012. Published by Elsevier Inc.

The Positively Charged Hyperbranched Polymers with Tunable Fluorescence and the Cell Imaging Application.

PubMed

Ma, Hengchang; Qin, Yanfang; Yang, Zenming; Yang, Manyi; Ma, Yucheng; Yin, Pei; Yang, Yuan; Wang, Tao; Lei, Ziqiang; Yao, Xiaoqiang

2018-04-25

Fluorescence-tunable materials are becoming increasingly attractive for their potential application in optics, electronics, and biomedical technology. Herein, a multi-color molecular pixel system is realized using simple copolymerization method. Bleeding both of complementary colors from blue and yellow fluorescence segments, reproduced a serious multicolor fluorescence materials. Interestingly, the emission colors of the polymers can be fine-tuned in solid state, solution phase, and in hydrogel state. More importantly, the positive fluorescent polymers exhibited cell-membrane permeable ability, and were found to accumulate on the cell nucleus, exhibiting remarkable selectivity to give bright fluorescence. The DNA/RNA selectivity experiments in vitro and in vivo verified that [tris(4-(pyridin-4-yl)phenyl)amine]-[1,8-dibromooctane] (TPPA-DBO) has prominent selectivity to DNA over RNA inside cells.

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method.

PubMed

Ishikawa, Sadamasa; Hiraga, Kou; Hiradate, Yuuki; Tanemura, Kentaro

2015-06-01

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.

Effect of ultrasonic irradiation on mammalian cells and chromosomes in vitro

NASA Technical Reports Server (NTRS)

Roseboro, J. A.; Buchanan, P.; Norman, A.; Stern, R.

1978-01-01

Human peripheral blood and HeLa cells were irradiated in vitro at the ultrasonic frequency of 65 kHz. The whole blood and HeLa cell suspensions were exposed to continuous and pulsed ultrasonic power levels of 0.12, 0.16, 0.72, 1.12 and 2.24 W for a period of one minute. The method of ultrasonic irradiation was carried out with the whole blood or HeLa cell suspensions coupled directly to a cylindrical transducer while heating of the cell suspensions in excess of 41 C was avoided. Irradiated and unirradiated peripheral blood lymphocyte chromosome cultures were prepared and scored for selected numerical and morphological aberrations. There was no significant difference in the frequency of chromosomal aberrations between irradiated and unirradiated cells.

Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker.

PubMed

Bakshi, Souvika; Saha, Bedabrata; Roy, Nand Kishor; Mishra, Sagarika; Panda, Sanjib Kumar; Sahoo, Lingaraj

2012-06-01

A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T₀ transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.

Ionene polymers for selectively inhibiting the vitro growth of malignant cells

NASA Technical Reports Server (NTRS)

Rembaum, Alan (Inventor)

1977-01-01

Ionene polymers of the structure ##STR1## WHERE X AND Y ARE INTEGERS FROM 3 TO 16, Z.sup.- is an anion such as a halogen and n is an integer from 50 to 150 are found to bind negatively charged mammalian cells such as malignant cells and can be utilized to selectively inhibit the growth of malignant cells in vitro.

A scanning electron microscopic evaluation of in vitro dentinal tubules penetration by selected anaerobic bacteria.

PubMed

Siqueira, J F; De Uzeda, M; Fonseca, M E

1996-06-01

In vitro root canal dentinal tubule invasion by selected anaerobic bacteria commonly isolated from endodontic infections was evaluated. Dentinal cylinders obtained from bovine incisors were inoculated with bacteria, and microbial penetration into tubules was demonstrated by scanning electron microscopy. The results indicated that all bacterial strains tested were able to penetrate into dentinal tubules, but to different extents.

Using in vitro/in silico data for consumer safety assessment of feed flavoring additives--A feasibility study using piperine.

PubMed

Thiel, A; Etheve, S; Fabian, E; Leeman, W R; Plautz, J R

2015-10-01

Consumer health risk assessment for feed additives is based on the estimated human exposure to the additive that may occur in livestock edible tissues compared to its hazard. We present an approach using alternative methods for consumer health risk assessment. The aim was to use the fewest possible number of animals to estimate its hazard and human exposure without jeopardizing the safety upon use. As an example we selected the feed flavoring substance piperine and applied in silico modeling for residue estimation, results from literature surveys, and Read-Across to assess metabolism in different species. Results were compared to experimental in vitro metabolism data in rat and chicken, and to quantitative analysis of residues' levels from the in vivo situation in livestock. In silico residue modeling showed to be a worst case: the modeled residual levels were considerably higher than the measured residual levels. The in vitro evaluation of livestock versus rodent metabolism revealed no major differences in metabolism between the species. We successfully performed a consumer health risk assessment without performing additional animal experiments. As shown, the use and combination of different alternative methods supports animal welfare consideration and provides future perspective to reducing the number of animals. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1.

PubMed

Huang, Yukun; Chen, Xiujuan; Duan, Nuo; Wu, Shijia; Wang, Zhouping; Wei, Xinlin; Wang, Yuanfeng

2015-01-01

Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selection and characterization of a DNA aptamer to crystal violet.

PubMed

Chen, Yang; Wang, Jine; Zhang, Yajie; Xu, Lijun; Gao, Tian; Wang, Bing; Pei, Renjun

2018-06-13

Aptamers are short single-stranded DNA or RNA, which can be selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). In order to develop novel light-up probes to substitute G-quadruplex (G4), we selected a DNA aptamer for crystal violet (CV), a triphenylmethane light-up dye, by a modified affinity chromatography-based SELEX. The ssDNA pool was first coupled on streptavidin-coated agarose beads through a biotin labeled complementary oligonucleotide, and then the aptamer sequences would be released from agarose beads by CV affinity. This method is simple, straightforward and effective. The aptamer sequence with a low micromolar dissociation constant (Kd) and good specificity was achieved after 11 rounds of selection. The light-up properties of the CV-aptamer were also investigated, and the CV showed dramatic fluorescence enhancement. The CV-aptamer pair could be further used as a novel light-up fluorescent probe to design biosensors.

Construction of human antibody gene libraries and selection of antibodies by phage display.

PubMed

Schirrmann, Thomas; Hust, Michael

2010-01-01

Recombinant antibodies as therapeutics offer new opportunities for the treatment of many tumor diseases. To date, 18 antibody-based drugs are approved for cancer treatment and hundreds of anti-tumor antibodies are under development. The first clinically approved antibodies were of murine origin or human-mouse chimeric. However, since murine antibody domains are immunogenic in human patients and could result in human anti-mouse antibody (HAMA) responses, currently mainly humanized and fully human antibodies are developed for therapeutic applications.Here, in vitro antibody selection technologies directly allow the selection of human antibodies and the corresponding genes from human antibody gene libraries. Antibody phage display is the most common way to generate human antibodies and has already yielded thousands of recombinant antibodies for research, diagnostics and therapy. Here, we describe methods for the construction of human scFv gene libraries and the antibody selection.

Noninvasive imaging systems for gametes and embryo selection in IVF programs: a review.

PubMed

Omidi, Marjan; Faramarzi, Azita; Agharahimi, Azam; Khalili, Mohammad Ali

2017-09-01

Optimizing the efficiency of the in vitro fertilization procedure by improving pregnancy rates and reducing the risks of multiple pregnancies simultaneously are the primary goals of the current assisted reproductive technology program. With the move to single embryo transfers, the need for more cost-effective and noninvasive methods for embryo selection prior to transfer is paramount. These aims require advancement in a more acquire gametes/embryo testing and selection procedures using high-tech devices. Therefore, the aim of the present review is to evaluate the efficacy of noninvasive imaging systems in the current literatures, focusing on the potential clinical application in infertile patients undergoing assisted reproductive technology treatments. In this regards, three advanced imaging systems of motile sperm organelle morphology examination, polarization microscopy and time-lapse monitoring for the best selection of the gametes and preimplantation embryos are introduced in full. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Formulation and optimization of doxorubicin loaded polymeric nanoparticles using Box-Behnken design: ex-vivo stability and in-vitro activity.

PubMed

Shaikh, Muhammad Vaseem; Kala, Manika; Nivsarkar, Manish

2017-03-30

Biodegradable nanoparticles (NPs) have gained tremendous interest for targeting chemotherapeutic drugs to the tumor environment. Inspite of several advances sufficient encapsulation along with the controlled release and desired size range have remained as considerable challenges. Hence, the present study examines the formulation optimization of doxorubicin loaded PLGA NPs (DOX-PLGA-NPs), prepared by single emulsion method for cancer targeting. Critical process parameters (CPP) were selected by initial screening. Later, Box-Behnken design (BBD) was used for analyzing the effect of the selected CPP on critical quality attributes (CQA) and to generate a design space. The optimized formulation was stabilized by lyophilization and was used for in-vitro drug release and in-vitro activity on A549 cell line. Moreover, colloidal stability of the NPs in the biological milieu was assessed. Amount of PLGA and PVA, oil:water ratio and sonication time were the selected independent factors for BBD. The statistical data showed that a quadratic model was fitted to the data obtained. Additionally, the lack of fit values for the models was not significant. The delivery system showed sustained release behavior over a period of 120h and was governed by Fickian diffusion. The multipoint analysis at 24, 48 and 72h showed gradual reduction in IC50 value of DOX-PLGA-NPs (p<0.05, Fig. 9). DOX-PLGA-NPs were found to be stable in the biological fluids indicating their in-vivo applicability. In conclusion, optimization of the DOX-PLGA-NPs by BBD yielded in a promising drug carrier for doxorubicin that could provide a novel treatment modality for cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

PubMed Central

Ueberberg, Sandra; Meier, Juris J.; Waengler, Carmen; Schechinger, Wolfgang; Dietrich, Johannes W.; Tannapfel, Andrea; Schmitz, Inge; Schirrmacher, Ralf; Köller, Manfred; Klein, Harald H.; Schneider, Stephan

2009-01-01

OBJECTIVE Noninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-cell–specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS We report the generation of SCAs that bind highly selective to either β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS Our data provide strong evidence that the presented SCAs are highly specific for pancreatic β-cells and enable imaging and quantification in vivo. PMID:19592622

Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.

PubMed

Choi, Jeong June; Park, Bo-Kyung; Park, Sunyoung; Yun, Chi-Young; Kim, Dong Hee; Kim, Jin Sook; Hwang, Eun Sook; Jin, Mirim

2009-03-01

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a wellknown antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

Selective androgen receptor modulators: in vitro and in vivo metabolism and analysis.

PubMed

de Rijke, Eva; Essers, Martien L; Rijk, Jeroen C W; Thevis, Mario; Bovee, Toine F H; van Ginkel, Leendert A; Sterk, Saskia S

2013-01-01

For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l⁻¹ and a detection capability of 0.025 µg l⁻¹ in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.

The test chemical selection procedure of the European Centre for the Validation of Alternative Methods for the EU Project ReProTect.

PubMed

Pazos, Patricia; Pellizzer, Cristian; Stummann, Tina C; Hareng, Lars; Bremer, Susanne

2010-08-01

The selection of reference compounds is crucial for a successful in vitro test development in order to proof the relevance of the test system. This publication describes the criteria and the selection strategy leading to a list of more than 130 chemicals suitable for test development within the ReProTect project. The presented chemical inventory aimed to support the development and optimization of in vitro tests that seek to fulfill ECVAM's criteria for entering into the prevalidation. In order to select appropriate substances, a primary database was established compiling information from existing databases. In a second step, predefined selection criteria have been applied to obtain a comprehensive list ready to undergo a peer review process from independent experts with industrial, academic and regulatory background. Finally, a peer reviewed chemical list containing 13 substances challenging endocrine disrupter tests, additional 50 substances serving as reference chemicals for various tests evaluating effects on male and female fertility, and finally 61 substances were identified as known to provoke effects on the early development of mammalian offspring. The final list aims to cover relevant and specific mode/site of actions as they are known to be relevant for various substance classes. However, the recommended list should not be interpreted as a list of reproductive toxicants, because such a description requires proven associations with adverse effects of mammalian reproduction, which are subject of regulatory decisions done by involved competent authorities. Copyright 2010 Elsevier Inc. All rights reserved.

Bayesian classification for the selection of in vitro human embryos using morphological and clinical data.

PubMed

Morales, Dinora Araceli; Bengoetxea, Endika; Larrañaga, Pedro; García, Miguel; Franco, Yosu; Fresnada, Mónica; Merino, Marisa

2008-05-01

In vitro fertilization (IVF) is a medically assisted reproduction technique that enables infertile couples to achieve successful pregnancy. Given the uncertainty of the treatment, we propose an intelligent decision support system based on supervised classification by Bayesian classifiers to aid to the selection of the most promising embryos that will form the batch to be transferred to the woman's uterus. The aim of the supervised classification system is to improve overall success rate of each IVF treatment in which a batch of embryos is transferred each time, where the success is achieved when implantation (i.e. pregnancy) is obtained. Due to ethical reasons, different legislative restrictions apply in every country on this technique. In Spain, legislation allows a maximum of three embryos to form each transfer batch. As a result, clinicians prefer to select the embryos by non-invasive embryo examination based on simple methods and observation focused on morphology and dynamics of embryo development after fertilization. This paper proposes the application of Bayesian classifiers to this embryo selection problem in order to provide a decision support system that allows a more accurate selection than with the actual procedures which fully rely on the expertise and experience of embryologists. For this, we propose to take into consideration a reduced subset of feature variables related to embryo morphology and clinical data of patients, and from this data to induce Bayesian classification models. Results obtained applying a filter technique to choose the subset of variables, and the performance of Bayesian classifiers using them, are presented.

EGFR targeted PLGA nanoparticles using gemcitabine for treatment of pancreatic cancer.

PubMed

Aggarwal, Sahil; Yadav, Sachin; Gupta, Swati

2011-02-01

The present study aimed to prepare and characterize anti EGFR monoclonal antibody (mab) conjugated Gemcitabine loaded PLGA nanoparticles for their selective delivery to pancreatic cells and evaluation of the systems in vitro. It was observed that direct covalent coupling of antibodies to glutaraldehyde activated nanoparticles is an appropriate method to achieve cell-type specific drug carrier systems based on polymeric nanoparticles that have potential to be applied for targeted chemotherapy in EGFR positive cancer.

Fitness of artemisinin-resistant Plasmodium falciparum in vitro

PubMed Central

Hott, Amanda; Tucker, Matthew S.; Casandra, Debora; Sparks, Kansas; Kyle, Dennis E.

2015-01-01

Objectives Drug resistance confers a fitness advantage to parasites exposed to frequent drug pressure, yet these mutations also may incur a fitness cost. We assessed fitness advantages and costs of artemisinin resistance in Plasmodium falciparum in vitro to understand how drug resistance will spread and evolve in a competitive environment. Methods Genotyping of SNPs, drug susceptibility assays and copy number determination were used to assess the impact of artemisinin resistance on parasite fitness. An artemisinin-resistant clone (C9) selected in vitro from an isogenic parental clone (D6) was used to conduct competitive growth studies to assess fitness of artemisinin resistance. The resistant and susceptible clones were mixed or grown alone in the presence and absence of drug pressure (dihydroartemisinin or pyrimethamine) to quantify the rate at which artemisinin resistance was gained or lost. Results We experimentally demonstrate for the first time that artemisinin resistance provides a fitness advantage that is selected for with infrequent exposure to drug, but is lost in the absence of exposure to artemisinin drugs. The best correlations with artemisinin resistance were decreased in vitro drug susceptibility to artemisinin derivatives, increased copy number of Pf3D7_1030100 and an SNP in Pf3D7_0307600. An SNP conferring an E208K mutation in the kelch gene (Pf3D7_1343700) was not associated with resistance. Furthermore, we observed second-cycle ring-stage dormancy induced by pyrimethamine, suggesting that dormancy is a fitness trait that provides an advantage for survival from antimalarial drug stress. Conclusions Artemisinin-resistant P. falciparum have a fitness advantage to survive and predominate in the population even in the face of infrequent exposure to artemisinin drugs. PMID:26203183

Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo.

PubMed

Arutla, Viswanath; Leal, Joseph; Liu, Xiaowei; Sokalingam, Sriram; Raleigh, Michael; Adaralegbe, Adejimi; Liu, Li; Pentel, Paul R; Hecht, Sidney M; Chang, Yung

2017-05-08

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC 50 ) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.

Interlaboratory Evaluation of in Vitro Cytotoxicity and Inflammatory Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

PubMed Central

Xia, Tian; Hamilton, Raymond F.; Bonner, James C.; Crandall, Edward D.; Elder, Alison; Fazlollahi, Farnoosh; Girtsman, Teri A.; Kim, Kwang; Mitra, Somenath; Ntim, Susana A.; Orr, Galya; Tagmount, Mani; Taylor, Alexia J.; Telesca, Donatello; Tolic, Ana; Vulpe, Christopher D.; Walker, Andrea J.; Wang, Xiang; Witzmann, Frank A.; Wu, Nianqiang; Xie, Yumei; Zink, Jeffery I.; Nel, Andre

2013-01-01

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity. PMID:23649538

Collecting near mature and immature orchid seeds for ex situ conservation: 'in vitro collecting' as a case study.

PubMed

Kendon, Jonathan P; Rajaovelona, Landy; Sandford, Helen; Fang, Rui; Bell, Jake; Sarasan, Viswambharan

2017-08-08

Lack of phenological information and efficient collecting methods are considered impediments for orchid seed collecting. This leads to opportunistic collecting as part of general seed collecting schedules that may last few weeks especially in remote areas. The study explored the feasibility of collecting near mature and immature seeds to support conservation action plans. Mature, near mature and immature seeds of orchids were collected from the wild in the Central Highlands of Madagascar (CHM). Seed capsules were collected in sterile culture medium in the wild, to prevent deterioration of seeds inside the capsule after collecting, later to be cultured under laboratory conditions. Seed capsules collected by the in vitro collecting (IVC) method were kept in very good condition for up to 4 weeks before germination under in vitro conditions. Significantly faster and higher germination rate (p < 0.001) than mature seeds was observed in a number of taxa collected during a 3 year-long study. In some taxa even immature seeds, with no apparent sign of testa covering the embryo, germinated following IVC where mature seeds failed to germinate. We propose that IVC method has potential to complement conventional seed collecting by increasing the germplasm that can be used in integrated conservation action plans. Improvements can be made in developing collections for taxa from biodiversity hotspots and remote areas where collecting requires considerable resources. This method can further be used on a wider selection of plants from different geographic areas and on embryo rescue programmes for economically important plants.

Formulation and evaluation of sublingual tablets containing Sumatriptan succinate

PubMed Central

Prajapati, Shailesh T; Patel, Parth B; Patel, Chhagan N

2012-01-01

Objective: Sumatriptan succinate is a selective 5-hydroxytryptamine-1 receptor agonist effective in the acute treatment of migraine headaches, having low bioavailability of about 15% orally due to first-pass metabolism. The purpose of this research was to mask the intensely bitter taste of Sumatriptan succinate and to formulate fast-acting, taste-masked sublingual tablet formulation. Materials and Methods: Taste masking was performed by solid dispersion method with mannitol and ion exchange with Kyron T 114 because it releases the drug in salivary pH. The resultant batches were evaluated for in-vivo taste masking as well compatability study (Fourier transform infrared (FTIR) and differential scanning calorimetry (DSC)). For a better feel in the mouth, menthol and sweetener Na saccharine were added to the tablet formulation. The tablets were prepared by direct compression and evaluated for weight variation, thickness, friability, drug content, hardness, disintegration time, wetting time, in vitro drug release, and in vitro permeation study. Results and Discussion: Optimized batches disintegrated in vitro within 28-34 s. Maximum drug release could be achieved with in 10 min for the solid dispersion batches and 14-15 min for the ion-exchange batches with Kyron T 114. The optimized tablet formulation showed better taste and the formulated sublingual tablets may act as a potential alternate for the Sumatriptan succinate oral tablet. Conclusion: Sumatriptan succinate can be successfully taste-masked by both the solid dispersion method using mannitol by the melting method and Ion exchange resin with Kyron T114. It was also concluded that prepared formulation improve bioavailability by prevention of first pass metabolism. PMID:23373008

Prospective performance evaluation of selected common virtual screening tools. Case study: Cyclooxygenase (COX) 1 and 2.

PubMed

Kaserer, Teresa; Temml, Veronika; Kutil, Zsofia; Vanek, Tomas; Landa, Premysl; Schuster, Daniela

2015-01-01

Computational methods can be applied in drug development for the identification of novel lead candidates, but also for the prediction of pharmacokinetic properties and potential adverse effects, thereby aiding to prioritize and identify the most promising compounds. In principle, several techniques are available for this purpose, however, which one is the most suitable for a specific research objective still requires further investigation. Within this study, the performance of several programs, representing common virtual screening methods, was compared in a prospective manner. First, we selected top-ranked virtual screening hits from the three methods pharmacophore modeling, shape-based modeling, and docking. For comparison, these hits were then additionally predicted by external pharmacophore- and 2D similarity-based bioactivity profiling tools. Subsequently, the biological activities of the selected hits were assessed in vitro, which allowed for evaluating and comparing the prospective performance of the applied tools. Although all methods performed well, considerable differences were observed concerning hit rates, true positive and true negative hits, and hitlist composition. Our results suggest that a rational selection of the applied method represents a powerful strategy to maximize the success of a research project, tightly linked to its aims. We employed cyclooxygenase as application example, however, the focus of this study lied on highlighting the differences in the virtual screening tool performances and not in the identification of novel COX-inhibitors. Copyright © 2015 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

In vitro testing of biological control agents on A1 and A2 isolates of Phytophthora ramorum

Treesearch

Marianne Elliott; Simon Shamoun

2008-01-01

Biological control products were tested in vitro with six isolates of Phytophthora ramorum. These isolates were geographically diverse and were selected based on their pathogenicity to detached Rhododendron leaves. In addition to five commercially available biocontrol products, nine species of Trichoderma were tested. The in vitro...

Accelerated in-vitro release testing methods for extended-release parenteral dosage forms.

PubMed

Shen, Jie; Burgess, Diane J

2012-07-01

This review highlights current methods and strategies for accelerated in-vitro drug release testing of extended-release parenteral dosage forms such as polymeric microparticulate systems, lipid microparticulate systems, in-situ depot-forming systems and implants. Extended-release parenteral dosage forms are typically designed to maintain the effective drug concentration over periods of weeks, months or even years. Consequently, 'real-time' in-vitro release tests for these dosage forms are often run over a long time period. Accelerated in-vitro release methods can provide rapid evaluation and therefore are desirable for quality control purposes. To this end, different accelerated in-vitro release methods using United States Pharmacopeia (USP) apparatus have been developed. Different mechanisms of accelerating drug release from extended-release parenteral dosage forms, along with the accelerated in-vitro release testing methods currently employed are discussed. Accelerated in-vitro release testing methods with good discriminatory ability are critical for quality control of extended-release parenteral products. Methods that can be used in the development of in-vitro-in-vivo correlation (IVIVC) are desirable; however, for complex parenteral products this may not always be achievable. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

Accelerated in vitro release testing methods for extended release parenteral dosage forms

PubMed Central

Shen, Jie; Burgess, Diane J.

2012-01-01

Objectives This review highlights current methods and strategies for accelerated in vitro drug release testing of extended release parenteral dosage forms such as polymeric microparticulate systems, lipid microparticulate systems, in situ depot-forming systems, and implants. Key findings Extended release parenteral dosage forms are typically designed to maintain the effective drug concentration over periods of weeks, months or even years. Consequently, “real-time” in vitro release tests for these dosage forms are often run over a long time period. Accelerated in vitro release methods can provide rapid evaluation and therefore are desirable for quality control purposes. To this end, different accelerated in vitro release methods using United States Pharmacopoeia (USP) apparatus have been developed. Different mechanisms of accelerating drug release from extended release parenteral dosage forms, along with the accelerated in vitro release testing methods currently employed are discussed. Conclusions Accelerated in vitro release testing methods with good discriminatory ability are critical for quality control of extended release parenteral products. Methods that can be used in the development of in vitro-in vivo correlation (IVIVC) are desirable, however for complex parenteral products this may not always be achievable. PMID:22686344

Towards well-defined gold nanomaterials via diafiltration and aptamer mediated synthesis

NASA Astrophysics Data System (ADS)

Sweeney, Scott Francis

Gold nanoparticles have garnered recent attention due to their intriguing size- and shape-dependent properties. Routine access to well-defined gold nanoparticle samples in terms of core diameter, shape, peripheral functionality and purity is required in order to carry out fundamental studies of their properties and to utilize these properties in future applications. For this reason, the development of methods for preparing well-defined gold nanoparticle samples remains an area of active research in materials science. In this dissertation, two methods, diafiltration and aptamer mediated synthesis, are explored as possible routes towards well-defined gold nanoparticle samples. It is shown that diafiltration has considerable potential for the efficient and convenient purification and size separation of water-soluble nanoparticles. The suitability of diafiltration for (i) the purification of water-soluble gold nanoparticles, (ii) the separation of a bimodal distribution of nanoparticles into fractions, (iii) the fractionation of a polydisperse sample and (iv) the isolation of [rimers from monomers and aggregates is studied. NMR, thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS) measurements demonstrate that diafiltration produces highly pure nanoparticles. UV-visible spectroscopic and transmission electron microscopic analyses show that diafiltration offers the ability to separate nanoparticles of disparate core size, including linked nanoparticles. These results demonstrate the applicability of diafiltration for the rapid and green preparation of high-purity gold nanoparticle samples and the size separation of heterogeneous nanoparticle samples. In the second half of the dissertation, the identification of materials specific aptamers and their use to synthesize shaped gold nanoparticles is explored. The use of in vitro selection for identifying materials specific peptide and oligonucleotide aptamers is reviewed, outlining the specific requirements of in vitro selection for materials and the ways in which the field can be advanced. A promising new technique, in vitro selection on surfaces (ISOS), is developed and the discovery using ISOS of RNA aptamers that bind to evaporated gold is discussed. Analysis of the isolated gold binding RNA aptamers indicates that they are highly structured with single-stranded polyadenosine binding motifs. These aptamers, and similarly isolated peptide aptamers, are briefly explored for their ability to synthesize gold nanoparticles. This dissertation contains both previously published and unpublished co-authored material.

The effect of red light irradiation on spermatozoa DNA

NASA Astrophysics Data System (ADS)

Chow, Kay W.; Preece, Daryl; Gomez-Godinez, Veronica; Berns, Michael W.

2016-09-01

A key goal in the conservation of endangered species is to increase successful reproduction. In cases where traditional methods of in vitro fertilization are unsuccessful, new methods of assisted reproduction are needed. One option is selective fertilization via optically trapped sperm. A more passive option is red light irradiation. Red light irradiation has been shown to increase sperm motility, thus increasing fertilizing potential. However, there is some concern that exposure to laser irradiation induces the production of oxidative species in cells, which can be damaging to DNA. In order to test the safety of irradiating sperm, sperm samples were exposed to 633 nm laser light and their DNA were tested for oxidative damage. Using fluorescence microscopy, antibody staining, and ELISA to detect oxidative DNA damage, it was concluded that red light irradiation does not pose a safety risk to sperm DNA. The use of red light on sperm has potential in both animal conservation and human reproduction techniques. This method can also be used in conjunction with optical trapping for viable sperm selection.

Informing Selection of Nanomaterial Concentrations for ...

EPA Pesticide Factsheets

Little justification is generally provided for selection of in vitro assay testing concentrations for engineered nanomaterials (ENMs). Selection of concentration levels for hazard evaluation based on real-world exposure scenarios is desirable. We reviewed published ENM concentrations measured in air in manufacturing and R&D labs to identify input levels for estimating ENM mass retained in the human lung using the Multiple-Path Particle Dosimetry (MPPD) model. Model input parameters were individually varied to estimate alveolar mass retained for different particle sizes (5-1000 nm), aerosol concentrations (0.1, 1 mg/m3), aspect ratios (2, 4, 10, 167), and exposure durations (24 hours and a working lifetime). The calculated lung surface concentrations were then converted to in vitro solution concentrations. Modeled alveolar mass retained after 24 hours is most affected by activity level and aerosol concentration. Alveolar retention for Ag and TiO2 nanoparticles and CNTs for a working lifetime (45 years) exposure duration is similar to high-end concentrations (~ 30-400 μg/mL) typical of in vitro testing reported in the literature. Analyses performed are generally applicable to provide ENM testing concentrations for in vitro hazard screening studies though further research is needed to improve the approach. Understanding the relationship between potential real-world exposures and in vitro test concentrations will facilitate interpretation of toxicological results

Irreversible 4-Aminopiperidine Transglutaminase 2 Inhibitors for Huntington's Disease.

PubMed

Prime, Michael E; Brookfield, Frederick A; Courtney, Stephen M; Gaines, Simon; Marston, Richard W; Ichihara, Osamu; Li, Marie; Vaidya, Darshan; Williams, Helen; Pedret-Dunn, Anna; Reed, Laura; Schaertl, Sabine; Toledo-Sherman, Leticia; Beconi, Maria; Macdonald, Douglas; Muñoz-Sanjuan, Ignacio; Dominguez, Celia; Wityak, John

2012-09-13

A new series of potent TG2 inhibitors are reported that employ a 4-aminopiperidine core bearing an acrylamide warhead. We establish the structure-activity relationship of this new series and report on the transglutaminase selectivity and in vitro ADME properties of selected compounds. We demonstrate that the compounds do not conjugate glutathione in an in vitro setting and have superior plasma stability over our previous series.

Development of an Extended-Release Formulation for Apremilast and a Level A in Vitro-in Vivo Correlation Study in Beagle Dogs.

PubMed

Tang, Meiqiong; Hu, Ping; Huang, Shigui; Zheng, Qiang; Yu, Hao; He, Yun

2016-11-01

The primary objective of the present study was to develop extended-release matrix formulations of apremilast for the oral delivery and to study their in vitro and in vivo correlation. Five extended-release formulations containing hydroxypropylmethylcellulose (HPMC) as the retarding excipient with different release rate of apremilast were prepared. Dissolution tests of all the formulated tablets were performed in water, pH 4.0 and 6.8 buffer solutions. The in vitro release kinetics was studied and supported by Korsmeyer-Peppas's equation as it presented highest values of correlation coefficients (r 2 up to 0.966). Among all formulated tablets, F2 (HPMC 25%) and F4 (HPMC 35%) were selected to perform an in vivo study in beagle dogs to obtain various pharmacokinetic parameters, i.e., peak plasma concentration (C max ), time to peak plasma concentration (t max ), area under the plasma-concentration vs. time curve (AUC). Higher t max and t 1/2 , lower C max and elimination coefficient (K e ) were observed for both extended formulations compared to marketed immediate-release products (Otezla ® ). Level A in vitro-in vivo correlations were created with the help of Wagner-Nelson and numeric deconvolution methods. Both formulations showed good in vitro-in vivo correlations whose accuracies were further verified by an internal validation.

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

PubMed Central

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study. PMID:26862534

Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining.

PubMed

Fathi, Mohamed; Ashry, Mohamed; Salama, Ali; Badr, Magdy R

2017-08-01

The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus-oocyte complexes (COCs) from BCB+, BCB- and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB- and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

Selective and self-guided micro-ablation of tissue with plasmonic nanobubbles.

PubMed

Lukianova-Hleb, Ekaterina Y; Koneva, Irina I; Oginsky, Alexander O; La Francesca, Saverio; Lapotko, Dmitri O

2011-03-01

The accuracy, selectivity, and safety of surgical and laser methods for tissue elimination are often limited at microscale. We developed a novel agent, the plasmonic nanobubble (PNB), for optically guided selective elimination of the target tissue with micrometer precision. PNBs were tested in vitro in the two different models of superficial tumors and vascular plaques. PNBs were selectively generated around gold nanoparticles (delivered to the target tissues) with short laser pulses. Monolayers of cancerous cells and atherosclerotic plaque tissue were eliminated with PNBs with micrometer accuracy and without thermal and mechanical damage to collateral normal tissues. The effect of the PNB was dynamically controlled through the fluence of laser pulses (532 nm, duration 0.5 and 10 ns) and was guided through the optical scattering by PNB. Plasmonic nanobubbles were shown to provide precise, tunable, selective, and guided ablation of tissue at a microscopic level and could be employed as a new generation of surgical tools. Copyright © 2011 Elsevier Inc. All rights reserved.

Masked Selection: A Straightforward and Flexible Approach for the Selection of Binders Against Specific Epitopes and Differentially Expressed Proteins by Phage Display*

PubMed Central

Even-Desrumeaux, Klervi; Nevoltris, Damien; Lavaut, Marie Noelle; Alim, Karima; Borg, Jean-Paul; Audebert, Stéphane; Kerfelec, Brigitte; Baty, Daniel; Chames, Patrick

2014-01-01

Phage display is a well-established procedure to isolate binders against a wide variety of antigens that can be performed on purified antigens, but also on intact cells. As selection steps are performed in vitro, it is possible to focus the outcome of the selection on relevant epitopes by performing some additional steps, such as depletion or competitive elutions. However in practice, the efficiency of these steps is often limited and can lead to inconsistent results. We have designed a new selection method named masked selection, based on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency and flexibility of this method by selecting single-domain antibodies against a specific portion of a fusion protein, by selecting binders against several members of the seven transmembrane receptor family using transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples. The relevance of this approach for antibody-based therapies was further validated by the identification of four of these markers, Epithelial cell adhesion molecule, Transferrin receptor 1, Metastasis cell adhesion molecule, and Sushi containing domain 2, using immunoprecipitation and mass spectrometry. This new phage display strategy can be applied to any type of antibody fragments or alternative scaffolds, and is especially suited for the rapid discovery and identification of cell surface markers. PMID:24361863

LC-MS/MS method for the determination of the prodrug aripiprazole lauroxil and its three metabolites in plasma and its application to in vitro biotransformation and animal pharmacokinetic studies.

PubMed

Sun, Yiying; Lu, Xiaoyan; Gai, Yunyun; Sha, Chunjie; Leng, Guangyi; Yang, Xiucheng; Liu, Wanhui

2018-04-01

A sensitive and selective LC-MS/MS method for determination of the prodrug aripiprazole lauroxil (AL) and the three metabolites (N-hydroxymethyl aripiprazole [NHA], aripiprazole [ARP], and dehydro aripiprazole [DHA]) in plasma was developed using ARP-d 8 as an internal standard. The analytes were determined on an AB Sciex Triple Quad™ 4500 system using positive ion electrospray ionization and selected multiple reaction monitoring mode. Solid phase extraction was applied for sample preparation for AL, ARP, and DHA, and protein precipitation for NHA. Chromatographic separation was performed on an Agilent Eclipse XDB-CN column (100 × 2.1 mm i.d., 3.5 μm) using the mobile phase of water and acetonitrile (25:75, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear ranges for AL, NHA, ARP, and DHA were 0.5-50 ng/mL, 1.0-50 ng/mL, 0.5-50 ng/mL, and 0.05-5.0 ng/mL, respectively. The average recovery in the plasma sample was stable and reproducible. The precision and accuracy of the intra- and inter-run were within assay variability criteria limits. The developed method was suitable for in vitro biotransformation studies in plasma and animal pharmacokinetic studies after intramuscular injection of AL formulations. Copyright © 2018. Published by Elsevier B.V.

PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

PubMed Central

Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

2015-01-01

Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

PubMed Central

Boogaard, Ivette; Smith, Melanie; Pulli, Kristiina; Szynol, Agnieszka; Albertus, Faywell; Lamers, Marieke B. A. C.; Dijkstra, Sipke; Kordt, Daniel; Reindl, Wolfgang; Herrmann, Frank; McAllister, George; Fischer, David F.; Munoz-Sanjuan, Ignacio

2014-01-01

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples. PMID:24816435

The accuracy of home glucose meters in hypoglycemia.

PubMed

Sonmez, Alper; Yilmaz, Zeynep; Uckaya, Gokhan; Kilic, Selim; Tapan, Serkan; Taslipinar, Abdullah; Aydogdu, Aydogan; Yazici, Mahmut; Yilmaz, Mahmut Ilker; Serdar, Muhittin; Erbil, M Kemal; Kutlu, Mustafa

2010-08-01

Home glucose meters (HGMs) may not be accurate enough to sense hypoglycemia. We evaluated the accuracy and the capillary and venous comparability of five different HGMs (Optium Xceed [Abbott Diabetes Care, Alameda, CA, USA], Contour TS [Bayer Diabetes Care, Basel, Switzerland], Accu-Chek Go [Roche Ltd., Basel, Switzerland], OneTouch Select [Lifescan, Milpitas, CA, USA], and EZ Smart [Tyson Bioresearch Inc., Chu-Nan, Taiwan]) in an adult population. The insulin hypoglycemia test was performed to 59 subjects (56 males; 23.6 +/- 3.2 years old). Glucose was measured from forearm venous blood and finger capillary samples both before and after regular insulin (0.1 U/kg) was injected. Venous samples were analyzed in the reference laboratory by the hexokinase method. In vitro tests for method comparison and precision analyses were also performed by spiking the glucose-depleted venous blood. All HGMs failed to sense hypoglycemia to some extend. EZ Smart was significantly inferior in critical error Zone D, and OneTouch Select was significantly inferior in the clinically unimportant error Zone B. Accu-Chek Go, Optium Xceed, and Contour TS had similar performances and were significantly better than the other two HGMs according to error grid analysis or International Organization for Standardization criteria. The in vitro tests were consistent with the above clinical data. The capillary and venous consistencies of Accu-Chek Go and OneTouch Select were better than the other HGMs. The present results show that not all the HGMs are accurate enough in low blood glucose levels. The patients and the caregivers should be aware of these restrictions of the HGMs and give more credit to the symptoms of hypoglycemia than the values obtained by the HGMs. Finally, these results indicate that there is a need for the revision of the accuracy standards of HGMs in low blood glucose levels.

Enhanced in Vitro Anti-Tumor Activity of 5-Azacytidine by Entrapment into Solid Lipid Nanoparticles

PubMed Central

Jahanfar, Farhad; Hasani, Akbar; Shanebandi, Dariush; Rahmati, Mohammad; Hamishehkar, Hamed

2016-01-01

Purpose: In this study the effectiveness of encapsulating of 5-azacytidine into the lipid nanoparticles was investigated and in vitro effect of encapsulated 5-azacytidine studied on MCF-7 cell lines Methods: 5-azacytidine -loaded solid lipid nanoparticles were produced by double emulsification (w/o/w) method by using stearic acid as lipid matrix, soy lecithin and poloxamer 407 as surfactant and co-surfactant respectively. Particle size, zeta potential, surface morphology, entrapment efficiency and kinetic of drug release were studied. In vitro effect of 5-azacytidine on MCF-7 cell line studied by MTT assay, DAPI staining, Rhodamine B relative uptake, and also Real time RT-PCR was performed for studying difference effect of free and encapsulated drug on expression of RARß2 gene. Results: The formulation F5 with 55.84±0.46 % of entrapment efficiency shows zero order kinetic of drug release and selected for in vitro studies; the cytotoxicity of free drug and encapsulated drug in 48 h of incubation have significant difference. DAPI staining shows morphology of apoptotic nucleus in both free and encapsulated drug, Rhodamine B labeled SLNs show time dependency and accumulation of SLNs in cytoplasm. Real time qRT-PCR doesn’t show any significant difference (p>0.05) in expression of RARß2 gene in both cells treated with free or encapsulated drug. Conclusion: The results of the present study indicated that the entrapment of 5-azacytidine into SLNs enhanced its cytotoxicity performance and may pave a way for the future design of a desired dosage form for 5-azacytidine. PMID:27766220

Foreskin-isolated keratinocytes provide successful extemporaneous autologous paediatric skin grafts.

PubMed

Mcheik, Jiad N; Barrault, Christine; Pedretti, Nathalie; Garnier, Julien; Juchaux, Franck; Levard, Guillaume; Morel, Franck; Lecron, Jean-Claude; Bernard, François-Xavier

2016-03-01

Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls. Copyright © 2013 John Wiley & Sons, Ltd.

Stem Cell Therapy for Healing Wounded Skin and Soft Tissues

DTIC Science & Technology

2012-07-01

changes of ASC surface markers due to repetitive in vitro sub-culturing. ASCs were harvested, washed in PBS to remove cell culture medium, and resuspended...Our in vitro and in vivo studies suggest that ASC and BM-MSC are not identical, though they have similar surface markers . We found that topically...ofpolybrene. Transduced cells were selected by treating 10 J.!g/rnl ofblasticidin. GFP expressing cells were further selected by flow cytometry using

In vitro validation of self designed "universal human Influenza A siRNA".

PubMed

Jain, Bhawana; Jain, Amita; Prakash, Om; Singh, Ajay Kr; Dangi, Tanushree; Singh, Mastan; Singh, K P

2015-08-01

The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.

Studies on the contributions of smoke constituents, individually and in mixtures, in a range of in vitro bioactivity assays.

PubMed

Stabbert, Regina; Dempsey, Ruth; Diekmann, Joerg; Euchenhofer, Christian; Hagemeister, Timo; Haussmann, Hans-Juergen; Knorr, Arno; Mueller, Boris P; Pospisil, Pavel; Reininghaus, Wolf; Roemer, Ewald; Tewes, Franz J; Veltel, Detlef J

2017-08-01

Tobacco smoke is a complex mixture with over 8700 identified constituents. Smoking causes many diseases including lung cancer, cardiovascular disease, and chronic obstructive pulmonary disease. However, the mechanisms of how cigarette smoke impacts disease initiation or progression are not well understood and individual smoke constituents causing these effects are not generally agreed upon. The studies reported here were part of a series of investigations into the contributions of selected smoke constituents to the biological activity of cigarette smoke. In vitro cytotoxicity measured by the neutral red uptake (NRU) assay and in vitro mutagenicity determined in the Ames bacterial mutagenicity assay (BMA) were selected because these assays are known to produce reproducible, quantitative results for cigarette smoke under standardized exposure conditions. In order to determine the contribution of individual cigarette smoke constituents, a fingerprinting method was developed to semi-quantify the mainstream smoke yields. For cytotoxicity, 90% of gas vapor phase (GVP) cytotoxicity of the Kentucky Reference cigarette 1R4F was explained by 3 aldehydes and 40% of the 1R4F particulate phase cytotoxicity by 10 smoke constituents, e.g., hydroquinone. In the microsuspension version of the BMA, 4 aldehydes accounted for approximately 70% of the GVP mutagenicity. Finally, the benefits of performing such studies along with the difficulties in interpretation in the context of smoking are discussed. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

In vitro screening techniques for reactive metabolites for minimizing bioactivation potential in drug discovery.

PubMed

Prakash, Chandra; Sharma, Raman; Gleave, Michelle; Nedderman, Angus

2008-11-01

Drug induced toxicity remains one of the major reasons for failures of new pharmaceuticals, and for the withdrawal of approved drugs from the market. Efforts are being made to reduce attrition of drug candidates, and to minimize their bioactivation potential in the early stages of drug discovery in order to bring safer compounds to the market. Therefore, in addition to potency and selectivity; drug candidates are now selected on the basis of acceptable metabolism/toxicology profiles in preclinical species. To support this, new approaches have been developed, which include extensive in vitro methods using human and animal hepatic cellular and subcellular systems, recombinant human drug metabolizing enzymes, increased automation for higher-throughput screens, sensitive analytical technologies and in silico computational models to assess the metabolism aspects of the new chemical entities. By using these approaches many compounds that might have serious adverse reactions associated with them are effectively eliminated before reaching clinical trials, however some toxicities such as those caused by idiosyncratic responses, are not detected until a drug is in late stages of clinical trials or has become available to the market. One of the proposed mechanisms for the development of idiosyncratic drug toxicity is the bioactivation of drugs to form reactive metabolites by drug metabolizing enzymes. This review discusses the different approaches to, and benefits of using existing in vitro techniques, for the detection of reactive intermediates in order to minimize bioactivation potential in drug discovery.

Aptamer Database

PubMed Central

Lee, Jennifer F.; Hesselberth, Jay R.; Meyers, Lauren Ancel; Ellington, Andrew D.

2004-01-01

The aptamer database is designed to contain comprehensive sequence information on aptamers and unnatural ribozymes that have been generated by in vitro selection methods. Such data are not normally collected in ‘natural’ sequence databases, such as GenBank. Besides serving as a storehouse of sequences that may have diagnostic or therapeutic utility, the database serves as a valuable resource for theoretical biologists who describe and explore fitness landscapes. The database is updated monthly and is publicly available at http://aptamer.icmb.utexas.edu/. PMID:14681367

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

Study of the photostability of 18 sunscreens in creams by measuring the SPF in vitro.

PubMed

Couteau, Céline; Faure, Aurélie; Fortin, June; Paparis, Eva; Coiffard, Laurence J M

2007-05-09

The target of this research was to evaluate the photostability of various sunscreen agents incorporated into an O/W emulsion. The concept of photostability is very important in the field of solar protection. The effectiveness of the anti-solar products is quantified using a universal indicator: the sun protection factor (SPF). This number which can be found on packaging can be given in two different ways: by methods in vivo (Colipa method) and in vitro. It is this last method which was adopted for this study. According to selected filter UVB (currently directive 76/768/EEC modified authorized 18 filters UVB), we can obtain more or less effective creams. We chose the irradiation of sun lotions formulated using the authorized filters, used with their maximum amount of employment, in a Suntest, with an irradiance of 650 W/m(2) throughout variable time. With interval of regular time, one carries out a measurement of SPF in order to establish for each filter the kinetics SPF=f(time). An indicator of stability (t(90)) is then given. In this way, we could classify the filters by order of increasing photostability.

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

PubMed

Kang, Homan; Jeong, Sinyoung; Jo, Ahla; Chang, Hyejin; Yang, Jin-Kyoung; Jeong, Cheolhwan; Kyeong, San; Lee, Youn Woo; Samanta, Animesh; Maiti, Kaustabh Kumar; Cha, Myeong Geun; Kim, Taek-Keun; Lee, Sukmook; Jun, Bong-Hyun; Chang, Young-Tae; Chung, Junho; Lee, Ho-Young; Jeong, Dae Hong; Lee, Yoon-Sik

2018-02-01

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Homogeneous Biosensing Based on Magnetic Particle Labels

PubMed Central

Schrittwieser, Stefan; Pelaz, Beatriz; Parak, Wolfgang J.; Lentijo-Mozo, Sergio; Soulantica, Katerina; Dieckhoff, Jan; Ludwig, Frank; Guenther, Annegret; Tschöpe, Andreas; Schotter, Joerg

2016-01-01

The growing availability of biomarker panels for molecular diagnostics is leading to an increasing need for fast and sensitive biosensing technologies that are applicable to point-of-care testing. In that regard, homogeneous measurement principles are especially relevant as they usually do not require extensive sample preparation procedures, thus reducing the total analysis time and maximizing ease-of-use. In this review, we focus on homogeneous biosensors for the in vitro detection of biomarkers. Within this broad range of biosensors, we concentrate on methods that apply magnetic particle labels. The advantage of such methods lies in the added possibility to manipulate the particle labels by applied magnetic fields, which can be exploited, for example, to decrease incubation times or to enhance the signal-to-noise-ratio of the measurement signal by applying frequency-selective detection. In our review, we discriminate the corresponding methods based on the nature of the acquired measurement signal, which can either be based on magnetic or optical detection. The underlying measurement principles of the different techniques are discussed, and biosensing examples for all techniques are reported, thereby demonstrating the broad applicability of homogeneous in vitro biosensing based on magnetic particle label actuation. PMID:27275824

In vitro antimicrobial activities of cinnamon bark oil, anethole, carvacrol, eugenol and guaiazulene against Mycoplasma hominis clinical isolates.

PubMed

Sleha, Radek; Mosio, Petra; Vydrzalova, Marketa; Jantovska, Alexandra; Bostikova, Vanda; Mazurova, Jaroslava

2014-06-01

The aim of this study was to evaluate the antimicrobial effects of five natural substances against 50 clinical isolates of Mycoplasma hominis. The in vitro activity of selected natural compounds, cinnamon bark oil, anethole, carvacrol, eugenol and guaiazulene, was investigated against 50 M. hominis isolates cultivated from cervical swabs by the broth dilution method. All showed valuable antimicrobial activity against the tested isolates. Oil from the bark of Cinnamomum zeylanicum (MBC90 = 500 µg/mL) however was found to be the most effective. Carvacrol (MBC90 = 600 µg/mL) and eugenol (MBC90 = 1000 µg/mL) also possessed strong antimycoplasmal activity. The results indicate that cinnamon bark oil, carvacrol and eugenol have strong antimycoplasmal activity and the potential for use as antimicrobial agents in the treatment of mycoplasmal infections.

An in-vitro-in-vivo model for the transdermal delivery of cholecalciferol for the purposes of rodent management.

PubMed

Davies, J; Ingham, A

2015-06-20

The natural selection of anticoagulant resistant rats has resulted in a need for an alternative to anticoagulant rodenticides which differs in both active ingredient and in the method of dosing. Cholecalciferol toxicity to rodents using the dermal route is demonstrated using a variety of penetration enhancing formulations in two in-vitro models and finally in-vivo. A 1 ml dose of 50/50 (v/v) DMSO/ethanol containing 15% (v/v) PEG 200 and 20% (w/v) cholecalciferol was judged as 'sufficiently effective' in line with the European Union's Biocidal Products Regulation (No. 528/2012) during in-vivo studies. This dose was found to cause 100% mortality in a rat population in 64.4h (± 22h). Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Chemical Retraction Agents - in vivo and in vitro Studies into their Physico-Chemical Properties, Biocompatibility with Gingival Margin Tissues and Compatibility with Elastomer Impression Materials.

PubMed

Nowakowska, Danuta; Saczko, Jolanta; Kulbacka, Julita; Wicckiewicz, Wlodzimierz

2017-01-01

Gingival margin retraction/displacement (GMR/D) is a commonly accepted procedure in restorative dentistry. Of the various retraction methods, the chemo-mechanical approach with retraction media and chemical retraction agents (ChRAs) is mostly used. Different local and/or systemic side effects were observed after "chemical attacks" from these retraction agents. Moreover, no consensus exists as to the compatibility of chemical agents with different impression materials. This paper reports the findings of in vivo and in vitro studies and we discuss the physico-chemical properties of chemical retraction agents, their undesirable clinical side effects, biological activity and compatibility with selected groups of elastomer impression materials. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Convergent evolution of adenosine aptamers spanning bacterial, human, and random sequences revealed by structure-based bioinformatics and genomic SELEX

PubMed Central

Vu, Michael M. K.; Jameson, Nora E.; Masuda, Stuart J.; Lin, Dana; Larralde-Ridaura, Rosa; Lupták, Andrej

2012-01-01

SUMMARY Aptamers are structured macromolecules in vitro evolved to bind molecular targets, whereas in nature they form the ligand-binding domains of riboswitches. Adenosine aptamers of a single structural family were isolated several times from random pools but they have not been identified in genomic sequences. We used two unbiased methods, structure-based bioinformatics and human genome-based in vitro selection, to identify aptamers that form the same adenosine-binding structure in a bacterium, and several vertebrates, including humans. Two of the human aptamers map to introns of RAB3C and FGD3 genes. The RAB3C aptamer binds ATP with dissociation constants about ten times lower than physiological ATP concentration, while the minimal FGD3 aptamer binds ATP only co-transcriptionally. PMID:23102219

Dissofilm: A Novel Approach for Delivery of Phenobarbital; Design and Characterization

PubMed Central

Yellanki, SK; Jagtap, S; Masareddy, R

2011-01-01

An attempt to develop and evaluate mouth-dissolving film of phenobarbital for quick effect in treatment of epilepsy occurring in pediatric population has been made in the present study. Suitable film formers and plasticizers are selected based on optimization studies. Effect of superdisintegrants in formulation of mouth dissolving films at different concentrations has been investigated. Films were prepared by solvent casting method. The prepared films were evaluated for physicochemical parameters, in vitro disintegration and dissolution time, in vitro release rate study, stability study, and in vivo animal safety study. The best formulation was found to be F3 showing 96.57% drug release in 14 min, following first-order kinetics. X-Ray diffraction studies show change in crystalline nature of drug in formulation. In vivo studies in hamster reports effective and safe use of formulation in animals. PMID:21897656

A Classic Near-Infrared Probe Indocyanine Green for Detecting Singlet Oxygen.

PubMed

Tang, Cheng-Yi; Wu, Feng-Yao; Yang, Min-Kai; Guo, Yu-Min; Lu, Gui-Hua; Yang, Yong-Hua

2016-02-06

The revelation of mechanisms of photodynamic therapy (PDT) at the cellular level as well as singlet oxygen (¹O₂) as a second messengers requires the quantification of intracellular ¹O₂. To detect singlet oxygen, directly measuring the phosphorescence emitted from ¹O₂ at 1270 nm is simple but limited for the low quantum yield and intrinsic efficiency of ¹O₂ emission. Another method is chemically trapping ¹O₂ and measuring fluorescence, absorption and Electron Spin Resonance (ESR). In this paper, we used indocyanine green (ICG), the only near-infrared (NIR) probe approved by the Food and Drug Administration (FDA), to detect ¹O₂ in vitro. Once it reacts with ¹O₂, ICG is decomposed and its UV absorption at 780 nm decreases with the laser irradiation. Our data demonstrated that ICG could be more sensitive and accurate than Singlet Oxygen Sensor Green reagent(®) (SOSG, a commercialized fluorescence probe) in vitro, moreover, ICG functioned with Eosin Y while SOSG failed. Thus, ICG would reasonably provide the possibility to sense ¹O₂ in vitro, with high sensitivity, selectivity and suitability to most photosensitizers.

Emergence of a replicating species from an in vitro RNA evolution reaction

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Antitubercular activity of ZnO nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel.

PubMed

Gopala Krishna, Prashanth; Paduvarahalli Ananthaswamy, Prashanth; Trivedi, Priyanka; Chaturvedi, Vinita; Bhangi Mutta, Nagabhushana; Sannaiah, Ananda; Erra, Amani; Yadavalli, Tejabhiram

2017-06-01

In this study, we report the synthesis, structural and morphological characteristics of zinc oxide (ZnO) nanoparticles using solution combustion synthesis method where lemon juice was used as the fuel. In vitro anti-tubercular activity of the synthesized ZnO nanoparticles and their biocompatibility studies, both in vitro and in vivo were carried out. The synthesized nanoparticles showed inhibition of Mycobacterium tuberculosis H37Ra strain at concentrations as low as 12.5μg/mL. In vitro cytotoxicity study performed with normal mammalian cells (L929, 3T3-L1) showed that ZnO nanoparticles are non-toxic with a Selectivity Index (SI) >10. Cytotoxicity performed on two human cancer cell lines DU-145 and Calu-6 indicated the anti-cancer activity of ZnO nanoparticles at varied concentrations. Results of blood hemolysis indicated the biocompatibility of ZnO nanoparticles. Furthermore, in vivo toxicity studies of ZnO nanoparticles conducted on Swiss albino mice (for 14days as per the OECD 423 guidelines) showed no evident toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro production of huperzine A, a promising drug candidate for Alzheimer's disease.

PubMed

Ma, Xiaoqiang; Gang, David R

2008-07-01

Alzheimer's disease (AD) is growing in impact on human health. With no known cure, AD is one of the most expensive diseases in the world to treat. Huperzine A (HupA), a anti-AD drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), has been shown to be a powerful and selective inhibitor of acetylcholinesterase and has attracted widespread attention because of its unique pharmacological activities and low toxicity. As a result, HupA is becoming an important lead compound for drugs to treat AD. HupA is obtained naturally from very limited and slowly growing natural resources, members of the Huperziaceae. Unfortunately, the content of HupA is very low in the raw plant material. This has led to strong interest in developing sources of HupA. We have developed a method to propagate in vitro tissues of Phlegmariurus squarrosus, a member of the Huperziaceae, that produce high levels of HupA. The in vitro propagated tissues produce even higher levels of HupA than the natural plant, and may represent an excellent source for HupA.

Antimicrobial Peptides of Meat Origin - An In silico and In vitro Analysis.

PubMed

Keska, Paulina; Stadnik, Joanna

2017-01-01

The aim of this study was to evaluate the antimicrobial activity of meat protein-derived peptides against selected Gram-positive and Gram-negative bacteria. The in silico and in vitro approach was combined to determine the potency of antimicrobial peptides derived from pig (Sus scrofa) and cow (Bos taurus) proteins. The in silico studies consisted of an analysis of the amino acid composition of peptides obtained from the CAMPR database, their molecular weight and other physicochemical properties (isoelectric point, molar extinction coefficient, instability index, aliphatic index, hydropathy index and net charge). The degree of similarity was estimated between the antimicrobial peptide sequences derived from the slaughtered animals and the main meat proteins. Antimicrobial activity of peptides isolated from dry-cured meat products was analysed (in vitro) against two strains of pathogenic bacteria using the disc diffusion method. There was no evidence of growthinhibitory properties of peptides isolated from dry-cured meat products against Escherichia coli K12 ATCC 10798 and Staphylococcus aureus ATCC 25923. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Ultra-high-performance liquid chromatography-tandem mass spectrometry measurement of climbazole deposition from hair care products onto artificial skin and human scalp.

PubMed

Chen, Guoqiang; Hoptroff, Michael; Fei, Xiaoqing; Su, Ya; Janssen, Hans-Gerd

2013-11-22

A sensitive and specific ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the measurement of climbazole deposition from hair care products onto artificial skin and human scalp. Deuterated climbazole was used as the internal standard. Atmospheric pressure chemical ionization (APCI) in positive mode was applied for the detection of climbazole. For quantification, multiple reaction monitoring (MRM) transition 293.0>69.0 was monitored for climbazole, and MRM transition 296.0>225.1 for the deuterated climbazole. The linear range ran from 4 to 2000 ng mL(-1). The limit of detection (LOD) and the limit of quantification (LOQ) were 1 ng mL(-1) and 4 ng mL(-1), respectively, which enabled quantification of climbazole on artificial skin and human scalp at ppb level (corresponding to 16 ng cm(-2)). For the sampling of climbazole from human scalp the buffer scrub method using a surfactant-modified phosphate buffered saline (PBS) solution was selected based on a performance comparison of tape stripping, the buffer scrub method and solvent extraction in in vitro studies. Using this method, climbazole deposition in in vitro and in vivo studies was successfully quantified. Copyright © 2013 Elsevier B.V. All rights reserved.

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...

In vitro antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms.

PubMed Central

Slee, A M; O'Connor, J R

1983-01-01

The antibacterial activity of octenidine dihydrochloride (WIN 41464-2) against intact preformed in vitro plaques of four indigenous oral plaque-forming microorganisms, Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus, and Actinomyces naeslundii, was studied. Both absolute (plaque bactericidal index) and relative (chlorhexidine coefficient) indices of antiplaque efficacy were established. Octenidine dihydrochloride compared favorably with chlorhexidine digluconate with respect to overall antiplaque potency in this in vitro plaque bactericidal model. These data indicate that prudent selection of treatment concentration and duration and frequency of exposure should provide an effective means to aid in controlling dental caries and Actinomyces-associated disease in vivo. PMID:6847170

Selection and maturation of antibodies by phage display through fusion to pIX.

PubMed

Tornetta, Mark; Reddy, Ramachandra; Wheeler, John C

2012-09-01

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM. Copyright © 2012 Elsevier Inc. All rights reserved.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

Micropropagation of Helleborus through axillary budding.

PubMed

Beruto, Margherita; Viglione, Serena; Bisignano, Alessandro

2013-01-01

Helleborus genus, belonging to the Ranunculaceae family, has 20 species of herbaceous perennial flowering plants. The commercial exploitation of this plant is dependent on the selection and propagation of appropriate lines. High propagation rate could be accomplished by using a suitable tissue culture method enabling the rapid introduction of valuable selections in the market. However, in vitro cultivation of Helleborus is still very difficult. Thereby the development of reliable in vitro propagation procedures is crucial for future production systems. Axillary buds cultured on agar-solidified Murashige and Skoog medium supplemented with 1 mg/L benzyladenine, 0.1 mg/L β-naphthoxyacetic acid, and 2 mg/L isopentenyl adenine develop shoots after 16 weeks of culture under 16 h light regime, 50-60 μmol/s/m(2), and 19 ± 1°C. The multiplication rate ranges from 1.4 to 2.1. However, the genotype and the number of subcultures affect the efficiency of the micropropagation process. The rooting of shoots is about 80% in solidified MS medium containing 1 mg/L 1-naphthaleneacetic acid and 3 mg/L indole-3-butyric acid. The described protocol provides information which can contribute to the commercial production of Helleborus plants.

Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

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

Nemoto, Naoto, E-mail: nemoto@fms.saitama-u.ac.jp; Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005; Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844

2012-04-27

Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library bymore » in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.« less

Degradation and epimerization of ergot alkaloids after baking and in vitro digestion.

PubMed

Merkel, Stefan; Dib, Baha; Maul, Ronald; Köppen, Robert; Koch, Matthias; Nehls, Irene

2012-11-01

The degradation and epimerization of ergot alkaloids (EAs) in rye flour were investigated after baking cookies and subsequently subjecting them to an in vitro digestion model. Different steps of digestion were analyzed using salivary, gastric, and duodenal juices. The degradation and bidirectional conversion of the toxicologically relevant (R)-epimers and the biologically inactive (S)-epimers for seven pairs of EAs were determined by a HPLC method coupled with fluorescence detection. Baking cookies resulted in degradation of EAs (2-30 %) and a shift in the epimeric ratio toward the (S)-epimer for all EAs. The applied digestion model led to a selective toxification of ergotamine and ergosine, two ergotamine-type EAs. The initial percentage of the toxic (R)-epimer in relation to the total toxin content was considerably increased after digestion of cookies. Ergotamine and ergosine increased from 32 to 51 % and 35 to 55 %, respectively. In contrast, EAs of the ergotoxine type (ergocornine, α- and β-ergocryptine, and ergocristine) showed an epimeric shift toward their biologically inactive (S)-epimers. Further experiments indicated that the selective epimerization of ergotamine EAs occurs in the duodenal juice only. These results demonstrate that toxification of EAs in the intestinal tract should be taken into consideration.

Screening of bacterial antagonists for biological control of Phytophthora blight of pepper.

PubMed

Rajkumar, M; Lee, Wang Hyu; Lee, Kui Jae

2005-01-01

The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici. ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Mechanisms of selective antitumor action of cold atmospheric plasma

NASA Astrophysics Data System (ADS)

Graves, David; Bauer, Georg

2016-09-01

Transformed (precancerous) cells are known to be subject to elimination through intercellular RONS-dependent apoptosis-inducing signaling. It is a remarkable fact that the chemical species utilized by apoptosis induction in transformed cells are essentially identical to chemical species created by cold atmospheric plasma (CAP) in aqueous solutions. The association between CAP-induced biochemistry and natural cell anti-tumor mechanisms offers the opportunity to establish a rationale for the observed successes of CAP in selectively eliminating tumor cells in vitro and in vivo. In particular, 1O2 appears to act to selectively induce apoptosis in tumor cells, and can also result in self-perpetuating, cell-to-cell apoptotic signaling. Various CAP-generated liquid phase species can react to form 1O2, thus providing a hypothetical mechanism to explain how CAP can trigger therapeutic apoptosis in tumors. The analysis of model experiments performed with defined RONS in vitro implies that CAP-derived 1O2 induces the mechanism through which CAP acts selectively against cancer cells in vitro and tumors in vivo. This hypothesis needs to be tested experimentally in order to establish its validity.

A computational proposal for designing structured RNA pools for in vitro selection of RNAs.

PubMed

Kim, Namhee; Gan, Hin Hark; Schlick, Tamar

2007-04-01

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

An Image Analysis Method for the Precise Selection and Quantitation of Fluorescently Labeled Cellular Constituents

PubMed Central

Agley, Chibeza C.; Velloso, Cristiana P.; Lazarus, Norman R.

2012-01-01

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored. PMID:22511600

Good cell culture practices &in vitro toxicology.

PubMed

Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

2017-12-01

Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selection of Lactic Acid Bacteria with Probiotic Potential Isolated from the Fermentation Process of "Cupuaçu" (Theobroma grandiflorum).

PubMed

Ornellas, Roberta Maria Santos; Santos, Tiza Teles; Arcucio, Leonardo Borges; Sandes, Sávio Henrique Cicco; Oliveira, Mayara Messias; Dias, Cristiano Villela; de Carvalho Silva, Samuel; Uetanabaro, Ana Paula Trovatti; Vinderola, Gabriel; Nicoli, Jacques Robert

2017-01-01

In the present study, nine lactic acid bacteria isolated from the fermentation process of "cupuaçu" (Theobroma grandiflorum) were selected for probiotic use. In vitro (resistance to gastrointestinal environment, in vitro antagonism and co-aggregation with pathogens) and in vivo (intestinal colonization and ex vivo antagonism in germ-free mice, cumulative mortality, translocation to liver and spleen, histopathological examination of liver and ileum and mRNA cytokine gene expression during an experimental infection with S. Typhimurium) assays were used. Among the nine Lactobacillus strains isolated from the "cupuaçu" fermentation, L. plantarum 81 and L. plantarum 90 were selected as potential probiotics based on better results obtained in in vitro evaluations (production of diffusible inhibitory compounds and co-aggregation) as well as in vivo experiments (resistance to gastrointestinal environment, ex vivo antagonism, higher survival after enteropathogen challenge, lower hepatic translocation of enteropathogen, lower histopathological lesions in ileum and liver and anti-inflammatory pattern of immunological response). Concluding, L. plantarum 81 and L. plantarum 90 showed in vitro and in vivo capacities for probiotic use through different mechanisms of protection and its origin would allow an easier adaptation in an alimentary matrix for its administration.

Design and optimization of a series of 1-sulfonylpyrazolo[4,3-b]pyridines as selective c-Met inhibitors.

PubMed

Ma, Yuchi; Sun, Guangqiang; Chen, Danqi; Peng, Xia; Chen, Yue-Lei; Su, Yi; Ji, Yinchun; Liang, Jin; Wang, Xin; Chen, Lin; Ding, Jian; Xiong, Bing; Ai, Jing; Geng, Meiyu; Shen, Jingkang

2015-03-12

c-Met has emerged as an attractive target for targeted cancer therapy because of its abnormal activation in many cancer cells. To identify high potent and selective c-Met inhibitors, we started with profiling the potency and in vitro metabolic stability of a reported hit 7. By rational design, a novel sulfonylpyrazolo[4,3-b]pyridine 9 with improved DMPK properties was discovered. Further elaboration of π-π stacking interactions and solvent accessible polar moieties led to a series of highly potent and selective type I c-Met inhibitors. On the basis of in vitro and in vivo pharmacological and pharmacokinetics studies, compound 46 was selected as a preclinical candidate for further anticancer drug development.

Exploiting Size-Dependent Drag and Magnetic Forces for Size-Specific Separation of Magnetic Nanoparticles

PubMed Central

Rogers, Hunter B.; Anani, Tareq; Choi, Young Suk; Beyers, Ronald J.; David, Allan E.

2015-01-01

Realizing the full potential of magnetic nanoparticles (MNPs) in nanomedicine requires the optimization of their physical and chemical properties. Elucidation of the effects of these properties on clinical diagnostic or therapeutic properties, however, requires the synthesis or purification of homogenous samples, which has proved to be difficult. While initial simulations indicated that size-selective separation could be achieved by flowing magnetic nanoparticles through a magnetic field, subsequent in vitro experiments were unable to reproduce the predicted results. Magnetic field-flow fractionation, however, was found to be an effective method for the separation of polydisperse suspensions of iron oxide nanoparticles with diameters greater than 20 nm. While similar methods have been used to separate magnetic nanoparticles before, no previous work has been done with magnetic nanoparticles between 20 and 200 nm. Both transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis were used to confirm the size of the MNPs. Further development of this work could lead to MNPs with the narrow size distributions necessary for their in vitro and in vivo optimization. PMID:26307980

Rapid isolation of choriocapillary endothelial cells by Lycopersicon esculentum-coated Dynabeads.

PubMed

Hoffmann, S; Spee, C; Murata, T; Cui, J Z; Ryan, S J; Hinton, D R

1998-10-01

In vitro studies of choroidal endothelial cells may be critical for understanding the pathogenesis of neovascularization in age-related macular degeneration, since endothelial cells from different sites are highly heterogeneous in their morphology and behavior. Isolation of choroidal endothelial cells is complicated and labor intensive because of the small size of the choroid and the difficulty of excluding contaminating cells. We describe a rapid, simplified method for the isolation of bovine choroidal endothelial cells using microdissection followed by the use of superparamagnetic beads (Dynabeads) coated with the endothelial cell-specific lectin Lycopersicon esculentum, which selectively binds to fucose residues on the endothelial cell surface. Cells bound to beads are isolated using a magnetic particle concentrator. Isolated cells grew to confluence in a monolayer with a cobblestone morphology and were shown to be endothelial cells by their greater than 95% immunoreactivity to von Willebrand factor and phagocytosis of dil-acetylated LDL. Isolated cells grew as tubes in three-dimensional cultures. This method markedly reduces the time needed for pure culture of cells and makes the in vitro study of choroidal endothelial cells practical and reproducible.

Creation of a Novel Class of Potent and Selective MutT Homologue 1 (MTH1) Inhibitors Using Fragment-Based Screening and Structure-Based Drug Design.

PubMed

Rahm, Fredrik; Viklund, Jenny; Trésaugues, Lionel; Ellermann, Manuel; Giese, Anja; Ericsson, Ulrika; Forsblom, Rickard; Ginman, Tobias; Günther, Judith; Hallberg, Kenth; Lindström, Johan; Persson, Lars Boukharta; Silvander, Camilla; Talagas, Antoine; Díaz-Sáez, Laura; Fedorov, Oleg; Huber, Kilian V M; Panagakou, Ioanna; Siejka, Paulina; Gorjánácz, Mátyás; Bauser, Marcus; Andersson, Martin

2018-03-22

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

Trypanocidal and Leishmanicidal Activities of Sesquiterpene Lactones from Ambrosia tenuifolia Sprengel (Asteraceae) ▿

PubMed Central

Sülsen, Valeria P.; Frank, Fernanda M.; Cazorla, Silvia I.; Anesini, Claudia A.; Malchiodi, Emilio L.; Freixa, Blanca; Vila, Roser; Muschietti, Liliana V.; Martino, Virginia S.

2008-01-01

Bioassay-guided fractionation of the organic extract of Ambrosia tenuifolia Sprengel (Asteraceae) led to the isolation of two bioactive sesquiterpene lactones with significant trypanocidal and leishmanicidal activities. By spectroscopic methods (1H- and 13C-nuclear magnetic resonance, distortionless enhancement by polarization transfer, correlated spectroscopy, heteronuclear multiple-quantum coherence, electron impact-mass spectrometry, and infrared spectroscopy), these compounds were identified as psilostachyin and peruvin. Both compounds showed a marked in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes with 50% inhibitory concentration (IC50) values of less than 2 μg/ml. Psilostachyin exerted a significant in vitro activity against the trypomastigote forms of T. cruzi (IC50, 0.76 μg/ml) and was selected for in vivo testing. Psilostachyin-treated mice had a survival of 100% and lower parasitemia values than control mice. Both compounds were also tested on Leishmania sp. promastigotes: psilostachyin (IC50, 0.12 μg/ml) and peruvin (IC50, 0.39 μg/ml) exerted significant leishmanicidal activities. This is the first time that the trypanocidal and leishmanicidal activities of these compounds have been reported. The selectivity index (SI) was employed to evaluate the cytotoxic effect of lactones on T lymphocytes. Although the SIs of both compounds were high for T. cruzi epimastigotes, psilostachyin was more selective against trypomastigotes (SI, 33.8) while peruvin showed no specificity for this parasite. Both compounds presented high selectivity for Leishmania spp. The results shown herein suggest that psilostachyin and peruvin could be considered potential candidates for the development of new antiprotozoal agents against Chagas' disease and leishmaniasis. PMID:18443111

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

PubMed

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 evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule. Copyright © 2013 Wiley-Liss, Inc.

An in vitro approach to investigate ocular metabolism of a topical, selective β1-adrenergic blocking agent, betaxolol.

PubMed

Bushee, Jennifer L; Dunne, Christine E; Argikar, Upendra A

2015-05-01

1. Topical glaucoma treatments have often been limited by poor absorption and bioavailability. Betaxolol, a selective β1-blocker, has been well studied for its pharmacokinetics and disposition. Limited ocular, betaxolol metabolism data is available despite a growing number of novel ocular treatments. 2. In vitro ocular fractions indicated the formation of an active metabolite, across rat, rabbit and human, which was only observed historically in the liver. 3. Ocular metabolic profiles of preclinical toxicology species, rat and rabbit, were not predictive of human in vitro ocular data. M1 was specific to human and only captured by the liver data. 4. Liver S9 over predicted the extent of ocular metabolism compared to ocular fractions. Rabbit liver S9 fractions demonstrated extensive glucuronidation and higher parent turn-over in 1 h as compared to other matrices. 5. This research assesses in vitro species and organ differences across preclinical species and human. The complex data set highlights the need for an in vitro ocular system to explore poorly documented ocular metabolism.

Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

PubMed Central

Pfeifer, N.; Baston-Büst, D. M.; Hirchenhain, J.; Friebe-Hoffmann, U.; Rein, D. T.; Krüssel, J. S.; Hess, A. P.

2012-01-01

Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies. PMID:22919324

Critical quality attributes, in vitro release and correlated in vitro skin permeation-in vivo tape stripping collective data for demonstrating therapeutic (non)equivalence of topical semisolids: A case study of "ready-to-use" vehicles.

PubMed

Ilić, Tanja; Pantelić, Ivana; Lunter, Dominique; Đorđević, Sanela; Marković, Bojan; Ranković, Dragana; Daniels, Rolf; Savić, Snežana

2017-08-07

This work aimed to prove the ability of "ready-to-use" topical vehicles based on alkyl polyglucoside-mixed emulsifier (with/without co-solvent modifications) to replace the conventionally used pharmacopoeial bases (e.g., non-ionic hydrophilic cream) in compounding practice. For this purpose, considering the regulatory efforts to establish alternative, scientifically valid methods for evaluating therapeutic equivalence of topical semisolids, we performed a comparative assessment of microstructure, selected critical quality attributes (CQAs) and in vitro/in vivo product performances, by utilizing aceclofenac as a model drug. The differences in composition between investigated samples have imposed remarkable variances in monitored CQAs (particularly in the amount of aceclofenac dissolved, rheological properties and water distribution mode), reflecting the distinct differences in microstructure formed, as partially observed by polarization microscopy and confocal Raman spectral imaging. Although not fully indicative of the in vivo performances, in vitro release data (vertical diffusion vs. immersion cells) proved the microstructure peculiarities, asserting the rheological properties as decisive factor for obtained liberation profiles. Contrary, in vitro permeation results obtained using pig ear epidermis correlated well with in vivo dermatopharmacokinetic data and distinguished unequivocally between tested formulations, emphasizing the importance of skin/vehicle interactions. In summary, suggested multi-faceted approach can provide adequate proof on topical semisolids therapeutic equivalence or lack thereof. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of oxybutynin selective sensors for monitoring the dissolution profile and assay of pharmaceutical dosage forms.

PubMed

El Hamshary, Marwa S; Salem, Omar H; El Nashar, Rasha M

2010-01-01

Two ion-selective sensors of the plastic membrane type were prepared for the determination of oxybutynin hydrochloride (OxCl). They depend on the incorporation of the ion-associates with phosphotungestic acid or phosphomolybdic acid in a PVC matrix. A comparative study is made between their performance characteristics in batch and FIA conditions. The sensors have nearly the same usable concentration, temperature and pH range. They have a wide range of selectivity and can be applied for the determination of the relevant drug with nearly the same precision and accuracy in vitro. Dissolution testing was applied using the sensors; this offers a simple, rapid, cheap way out of sophisticated and high cost instruments used in the pharmacopeial method using HPLC. The investigated drug was determined in its pure and pharmaceutical preparations. The results were accurate and precise, as indicated by the recovery values and coefficients of variation.

A novel approach for in vitro meat production.

PubMed

Pandurangan, Muthuraman; Kim, Doo Hwan

2015-07-01

The present review describes the possibility of in vitro meat production with the help of advanced co-culturing methods. In vitro meat production method could be a possible alternative for the conventional meat production. Originally, the research on in vitro meat production was initiated by the National Aeronautics and Space Administration (NASA) for space voyages. The required key qualities for accepting in vitro meat for consumption would be good efficiency ratio, increased protein synthesis rate in skeletal muscles, and mimicking the conventional meat qualities. In vitro culturing of meat is possible with the use of skeletal muscle tissue engineering, stem cell, cell co-culture, and tissue culture methods. Co-culture of myoblast and fibroblast is believed as one of the major techniques for in vitro meat production. In our lab, we have co-cultured myoblast and fibroblast. We believe that a billion pounds of in vitro meat could be produced from one animal for consumption. However, we require a great deal of research on in vitro meat production.

Spectrofluorimetric determination of 3-methylflavone-8-carboxylic acid, the main active metabolite of flavoxate hydrochloride in human urine.

PubMed

Zaazaa, Hala E; Mohamed, Afaf O; Hawwam, Maha A; Abdelkawy, Mohamed

2015-01-05

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of 3-methylflavone-8-carboxylic acid as the main active metabolite of flavoxate hydrochloride in human urine. The proposed method was based on the measurement of the native fluorescence of the metabolite in methanol at an emission wavelength 390 nm, upon excitation at 338 nm. Moreover, the urinary excretion pattern has been calculated using the proposed method. Taking the advantage that 3-methylflavone-8-carboxylic acid is also the alkaline degradate, the proposed method was applied to in vitro determination of flavoxate hydrochloride in tablets dosage form via the measurement of its corresponding degradate. The method was validated in accordance with the ICH requirements and statistically compared to the official method with no significant difference in performance. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro bacterial isolate susceptibility to empirically selected antimicrobials in 111 dogs with bacterial pneumonia.

PubMed

Proulx, Alexandre; Hume, Daniel Z; Drobatz, Kenneth J; Reineke, Erica L

2014-01-01

To determine the proportion of airway bacterial isolates resistant to both empirically selected and recently administered antimicrobials, and to assess the impact of inappropriate initial empiric antimicrobials selection on length of hospital stay and survival to discharge in dogs with bacterial pneumonia. Retrospective study. University veterinary teaching hospital. One hundred and eleven dogs with a clinical diagnosis of bacterial pneumonia that had aerobic bacterial culture and susceptibility testing performed from a tracheal wash sample. None. Overall, 26% (29/111) of the dogs had at least 1 bacterial isolate that was resistant to empirically selected antimicrobials. In dogs with a history of antimicrobial administration within the preceding 4 weeks, a high incidence (57.4%, 31/54) of in vitro bacterial resistance to those antimicrobials was found: 64.7% (11/17) in the community-acquired pneumonia group, 55.2% (16/29) in the aspiration pneumonia group, and 50.0% (4/8) in the other causes of bacterial pneumonia group. No statistically significant association was found between bacterial isolate resistance to empirically selected antimicrobials and length of hospital stay or mortality. The high proportion of in vitro airway bacterial resistance to empiric antimicrobials would suggest that airway sampling for bacterial culture and susceptibility testing may be helpful in guiding antimicrobial therapy and recently administered antimicrobials should be avoided when empirically selecting antimicrobials. Although no relationship was found between inappropriate initial empiric antimicrobial selection and length of hospital stay or mortality, future prospective studies using standardized airway-sampling techniques, treatment modalities, and stratification of disease severity based on objective values, such as arterial blood gas analysis in all dogs with pneumonia, would be needed to determine if a clinical effect of in vitro bacterial resistance to empirically administered antimicrobials truly exists or not. © Veterinary Emergency and Critical Care Society 2013.

Engineering Tocopherol Selectivity in α-TTP: A Combined In Vitro/In Silico Study

PubMed Central

Helbling, Rachel E.; Aeschimann, Walter; Simona, Fabio; Stocker, Achim; Cascella, Michele

2012-01-01

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of -tocopherol transfer protein (-TTP) towards -tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for -tocopherol to -TTP 8.262.13 kcal mol lower than that of -tocopherol. Our calculations show that -tocopherol binds to -TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of -TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to -tocopherol, with striking structural similarities to the wild-type--tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of -TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a -tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for -tocopherol selection in omnivorous animals. PMID:23152872

Testing the mutant selection window hypothesis with Escherichia coli exposed to levofloxacin in a rabbit tissue cage infection model.

PubMed

Ni, W; Song, X; Cui, J

2014-03-01

The purpose of this study was to test the mutant selection window (MSW) hypothesis with Escherichia coli exposed to levofloxacin in a rabbit model and to compare in vivo and in vitro exposure thresholds that restrict the selection of fluoroquinolone-resistant mutants. Local infection with E. coli was established in rabbits, and the infected animals were treated orally with various doses of levofloxacin once a day for five consecutive days. Changes in levofloxacin concentration and levofloxacin susceptibility were monitored at the site of infection. The MICs of E. coli increased when levofloxacin concentrations at the site of infection fluctuated between the lower and upper boundaries of the MSW, defined in vitro as the minimum inhibitory concentration (MIC99) and the mutant prevention concentration (MPC), respectively. The pharmacodynamic thresholds at which resistant mutants are not selected in vivo was estimated as AUC24/MPC > 20 h or AUC24/MIC > 60 h, where AUC24 is the area under the drug concentration time curve in a 24-h interval. Our finding demonstrated that the MSW existed in vivo. The AUC24/MPC ratio that prevented resistant mutants from being selected estimated in vivo is consistent with that observed in vitro, indicating it might be a reliable index for guiding the optimization of antimicrobial treatment regimens for suppression of the selection of antimicrobial resistance.

Biofilms of vaginal Lactobacillus in vitro test.

PubMed

Wei, Xiao-Yu; Zhang, Rui; Xiao, Bing-Bing; Liao, Qin-Ping

2017-01-01

This paper focuses on biofilms of Lactobacillus spp. - a type of normal flora isolated from healthy human vaginas of women of childbearing age; thereupon, it broadens the research scope of investigation of vaginal normal flora. The static slide culture method was adopted to foster biofilms, marked by specific fluorescence staining. Laser scanning confocal and scanning electron microscopy were used to observe the microstructure of the biofilms. Photographs taken from the microstructure were analysed to calculate the density of the biofilms. The body of Lactobacillus spp., though red, turned yellow when interacting with the green extracellular polysaccharides. The structure of the biofilm and aquaporin within the biofilm were imaged. Lactobacillus density increases over time. This study provides convincing evidence that Lactobacillus can form biofilms and grow over time in vitro. This finding establishes an important and necessary condition for selecting proper strains for the pharmaceutics of vaginal ecology.

3-(1,3,4-Thiadiazole-2-yl)quinoline derivatives: synthesis, characterization and anti-microbial activity.

PubMed

Bhat, Abdul R; Tazeem; Azam, Amir; Choi, Inho; Athar, Fareeda

2011-07-01

A new series of thiadiazoles and intermediate thiosemicarbazones were synthesized from the chloroquinone molecule, with an aim to explore their effect on in vitro growth of microorganisms causing microbial infection. The chemical structures of the compound were elucidated by elemental analysis, FTIR, 1H and 13C NMR and ESI-MS spectral data. In vitro anti-microbial activity was performed against Staphylococcusaureus, Streptococcuspyogenes, Salmonellatyphimurium, and Escherichiacoli. The MIC was detected using the double dilution method. The results were compared by calculating percent inhibit area/μg of the compounds and the standard "amoxicillin". The selected compounds were tested for cytotoxic results using MTT assay H9c2 cardiac myoblasts cell line and the results showed that all the compounds offered remarkable >80% viability to a concentration of 200 μg/mL. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Setting the proportion of CD4+ and CD8+ T-cells co-cultured with canine macrophages infected with Leishmania chagasi.

PubMed

Viana, Kelvinson Fernandes; Aguiar-Soares, Rodrigo Dian Oliveira; Ker, Henrique Gama; Resende, Lucilene Aparecida; Souza-Fagundes, Elaine Maria; Dutra, Walderez Ornelas; Fujiwara, Ricardo Toshio; da Silveira-Lemos, Denise; Sant'Ana, Rita de Cássia Oliveira; Wardini, Amanda Brito; Araújo, Márcio Sobreira Silva; Martins-Filho, Olindo Assis; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

2015-07-30

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions. Copyright © 2015 Elsevier B.V. All rights reserved.

Prediction of Chemical Respiratory Sensitizers Using GARD, a Novel In Vitro Assay Based on a Genomic Biomarker Signature

PubMed Central

Albrekt, Ann-Sofie; Borrebaeck, Carl A. K.; Lindstedt, Malin

2015-01-01

Background Repeated exposure to certain low molecular weight (LMW) chemical compounds may result in development of allergic reactions in the skin or in the respiratory tract. In most cases, a certain LMW compound selectively sensitize the skin, giving rise to allergic contact dermatitis (ACD), or the respiratory tract, giving rise to occupational asthma (OA). To limit occurrence of allergic diseases, efforts are currently being made to develop predictive assays that accurately identify chemicals capable of inducing such reactions. However, while a few promising methods for prediction of skin sensitization have been described, to date no validated method, in vitro or in vivo, exists that is able to accurately classify chemicals as respiratory sensitizers. Results Recently, we presented the in vitro based Genomic Allergen Rapid Detection (GARD) assay as a novel testing strategy for classification of skin sensitizing chemicals based on measurement of a genomic biomarker signature. We have expanded the applicability domain of the GARD assay to classify also respiratory sensitizers by identifying a separate biomarker signature containing 389 differentially regulated genes for respiratory sensitizers in comparison to non-respiratory sensitizers. By using an independent data set in combination with supervised machine learning, we validated the assay, showing that the identified genomic biomarker is able to accurately classify respiratory sensitizers. Conclusions We have identified a genomic biomarker signature for classification of respiratory sensitizers. Combining this newly identified biomarker signature with our previously identified biomarker signature for classification of skin sensitizers, we have developed a novel in vitro testing strategy with a potent ability to predict both skin and respiratory sensitization in the same sample. PMID:25760038

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

PubMed

Gettings, S D; Lordo, R A; Hintze, K L; Bagley, D M; Casterton, P L; Chudkowski, M; Curren, R D; Demetrulias, J L; Dipasquale, L C; Earl, L K; Feder, P I; Galli, C L; Glaza, S M; Gordon, V C; Janus, J; Kurtz, P J; Marenus, K D; Moral, J; Pape, W J; Renskers, K J; Rheins, L A; Roddy, M T; Rozen, M G; Tedeschi, J P; Zyracki, J

1996-01-01

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Study of the in vitro antiplasmodial, antileishmanial and antitrypanosomal activities of medicinal plants from Saudi Arabia.

PubMed

Al-Musayeib, Nawal M; Mothana, Ramzi A; Al-Massarani, Shaza; Matheeussen, An; Cos, Paul; Maes, Louis

2012-09-25

The present study investigated the in vitro antiprotozoal activity of sixteen selected medicinal plants. Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC₅₀ < 10 μg/mL (<5 μg/mL for T. brucei) and a selectivity index of ≥4. Antiplasmodial activity was found in the extracts of Prosopis juliflora and Punica granatum. Antileishmanial activity against L. infantum was demonstrated in Caralluma sinaica and Periploca aphylla. Amastigotes of T. cruzi were affected by the methanol extract of Albizia lebbeck pericarp, Caralluma sinaica, Periploca aphylla and Prosopius juliflora. Activity against T. brucei was obtained in Prosopis juliflora. Cytotoxicity (MRC-5 IC₅₀ < 10 μg/mL) and hence non-specific activities were observed for Conocarpus lancifolius.

Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Targeting-Free Cancer Cell Screening and Killing.

PubMed

Chen, Limei; Li, Haijuan; He, Haili; Wu, Haoxi; Jin, Yongdong

2015-07-07

Fast and accurate identification of cancer cells from healthy normal cells in a simple, generic way is very crucial for early cancer detection and treatment. Although functional nanoparticles, like fluorescent quantum dots and plasmonic Au nanoparticles (NPs), have been successfully applied for cancer cell imaging and photothermal therapy, they suffer from the main drawback of needing time-consuming targeting preparation for specific cancer cell detection and selective ablation. The lack of a generic and effective method therefore limits their potential high-throughput cancer cell preliminary screening and theranostic applications. We report herein a generic in vitro method for fast, targeting-free (avoiding time-consuming preparations of targeting moiety for specific cancer cells) visual screening and selective killing of cancer cells from normal cells, by using glucose-responsive/-sensitive glucose oxidase-modified Ag/Au nanoshells (Ag/Au-GOx NSs) as a smart plasmonic theranostic agent. The method is generic to some extent since it is based on the distinct localized surface plasmon resonance (LSPR) responses (and colors) of the smart nanoprobe with cancer cells (typically have a higher glucose uptake level) and normal cells.

Statistical Considerations Concerning Dissimilar Regulatory Requirements for Dissolution Similarity Assessment. The Example of Immediate-Release Dosage Forms.

PubMed

Jasińska-Stroschein, Magdalena; Kurczewska, Urszula; Orszulak-Michalak, Daria

2017-05-01

When performing in vitro dissolution testing, especially in the area of biowaivers, it is necessary to follow regulatory guidelines to minimize the risk of an unsafe or ineffective product being approved. The present study examines model-independent and model-dependent methods of comparing dissolution profiles based on various compared and contrasted international guidelines. Dissolution profiles for immediate release solid oral dosage forms were generated. The test material comprised tablets containing several substances, with at least 85% of the labeled amount dissolved within 15 min, 20-30 min, or 45 min. Dissolution profile similarity can vary with regard to the following criteria: time point selection (including the last time point), coefficient of variation, and statistical method selection. Variation between regulatory guidance and statistical methods can raise methodological questions and result potentially in a different outcome when reporting dissolution profile testing. The harmonization of existing guidelines would address existing problems concerning the interpretation of regulatory recommendations and research findings. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

More Genetic Engineering With Cloned Hemoglobin Genes

NASA Technical Reports Server (NTRS)

Bailey, James E.

1992-01-01

Cells modified to enhance growth and production of proteins. Method for enhancing both growth of micro-organisms in vitro and production of various proteins or metalbolites in these micro-organisms provides for incorporation of selected chromosomal or extrachormosomal deoxyribonucleic acid (DNA) sequences into micro-organisms from other cells or from artificial sources. Incorporated DNA includes parts encoding desired product(s) or characteristic(s) of cells and parts that control expression of productor characteristic-encoding parts in response to variations in environment. Extended method enables increased research into growth of organisms in oxygen-poor environments. Industrial applications found in enhancement of processing steps requiring oxygen in fermentation, enzymatic degradation, treatment of wastes containing toxic chemicals, brewing, and some oxidative chemical reactions.

Repositioning FDA Drugs as Potential Cruzain Inhibitors from Trypanosoma cruzi: Virtual Screening, In Vitro and In Vivo Studies.

PubMed

Palos, Isidro; Lara-Ramirez, Edgar E; Lopez-Cedillo, Julio Cesar; Garcia-Perez, Carlos; Kashif, Muhammad; Bocanegra-Garcia, Virgilio; Nogueda-Torres, Benjamin; Rivera, Gildardo

2017-06-18

Chagas disease (CD) is a neglected disease caused by the parasite Trypanosoma cruzi , which affects underdeveloped countries. The current drugs of choice are nifurtimox and benznidazole, but both have severe adverse effects and less effectivity in chronic infections; therefore, the need to discover new drugs is essential. A computer-guided drug repositioning method was applied to identify potential FDA drugs (approved and withdrawn) as cruzain (Cz) inhibitors and trypanocidal effects were confirmed by in vitro and in vivo studies. 3180 FDA drugs were virtually screened using a structure-based approach. From a first molecular docking analysis, a set of 33 compounds with the best binding energies were selected. Subsequent consensus affinity binding, ligand amino acid contact clustering analysis, and ranked position were used to choose four known pharmacological compounds to be tested in vitro. Mouse blood samples infected with trypomastigotes from INC-5 and NINOA strains were used to test the trypanocidal effect of four selected compounds. Among these drugs, one fibrate antilipemic (etofyllin clofibrate) and three β-lactam antibiotics (piperacillin, cefoperazone, and flucloxacillin) showed better trypanocidal effects (LC 50 range 15.8-26.1 μg/mL) in comparison with benznidazole and nifurtimox (LC 50 range 33.1-46.7 μg/mL). A short-term in vivo evaluation of these compounds showed a reduction of parasitemia in infected mice (range 90-60%) at 6 h, but this was low compared to benznidazole (50%). This work suggests that four known FDA drugs could be used to design and obtain new trypanocidal agents.

In vitro and in vivo investigation of taste-masking effectiveness of Eudragit E PO as drug particle coating agent in orally disintegrating tablets.

PubMed

Drašković, Milica; Medarević, Djordje; Aleksić, Ivana; Parojčić, Jelena

2017-05-01

Considering that bitter taste of drugs incorporated in orally disintegrating tablets (ODTs) can be the main reason for avoiding drug therapy, it is of the utmost importance to achieve successful taste-masking. The evaluation of taste-masking effectiveness is still a major challenge. The objective of this study was to mask bitter taste of the selected model drugs by drug particle coating with Eudragit ® E PO, as well as to evaluate taste-masking effectiveness of prepared ODTs using compendial dissolution testing, dissolution in the small-volume shake-flask assembly and trained human taste panel. Model drugs were coated in fluidized bed. Disintequik™ ODT was used as a novel co-processed excipient for ODT preparation. Selected formulations were investigated in vitro and in vivo using techniques for taste-masking assessment. Significantly slower drug dissolution was observed from tablets with coated drug particles during the first 3 min of investigation. Results of in vivo taste-masking assessment demonstrated significant improvement in drug bitterness suppression in formulations with coated drug. Strong correlation between the results of drug dissolution in the small-volume shake-flask assembly and in vivo evaluation data was established (R ≥ 0.970). Drug particle coating with Eudragit ® E PO can be a suitable approach for bitter taste-masking. Strong correlation between in vivo and in vitro results implicate that small-volume dissolution method may be used as surrogate for human panel taste-masking assessment, in the case of physical taste-masking approach application.

In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

PubMed

Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

2013-01-01

Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

Development and application of a biorelevant dissolution method using USP apparatus 4 in early phase formulation development.

PubMed

Fang, Jiang B; Robertson, Vivian K; Rawat, Archana; Flick, Tawnya; Tang, Zhe J; Cauchon, Nina S; McElvain, James S

2010-10-04

Dissolution testing is frequently used to determine the rate and extent at which a drug is released from a dosage form, and it plays many important roles throughout drug product development. However, the traditional dissolution approach often emphasizes its application in quality control testing and usually strives to obtain 100% drug release. As a result, dissolution methods are not necessarily biorelevant and meaningful application of traditional dissolution methods in the early phases of drug product development can be very limited. This article will describe the development of a biorelevant in vitro dissolution method using USP apparatus 4, biorelevant media, and real-time online UV analysis. Several case studies in the areas of formulation selection, lot-to-lot variability, and food effect will be presented to demonstrate the application of this method in early phase formulation development. This biorelevant dissolution method using USP apparatus 4 provides a valuable tool to predict certain aspects of the in vivo drug release. It can be used to facilitate the formulation development/selection for pharmacokinetic (PK) and clinical studies. It may also potentially be used to minimize the number of PK studies, and to aid in the design of more efficient PK and clinical studies.

Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis.

PubMed

Rençber, Seda; Karavana, Sinem Yaprak; Yılmaz, Fethiye Ferda; Eraç, Bayri; Nenni, Merve; Özbal, Seda; Pekçetin, Çetin; Gurer-Orhan, Hande; Hoşgör-Limoncu, Mine; Güneri, Pelin; Ertan, Gökhan

2016-01-01

This study aimed to develop a suitable buccal mucoadhesive nanoparticle (NP) formulation containing fluconazole for the local treatment of oral candidiasis. The suitability of the prepared formulations was assessed by means of particle size (PS), polydispersity index, and zeta potential measurements, morphology analysis, mucoadhesion studies, drug entrapment efficiency (EE), in vitro drug release, and stability studies. Based on the optimum NP formulation, ex vivo drug diffusion and in vitro cytotoxicity studies were performed. Besides, evaluation of the antifungal effect of the optimum formulation was evaluated using agar diffusion method, fungicidal activity-related in vitro release study, and time-dependent fungicidal activity. The effect of the optimum NP formulation on the healing of oral candidiasis was investigated in an animal model, which was employed for the first time in this study. The zeta potential, mucoadhesion, and in vitro drug release studies of various NP formulations revealed that chitosan-coated NP formulation containing EUDRAGIT(®) RS 2.5% had superior properties than other formulations. Concerning the stability study of the selected formulation, the formulation was found to be stable for 6 months. During the ex vivo drug diffusion study, no drug was found in receptor phase, and this is an indication of local effect. The in vitro antifungal activity studies showed the in vitro efficacy of the NP against Candida albicans for an extended period. Also, the formulation had no cytotoxic effect at the tested concentration. For the in vivo experiments, infected rabbits were successfully treated with local administration of the optimum NP formulation once a day. This study has shown that the mucoadhesive NP formulation containing fluconazole is a promising candidate with once-a-day application for the local treatment of oral candidiasis.

Analysis of the sensitivity of in vitro bioassays for androgenic, progestagenic, glucocorticoid, thyroid and estrogenic activity: Suitability for drinking and environmental waters.

PubMed

Leusch, Frederic D L; Neale, Peta A; Hebert, Armelle; Scheurer, Marco; Schriks, Merijn C M

2017-02-01

The presence of endocrine disrupting chemicals in the aquatic environment poses a risk for ecosystem health. Consequently there is a need for sensitive tools, such as in vitro bioassays, to monitor endocrine activity in environmental waters. The aim of the current study was to assess whether current in vitro bioassays are suitable to detect endocrine activity in a range of water types. The reviewed assays included androgenic (n=11), progestagenic (n=6), glucocorticoid (n=5), thyroid (n=5) and estrogenic (n=8) activity in both agonist and antagonist mode. Existing in vitro bioassay data were re-evaluated to determine assay sensitivity, with the calculated method detection limit compared with measured hormonal activity in treated wastewater, surface water and drinking water to quantify whether the studied assays were sufficiently sensitive for environmental samples. With typical sample enrichment, current in vitro bioassays are sufficiently sensitive to detect androgenic activity in treated wastewater and surface water, with anti-androgenic activity able to be detected in most environmental waters. Similarly, with sufficient enrichment, the studied mammalian assays are able to detect estrogenic activity even in drinking water samples. Fewer studies have focused on progestagenic and glucocorticoid activity, but some of the reviewed bioassays are suitable for detecting activity in treated wastewater and surface water. Even less is known about (anti)thyroid activity, but the available data suggests that the more sensitive reviewed bioassays are still unlikely to detect this type of activity in environmental waters. The findings of this review can help provide guidance on in vitro bioassay selection and required sample enrichment for optimised detection of endocrine activity in environmental waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells

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

Driscoll, K.E.; Deyo, L.C.; Howard, B.W.

1995-12-31

A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H{sub 2}O{sub 2},more » and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H{sub 2}O produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or {alpha}-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or {alpha}-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H{sub 2}O{sub 2} in vitro and ENU and {alpha}-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation. 41 refs., 4 figs., 3 tabs.« less

In vitro gametogenesis: just another way to have a baby?

PubMed Central

Suter, Sonia M.

2015-01-01

Advances in science have made possible the derivation of reproductively viable gametes in vitro from mice. The research on human cells suggests that in vitro gametogenesis (“IVG”) with reproductive potential may one day be possible with humans. This technology would allow same-sex couples to have children who are biologically related to both of them; allow single individuals to procreate without the genetic contribution of another individual; and facilitate “multiplex” parenting, where groups of more than two individuals procreate together, producing children who are the genetic progeny of them all. IVG could also make prenatal selection a much more refined and comprehensive process than it is today, allowing for the selection of embryos on the basis of multiple factors. Evaluating IVG under a relational autonomy framework, this article argues that the potential benefits or harms of IVG depend on the social, scientific, and legal context in which it is situated and how it is used. It concludes that IVG is preferable to some forms of assisted reproductive technologies in certain instances and substantially more problematic in others. Finally, it suggests that its capacity to “perfect” prenatal selection in many ways exacerbates the problematic aspects of increasingly expansive prenatal selection. PMID:27774234

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

PubMed Central

2013-01-01

Background Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Conclusions Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery. PMID:23773766

Analytical and Biological Methods for Probing the Blood-Brain Barrier

PubMed Central

Sloan, Courtney D. Kuhnline; Nandi, Pradyot; Linz, Thomas H.; Aldrich, Jane V.; Audus, Kenneth L.; Lunte, Susan M.

2013-01-01

The blood-brain barrier (BBB) is an important interface between the peripheral and central nervous systems. It protects the brain against the infiltration of harmful substances and regulates the permeation of beneficial endogenous substances from the blood into the extracellular fluid of the brain. It can also present a major obstacle in the development of drugs that are targeted for the central nervous system. Several methods have been developed to investigate the transport and metabolism of drugs, peptides, and endogenous compounds at the BBB. In vivo methods include intravenous injection, brain perfusion, positron emission tomography, and microdialysis sampling. Researchers have also developed in vitro cell-culture models that can be employed to investigate transport and metabolism at the BBB without the complication of systemic involvement. All these methods require sensitive and selective analytical methods to monitor the transport and metabolism of the compounds of interest at the BBB. PMID:22708905

Synthesis and in Vitro and in Vivo Characterization of Highly β1-Selective β-Adrenoceptor Partial Agonists

PubMed Central

2013-01-01

β-Adrenoceptor antagonists boast a 50-year use for symptomatic control in numerous cardiovascular diseases. One might expect highly selective antagonists are available for the human β-adrenoceptor subtype involved in these diseases, yet few truly β1-selective molecules exist. To address this clinical need, we re-evaluated LK 204-545 (1),1 a selective β1-adrenoceptor antagonist, and discovered it possessed significant partial agonism. Removal of 1’s aromatic nitrile afforded 19, a ligand with similar β1-adrenoceptor selectivity and partial agonism (log KD of −7.75 and −5.15 as an antagonist of functional β1- and β2-mediated responses, respectively, and 34% of the maximal response of isoprenaline (β1)). In vitro β-adrenoceptor selectivity and partial agonism of 19 were mirrored in vivo. We designed analogues of 19 to improve affinity, selectivity, and partial agonism. Although partial agonism could not be fully attenuated, SAR suggests that an extended alkoxyalkoxy side chain, alongside substituents at the meta- or para-positions of the phenylurea, increases ligand affinity and β1-selectivity. PMID:23614528

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

PubMed

Kieffer, Charline; Cohen, Anita; Verhaeghe, Pierre; Paloque, Lucie; Hutter, Sébastien; Castera-Ducros, Caroline; Laget, Michèle; Rault, Sylvain; Valentin, Alexis; Rathelot, Pascal; Azas, Nadine; Vanelle, Patrice

2015-05-15

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interlaboratory Evaluation of in Vitro Cytotoxicity and Inflammatory Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

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

Xia, Tian; Hamilton, Raymond F.; Bonner, James C.

2013-06-01

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different speciesmore » (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μ g/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.« less

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid.

PubMed

Metz, Bernard; Tilstra, Wichard; van der Put, Robert; Spruit, Nanda; van den Ijssel, Jan; Robert, Jolanda; Hendriksen, Coenraad; Kersten, Gideon

2013-07-01

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

A preliminary study for the development and optimization by experimental design of an in vitro method for prediction of drug buccal absorption.

PubMed

Mura, Paola; Orlandini, Serena; Cirri, Marzia; Maestrelli, Francesca; Mennini, Natascia; Casella, Giada; Furlanetto, Sandra

2018-06-15

The work was aimed at developing an in vitro method able to provide rapid and reliable evaluation of drug absorption through buccal mucosa. Absorption simulator apparatus endowed with an artificial membrane was purposely developed by experimental design. The apparent permeation coefficient (P app ) through excised porcine buccal mucosa of naproxen, selected as model drug, was the target value to obtain with the artificial membrane. The multivariate approach enabled systematic evaluation of the effect on the response (P app ) of simultaneous variations of the variables (kind of lipid components for support impregnation and relative amounts). A screening phase followed by a response-surface study allowed optimization of the lipid-mixture composition to obtain the desired P app value, and definition of a design space where all mixture components combinations fulfilled the desired target at a fixed probability level. The method offers a useful tool for a quick screening in the early stages of drug discovery and/or in preformulation studies, improving efficiency and chance of success in the development of buccal delivery systems. Further studies with other model drugs are planned to confirm the buccal absorption predictive capacity of the developed membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials.

PubMed

Biksi, I; Major, Andrea; Fodor, L; Szenci, O; Vetési, F

2003-01-01

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125 micrograms/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5 micrograms/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100 micrograms/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p < 0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.

In vitro eye irritation testing using the open source reconstructed hemicornea - a ring trial.

PubMed

Mewes, Karsten R; Engelke, Maria; Zorn-Kruppa, Michaela; Bartok, Melinda; Tandon, Rashmi; Brandner, Johanna M; Petersohn, Dirk

2017-01-01

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers' laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer's instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

An in vitro digestion method adapted for carotenoids and carotenoid esters: moving forward towards standardization.

PubMed

Rodrigues, Daniele Bobrowski; Mariutti, Lilian Regina Barros; Mercadante, Adriana Zerlotti

2016-12-07

In vitro digestion methods are a useful approach to predict the bioaccessibility of food components and overcome some limitations or disadvantages associated with in vivo methodologies. Recently, the INFOGEST network published a static method of in vitro digestion with a proposal for assay standardization. The INFOGEST method is not specific for any food component; therefore, we aimed to adapt this method to assess the in vitro bioaccessibility of carotenoids and carotenoid esters in a model fruit (Byrsonima crassifolia). Two additional steps were coupled to the in vitro digestion procedure, centrifugation at 20 000g for the separation of the aqueous phase containing mixed micelles and exhaustive carotenoid extraction with an organic solvent. The effect of electrolytes, enzymes and bile acids on carotenoid micellarization and stability was also tested. The results were compared with those found with a simpler method that has already been used for carotenoid bioaccessibility analysis. These values were in the expected range for free carotenoids (5-29%), monoesters (9-26%) and diesters (4-28%). In general, the in vitro bioaccessibility of carotenoids assessed by the adapted INFOGEST method was significantly higher (p < 0.05) than those assessed by the simplest protocol, with or without the addition of simulated fluids. Although no trend was observed, differences in bioaccessibility values depended on the carotenoid form (free, monoester or diester), isomerization (Z/E) and the in vitro digestion protocol. To the best of our knowledge, it was the first time that a systematic identification of carotenoid esters by HPLC-DAD-MS/MS after in vitro digestion using the INFOGEST protocol was carried out.

Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15-O-acetate as the main active compound

PubMed Central

2012-01-01

Background Natural products could play an important role in the challenge to discover new anti-malarial drugs. In a previous study, Dicoma tomentosa (Asteraceae) was selected for its promising anti-plasmodial activity after a preliminary screening of several plants traditionally used in Burkina Faso to treat malaria. The aim of the present study was to further investigate the anti-plasmodial properties of this plant and to isolate the active anti-plasmodial compounds. Methods Eight crude extracts obtained from D. tomentosa whole plant were tested in vitro against two Plasmodium falciparum strains (3D7 and W2) using the p-LDH assay (colorimetric method). The Peters’ four-days suppressive test model (Plasmodium berghei-infected mice) was used to evaluate the in vivo anti-plasmodial activity. An in vitro bioguided fractionation was undertaken on a dichloromethane extract, using preparative HPLC and TLC techniques. The identity of the pure compound was assessed using UV, MS and NMR spectroscopic analysis. In vitro cytotoxicity against WI38 human fibroblasts (WST-1 assay) and haemolytic activity were also evaluated for extracts and pure compounds in order to check selectivity. Results The best in vitro anti-plasmodial results were obtained with the dichloromethane, diethylether, ethylacetate and methanol extracts, which exhibited a high activity (IC50 ≤ 5 μg/ml). Hot water and hydroethanolic extracts also showed a good activity (IC50 ≤ 15 μg/ml), which confirmed the traditional use and the promising anti-malarial potential of the plant. The activity was also confirmed in vivo for all tested extracts. However, most of the active extracts also exhibited cytotoxic activity, but no extract was found to display any haemolytic activity. The bioguided fractionation process allowed to isolate and identify a sesquiterpene lactone (urospermal A-15-O-acetate) as the major anti-plasmodial compound of the plant (IC50 < 1 μg/ml against both 3D7 and W2 strains). This was also found to be the main cytotoxic compound (SI = 3.3). While this melampolide has already been described in the plant, this paper is the first report on the biological properties of this compound. Conclusions The present study highlighted the very promising anti-plasmodial activity of D. tomentosa and enabled to identify its main active compound, urospermal A-15-O-acetate. The high anti-plasmodial activity of this compound merits further study about its anti-plasmodial mechanism of action. The active extracts of D. tomentosa, as well as urospermal A 15-O-acetate, displayed only a moderate selectivity, and further studies are needed to assess the safety of the use of the plant by the local population. PMID:22909422

One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

PubMed

Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

2011-09-12

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Impact of microparticle formulation approaches on drug burst release: a level A IVIVC.

PubMed

Ishak, Rania A H; Mortada, Nahed D; Zaki, Noha M; El-Shamy, Abd El-Hamid A; Awad, Gehanne A S

2014-01-01

To study the effect of poly(d,l-lactic-co-glycolic acid) (PLGA) microparticles (MPs) preparation techniques on particle physical characterization with special emphasis on burst drug release. A basic drug clozapine was used in combination with acid-terminated PLGA. Two approaches for MP preparation were compared; the in situ forming microparticle (ISM) and the emulsion-solvent evaporation (ESE) methods using an experimental design. The MPs obtained were compared according to their physical characterization, burst release and T80%. An in vivo pharmacokinetic study with in vitro-in vivo correlation (IVIVC) was also performed for the selected formula. Both methods were able to sustain drug release for three weeks. ISM produced more porous particles and was not effective as ESE for controlling burst release. A good IVIVC (R(2) = 0.9755) was attained when injecting the selected formula into rats. MPs prepared with ESE showed a minimum burst release and a level A IVIVC was obtained when administered to rats.

A Novel Source of Cultured Podocytes

PubMed Central

Da Sacco, Stefano; Lemley, Kevin V.; Sedrakyan, Sargis; Zanusso, Ilenia; Petrosyan, Astgik; Peti-Peterdi, Janos; Burford, James; De Filippo, Roger E.; Perin, Laura

2013-01-01

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies. PMID:24349133

In vitro studies of serum albumin interaction with poly(D,L-lactide) nanospheres loaded by hydrophobic cargo.

PubMed

Pietkiewicz, Jadwiga; Wilk, Kazimiera A; Bazylińska, Urszula

2016-01-05

The various polymer-based nanocarriers are very attractive for in vitro and in vivo bioapplications. A new type of a promising drug delivery systems for cancer tissues-poly(D,L-lactide) nanospheres stabilized with Cremophor EL and loaded with hydrophobic cyanines (IR-780 or ZnPc) or curcumin (CUR) were fabricated by the nanoprecipitation method. The Cremophor EL/PLA/water nanospheres demonstrated regular shape, low polydispersity (PdI<0.3) and high entrapment efficiency of selected cargo (over 90%). The size of those nanoconstructs below 130 nm are in the desired nanocarriers size range for tumor delivery. Low level of in vitro drug release from loaded nanospheres after long-time storage indicates their good stability. The half-life of nanocarriers in the circulation, and their biodistribution after parenteral administration are associated with the ability of plasma proteins adsorption. For these reasons the affinity of obtained nanospheres for albumin as a major plasma protein was in vitro investigated. The binding of nanocarrier containing cyanine IR-780 with albumin immobilized in the wells of polystyrene plate occurred with lower efficiency than analogs loaded with ZnPc or CUR. Similar relationships were observed after UV-vis spectra analysis of nanospheres in the presence of albumin at various protein concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

At the Crossroads of Nanotoxicology: Past Achievements and Current Challenges

DTIC Science & Technology

2015-01-01

rates of ionic dissolution, improving in vitro to in vivo predictive efficiencies, and establishing safety exposure limits. This Review will discuss...Oberdörster et al., 2005a), which drove the focus of in vitro and in vivo model selection to accommodate these areas of higher NM exposure. Most...Accordingly, a current challenge is the design of simple, in vitro models that reliably predict in vivo effects following a NM challenge. In order

In Vitro Evaluation and Mechanism Analysis of the Fiber Shedding Property of Textile Pile Debridement Materials

PubMed Central

Fu, Yijun; Xie, Qixue; Lao, Jihong; Wang, Lu

2016-01-01

Fiber shedding is a critical problem in biomedical textile debridement materials, which leads to infection and impairs wound healing. In this work, single fiber pull-out test was proposed as an in vitro evaluation for the fiber shedding property of a textile pile debridement material. Samples with different structural design (pile densities, numbers of ground yarns and coating times) were prepared and estimated under this testing method. Results show that single fiber pull-out test offers an appropriate in vitro evaluation for the fiber shedding property of textile pile debridement materials. Pull-out force for samples without back-coating exhibited a slight escalating trend with the supplement in pile density and number of ground yarn plies, while back-coating process significantly raised the single fiber pull-out force. For fiber shedding mechanism analysis, typical pull-out behavior and failure modes of the single fiber pull-out test were analyzed in detail. Three failure modes were found in this study, i.e., fiber slippage, coating point rupture and fiber breakage. In summary, to obtain samples with desirable fiber shedding property, fabric structural design, preparation process and raw materials selection should be taken into full consideration. PMID:28773428

Models of Protocellular Structure, Function and Evolution

NASA Technical Reports Server (NTRS)

New, Michael H.; Pohorille, Andrew; Szostak, Jack W.; Keefe, Tony; Lanyi, Janos K.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

In the absence of any record of protocells, the most direct way to test our understanding, of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard Medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids in which a small number of functional molecules are selected from a large, random population of candidates. The selected molecules are next vastly multiplied using the polymerase chain reaction.

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells

NASA Astrophysics Data System (ADS)

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells.

PubMed

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

In Vitro Sensitivity and Resistance of 46 Leptospira Strains Isolated from Rats in the Philippines to 14 Antimicrobial Agents▿ †

PubMed Central

Chakraborty, Antara; Miyahara, Satoshi; Villanueva, Sharon Y. A. M.; Gloriani, Nina G.; Yoshida, Shin-ichi

2010-01-01

The in vitro susceptibilities of 46 Leptospira isolates from rats to 14 antimicrobial agents were tested. All of the strains were found to be sensitive to ampicillin, cefotaxime, ciprofloxacin, norfloxacin, doxycycline, erythromycin, and streptomycin. In contrast, the tested isolates showed resistance to amphotericin B, 5-fluorouracil, fosfomycin, trimethoprim, sulfamethoxazole, neomycin, and vancomycin. These findings will help in selecting effective and ineffective antimicrobials for treatment of leptospirosis and for the development of new selective media, respectively. PMID:20855741

From Cell to Beak: In-Vitro and In-Vivo Characterization of Chicken Bitter Taste Thresholds.

PubMed

Cheled-Shoval, Shira; Behrens, Maik; Korb, Ayelet; Di Pizio, Antonella; Meyerhof, Wolfgang; Uni, Zehava; Niv, Masha Y

2017-05-17

Bitter taste elicits an aversive reaction, and is believed to protect against consuming poisons. Bitter molecules are detected by the Tas2r family of G-protein-coupled receptors, with a species-dependent number of subtypes. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors-ggTas2r1, ggTas2r2 and ggTas2r7. This minimalistic bitter taste system in chickens was used to determine relationships between in-vitro (measured in heterologous systems) and in-vivo (behavioral) detection thresholds. ggTas2r-selective ligands, nicotine (ggTas2r1), caffeine (ggTas2r2), erythromycin and (+)-catechin (ggTas2r7), and the Tas2r-promiscuous ligand quinine (all three ggTas2rs) were studied. Ligands of the same receptor had different in-vivo:in-vitro ratios, and the ggTas2r-promiscuous ligand did not exhibit lower in-vivo:in-vitro ratios than ggTas2r-selective ligands. In-vivo thresholds were similar or up to two orders of magnitude higher than the in-vitro ones.

Is it time for a paradigm shift in understanding embryo selection?

PubMed

Gleicher, Norbert; Kushnir, Vitaly A; Barad, David H

2015-01-11

Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF. We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients. Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.

In Vitro Effects of Cooking Methods on Digestibility of Lipids and Formation of Cholesterol Oxidation Products in Pork

PubMed Central

Moon, Sung Sil

2014-01-01

This study investigated the effects of cooking methods on the digestibility of lipids and formation of cholesterol oxidation products (COPs) in pork, during in vitro human digestion. Pork patties were cooked using four different methods (oven cooking, pan frying, boiling, and microwaving), to an internal temperature of approximately 85℃. The digestibility of pork patties were then evaluated, using the in vitro human digestion model that simulated the composition (pH, minerals, surfaceactive components, and enzymes) of digestive juices in the human mouth, stomach, and small intestine. The total lipid digestibility was higher after microwave cooking, whereas pan-frying resulted in lower in vitro digestibility, compared to the other cooking methods. The microwaving method followed by in vitro digestion also showed significantly higher content of free fatty acids and thiobarbituric acid reactive substances (TBARS), compared to the other cooking methods; whereas, the pan frying and boiling methods showed the lowest. Cholesterol content was not significantly different among the cooked samples before, and after in vitro human digestion. The formation of COPs was significantly higher in the microwave-treated pork samples, compared to those cooked by the other methods, which was consistent with the trend for lipid peroxidation (TBARS). We propose that from the point of view of COPs formation and lipid oxidation, the pan-frying or boiling methods would be useful. PMID:26761168

Isolation and in vitro selection of actinomycetes strains as potential probiotics for aquaculture

PubMed Central

Bernal, Milagro García; Campa-Córdova, Ángel Isidro; Saucedo, Pedro Enrique; González, Marlen Casanova; Marrero, Ricardo Medina; Mazón-Suástegui, José Manuel

2015-01-01

Aim: This study was designed to describe a series of in vitro tests that may aid the discovery of probiotic strains from actinomycetes. Materials and Methods: Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis of human blood cells, respectively. Extracellular enzymatic production was monitored by the hydrolysis of proteins, lipids and carbohydrates. Tolerance to different pH values and salt concentrations was also determined, followed by hydrophobicity analysis and genetic identification of the most promising strains. Results: Five out of 31 isolated strains showed antimicrobial activity against three Vibrio species. Three non-hemolytic strains (N7, RL8 and V4) among these active isolates yielded positive results in hydrophobicity tests and exhibited good growth at salt concentrations ranging from 0% to 10%, except strain RL8, which required a salt concentration >0.6%. Although these strains did not grow at pH<3, they showed different enzymatic activities. Phylogenetic analysis revealed that strains N7 and V4 have more than 99% identity with several Streptomyces species, whereas the closest matches to strain RL8 are Streptomyces panacagri and Streptomyces flocculus, with 98% and 98.2% similarity, respectively. Conclusion: Three actinomycetes strains showing probiotic-like properties were discovered using several in vitro tests that can be easily implemented in different institutions around the world. PMID:27047067

Sirc-cvs cytotoxicity test: an alternative for predicting rodent acute systemic toxicity.

PubMed

Kitagaki, Masato; Wakuri, Shinobu; Hirota, Morihiko; Tanaka, Noriho; Itagaki, Hiroshi

2006-10-01

An in vitro crystal violet staining method using the rabbit cornea-derived cell line (SIRC-CVS) has been developed as an alternative to predict acute systemic toxicity in rodents. Seventy-nine chemicals, the in vitro cytotoxicity of which was already reported by the Multicenter Evaluation of In vitro Toxicity (MEIC) and ICCVAM/ECVAM, were selected as test compounds. The cells were incubated with the chemicals for 72 hrs and the IC(50) and IC(35) values (microg/mL) were obtained. The results were compared to the in vivo (rat or mouse) "most toxic" oral, intraperitoneal, subcutaneous and intravenous LD(50) values (mg/kg) taken from the RTECS database for each of the chemicals by using Pearson's correlation statistics. The following parameters were calculated: accuracy, sensitivity, specificity, prevalence, positive predictability, and negative predictability. Good linear correlations (Pearson's coefficient; r>0.6) were observed between either the IC(50) or the IC(35) values and all the LD(50) values. Among them, a statistically significant high correlation (r=0.8102, p<0.001) required for acute systemic toxicity prediction was obtained between the IC(50) values and the oral LD(50) values. By using the cut-off concentrations of 2,000 mg/kg (LD(50)) and 4,225 microg/mL (IC(50)), no false negatives were observed, and the accuracy was 84.8%. From this, it is concluded that this method could be used to predict the acute systemic toxicity potential of chemicals in rodents.

Antioxidative and in vitro antiproliferative activity of Arctium lappa root extracts

PubMed Central

2011-01-01

Background Arctium lappa, known as burdock, is widely used in popular medicine for hypertension, gout, hepatitis and other inflammatory disorders. Pharmacological studies indicated that burdock roots have hepatoprotective, anti-inflammatory, free radical scavenging and antiproliferative activities. The aim of this study was to evaluate total phenolic content, radical scavenging activity by DPPH and in vitro antiproliferative activity of different A. lappa root extracts. Methods Hot and room temperature dichloromethanic, ethanolic and aqueous extracts; hydroethanolic and total aqueous extract of A. lappa roots were investigated regarding radical scavenging activity by DPPH, total phenolic content by Folin-Ciocalteau method and antiproliferative in vitro activity was evaluated in human cancer cell lines. The hydroethanolic extract analyzed by high-resolution electrospray ionization mass spectroscopy. Results Higher radical scavenging activity was found for the hydroethanolic extract. The higher phenolic contents were found for the dichloromethane, obtained both by Soxhlet and maceration extraction and hydroethanolic extracts. The HRESI-MS demonstrated the presence of arctigenin, quercetin, chlorogenic acid and caffeic acid compounds, which were identified by comparison with previous data. The dichloromethane extracts were the only extracts that exhibited activity against cancer cell lines, especially for K562, MCF-7 and 786-0 cell lines. Conclusions The hydroethanolic extracts exhibited the strongest free radical scavenging activity, while the highest phenolic content was observed in Soxhlet extraction. Moreover, the dichloromethanic extracts showed selective antiproliferative activity against K562, MCF-7 and 786-0 human cancer cell lines. PMID:21429215

RNA mediated assembly of nanostructures

NASA Astrophysics Data System (ADS)

Rouge, Jessica Lynn

The first chapter of this work presents a comprehensive look at RNA mediated nanoparticle formation. The overall goal of this research is to gain a deeper understanding of the RNA-particle formation mechanism and the basic properties of the materials selected by modified RNA molecules. Understanding such RNA-substrate interactions and how they translate into the physical and chemical characteristics of the nanoparticles they create are important fundamental concepts when considering these biotemplated materials as potential chemical catalysts. The RNA sequences discussed in the first chapter (referred to as Pdases) were discovered using RNA in vitro selection techniques. These Pdases were found to be capable of forming inorganic palladium (Pd) containing nanoparticles with impressive control over an individual particle's size and shape, despite incubation with the same organometallic precursor. This discovery held exciting implications for inorganic nanoparticle design while also generating numerous questions regarding the mechanism of RNA mediated particle growth. The central question that arose after this initial discovery was how could a biomolecule be used to tailor the physical size and shape of inorganic materials? Starting with a chemical proof designed to uncover the composition of the nanoparticles formed by RNA mediation, this chapter investigates the basic material properties of the nanoparticles while also introducing surprising results regarding the effect of multiple sequences on nanoparticle growth outcomes. In the second chapter, the experiments shift to developing methods to investigate nanoparticle growth mechanisms by fluorescence spectroscopy. A fluorescence polarization anisotropy (FPA) assay is presented in which the strengths of the technique are adapted for studying the formation of RNA mediated Pd nanoparticles in real time. This is a unique application of FPA, as it has been adapted to encompass both the biochemical and materials analysis of a single dynamic system. Although the initial studies described in chapter two focus on the growth kinetics of selected Pdases and their organometallic substrate (Pd2DBA3), it is envisioned that this technique can be used to study a variety of biotemplated systems in a similar fashion. For the experiments described, a key interest was to understand if the RNA governed the rates associated with nanoparticle formation and to gain deeper insight in to the potential growth mechanisms of RNA-nanoparticle constructs. Understanding such interactions could help identify the role RNA play in forming materials while also helping to shape the experimental design of future in vitro selections between RNA and materials. The strengths of these FPA experiments are described as well the associated kinetics observed for RNA mediated particle growth. In chapter three, the fundamental concepts surrounding RNA-nanoparticle interactions shifts to the first application-oriented study of RNA mediated nanoparticle formation for chemical catalysis. The product of a second materials selection is presented in which platinum (Pt) rich nanoparticles are formed using pyridyl modified RNA sequences. These RNA-Pt nanoparticle constructs are interfaced with cadmium sulfide (CdS) quantum dots in an effort to assess the ability of the RNA-Pt nanoparticles to serve as functional catalyst for the photocatalytic production of metal hydrides from aqueous solutions at neutral pH. Metal hydride formation is a crucial step in the challenging chemical reaction of water splitting. The results of this hybrid RNA-Pt/CdS water splitting catalyst are described and compared to more traditional catalyst designs. In the final chapter, the combination of concepts and insights gained as presented in chapters 1-3 are systematically combined into the first RNA in vitro selection for photochemically active materials. This novel selection utilizes an RNA library that is chemically modified such that it can both find and assess the ability of a material to perform photon-driven oxidation chemistry in a complex mixture. In order to conduct such a selection, a new DNA phosphoramidite was synthesized and attached to the RNA library prior to beginning the selection. The details of this synthesis are described. Later in this chapter, the results of this complex yet powerful in vitro selection are outlined. In closing, the prospect of using in vitro selection techniques for discovering other chemically active materials is discussed.

In search of the skeletal stem cell: isolation and separation strategies at the macro/micro scale for skeletal regeneration.

PubMed

Gothard, David; Tare, Rahul S; Mitchell, Peter D; Dawson, Jonathan I; Oreffo, Richard O C

2011-04-07

Skeletal stem cells (SSCs) show great capacity for bone and cartilage repair however, current in vitro cultures are heterogeneous displaying a hierarchy of differentiation potential. SSCs represent the diminutive true multipotent stem cell fraction of bone marrow mononuclear cell (BMMNC) populations. Endeavours to isolate SSCs have generated a multitude of separation methodologies. SSCs were first identified and isolated by their ability to adhere to culture plastic. Once isolated, further separation is achieved via culture in selective or conditioned media (CM). Indeed, preferential SSC growth has been demonstrated through selective in vitro culture conditions. Other approaches have utilised cell morphology (size and shape) as selection criteria. Studies have also targeted SSCs based on their preferential adhesion to specified compounds, individually or in combination, on both macro and microscale platforms. Nevertheless, most of these methods which represent macroscale function with relatively high throughput, yield insufficient purity. Consequently, research has sought to downsize isolation methodologies to the microscale for single cell analysis. The central approach is identification of the requisite cell populations of SSC-specific surface markers that can be targeted for isolation by either positive or negative selection. SELEX and phage display technology provide apt means to sift through substantial numbers of candidate markers. In contrast, single cell analysis is the paramount advantage of microfluidics, a relatively new field for cell biology. Here cells can be separated under continuous or discontinuous flow according to intrinsic phenotypic and physicochemical properties. The combination of macroscale quantity with microscale specificity to generate robust high-throughput (HT) technology for pure SSC sorting, isolation and enrichment offers significant implications therein for skeletal regenerative strategies as a consequence of lab on chip derived methodology.

Immunophototherapy for the treatment of AIDS and AIDS-related infections

NASA Astrophysics Data System (ADS)

Schlager, Kenneth J.

1992-06-01

Immunophototherapy (IPT) is an experimental method of medical treatment that seeks to provide for the selective destruction of diseased cells and microbes such as human immunodeficiency virus (HIV)-T4 cells and the rapid elimination of their toxic by-products from the human body. Photosensitive monoclonal or polyclonal antibody fragments, which are specific to the diseased cell or microbe, will be used to treat acquired immunodeficiency syndrome (AIDS) and related infections. These antibody fragments are tagged with photosensitive compounds and metal colloids and then intravenously injected into the patient. The tagged antibodies quickly and selectively bind to the diseased cells or microbes in the blood stream and affected organs. These cells or microbes are then selectively destroyed by irradiation of these complexes with light of the proper wavelength. This light activates the photosensitive material which then creates singlet oxygen that destroys the microbe or cell. Toxic products of lysis are quickly discharged from the body by activation of the reticuloendothelial system. IPT has been demonstrated by Biotronics to be very effective in the in vitro selective destruction of specified cell types. In a proposed AIDS-treatment research program, IPT will be first demonstrated in vitro for a set of infected blood samples using commercially-available antibodies labeled with appropriate photosensitizers. Efficacy will be determined by a p24 antigen immunodiagnostic test that will indicate the % inhibition in comparison to controls and samples treated with the drug AZT. Subcontracted animal efficacy studies will use a SCID-hu mouse model and PCR/DNA-RNA for endpoint analysis. Toxicity studies of animal (rat) models will be based on post-treatment investigations of lymph nodes, spleen, liver and other organs.

In vitro antioxidant activities of edible artichoke (Cynara scolymus L.) and effect on biomarkers of antioxidants in rats.

PubMed

Jiménez-Escrig, Antonio; Dragsted, Lars Ove; Daneshvar, Bahram; Pulido, Raquel; Saura-Calixto, Fulgencio

2003-08-27

Artichoke (Cynara scolymus L.), an edible vegetable from the Mediterranean area, is a good source of natural antioxidants such as vitamin C, hydroxycinnamic acids, and flavones. The antioxidant activity of aqueous-organic extracts of artichoke were determined using three methods: (a) free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) scavenging, (b) ferric-reducing antioxidant power (FRAP), and (c) inhibition of copper(II)-catalyzed in vitro human low-density lipoprotein (LDL) oxidation. In addition, the present study was performed to investigate the ability of the edible portion of artichoke to alter in vivo antioxidative defense in male rats using selected biomarkers of antioxidant status. One gram (dry matter) had a DPPH(*) activity and a FRAP value in vitro equivalent to those of 29.2 and 62.6 mg of vitamin C and to those of 77.9 and 159 mg of vitamin E, respectively. Artichoke extracts showed good efficiency in the inhibition in vitro of LDL oxidation. Neither ferric-reducing ability nor 2,2'-azinobis(3-ethylbenzothiazolin-6-sulfonate) radical scavenging activity was modified in the plasma of the artichoke group with respect to the control group. Among different antioxidant enzymes measured (superoxide dismutase, gluthatione peroxidase, gluthatione reductase, and catalase) in erythrocytes, only gluthatione peroxidase activity was elevated in the artichoke group compared to the control group. 2-Aminoadipic semialdehyde, a protein oxidation biomarker, was decreased in plasma proteins and hemoglobin in the artichoke-fed group versus the control group. In conclusion, the in vitro protective activity of artichoke was confirmed in a rat model.

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.

Coating solid dispersions on microneedles via a molten dip coating method: development and in vitro evaluation for transdermal delivery of a water insoluble drug

PubMed Central

Ma, Yunzhe; Gill, Harvinder S.

2014-01-01

This study demonstrates for the first time the ability to coat solid dispersions on microneedles as a means to deliver water-insoluble drugs through the skin. Polyethylene glycol (PEG) was selected as the hydrophilic matrix, and lidocaine base was selected as the model hydrophobic drug to create the solid dispersion. First, thermal characterization and viscosity measurements of the PEG-lidocaine mixture at different mass fractions were performed. The results show that lidocaine can remain stable at temperatures up to ~130 °C, and that viscosity of the PEG-lidocaine molten solution increases as the mass fraction of lidocaine decreases. Differential scanning calorimetry demonstrated that at lidocaine mass fraction less than or equal to 50%, lidocaine is well dispersed in the PEG-lidocaine mixture. Uniform coatings were obtained on microneedle surfaces. In vitro dissolution studies in porcine skin showed that microneedles coated with PEG-lidocaine dispersions resulted in significantly higher delivery of lidocaine in just 3 min compared to 1 h topical application of 0.15 g EMLA®, a commercial lidocaine-prilocaine cream. In conclusion, the molten coating process we introduce here offers a practical approach to coat water-insoluble drugs on microneedles for transdermal delivery. PMID:25213295

A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication.

PubMed

Thayanithy, Venugopal; O'Hare, Patrick; Wong, Phillip; Zhao, Xianda; Steer, Clifford J; Subramanian, Subbaya; Lou, Emil

2017-11-13

Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

A method for deriving homogenous population of oligodendrocytes from mouse embryonic stem cells.

PubMed

Neman, J; de Vellis, J

2012-06-01

There is a pressing need for new therapeutics for the generation and transplantation of oligodendrocyte to the white matter to help replace and render injured cells that are lost in demyelinating disease. There are a few protocols describing a homogenous derivation of non-manipulated mouse embryonic stem cells to oligodendrocytes (ES-OL). Moreover, protocols that are successful in producing ES-OL do so with low efficiency. Therefore, we describe clear methodology for differentiation of mouse ES cells to oligodendrocyte to a high degree of homogenity and reproducibility in vitro. In addition, taking advantage of three defined media, we can generate a defined ES to oligodendrocyte lineage while selecting against neurons and astrocytes. More specifically, (1) Glial stem cell defining media (GSCDM), supplemented with appropriate combination of SHH and RA support pro-oligodendrocyte developing neural spheres from ES cells, (2) Oligodendrocyte differentiating media, induces lineage selection of oligodendrocytes progenitors from neural stem cells, and (3) Oligodendrocyte maturation media, supports oligodendrocytes progenitor maturation. Moreover, the ES cell derived oligodendrocytes display mature properites in the prescence of rat dorsal root gangila in vitro. Thus confirming thier potential for use to invesitgate developmental pathways and future potential use of cells in transplantation towards myelin repair. Copyright © 2012 Wiley Periodicals, Inc.

Tooth wear against ceramic crowns in posterior region: a systematic literature review

PubMed Central

Hmaidouch, Rim; Weigl, Paul

2013-01-01

The objective of this systematic review was to assess tooth wear against ceramic crowns in posterior region in vitro and in vivo. An electronic PubMed search was conducted to identify studies on tooth wear against ceramic crowns in posterior region. The selected studies were analyzed in regard to type of crowns, natural antagonist, measuring protocol and outcome. From a yield of 1 000 titles, 43 articles were selected for full-text analysis; finally, no in vitro and only five in vivo studies met the inclusion criteria. As there is heterogeneity in design, used measuring method, ceramics and analysis-form, a meta-analysis was not possible. Results of these studies are very controversial which makes a scientifically valid comparison impossible. This review indicated that some all-ceramic crowns are as wear friendly as metal-ceramic crowns. Up to now, it has been impossible to associate tooth wear with any specific causal agent. The role of ceramic surface treatment that might be responsible for the changing in rate of tooth wear seems undetermined as yet through clinical trials. The literature reveals that studies on this topic are subject to a substantial amount of bias. Therefore, additional clinical studies, properly designed to diminish bias, are warranted. PMID:24136675

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

PubMed

Rouet, Romain; Thuma, Benjamin A; Roy, Marc D; Lintner, Nathanael G; Rubitski, David M; Finley, James E; Wisniewska, Hanna M; Mendonsa, Rima; Hirsh, Ariana; de Oñate, Lorena; Compte Barrón, Joan; McLellan, Thomas J; Bellenger, Justin; Feng, Xidong; Varghese, Alison; Chrunyk, Boris A; Borzilleri, Kris; Hesp, Kevin D; Zhou, Kaihong; Ma, Nannan; Tu, Meihua; Dullea, Robert; McClure, Kim F; Wilson, Ross C; Liras, Spiros; Mascitti, Vincent; Doudna, Jennifer A

2018-05-30

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Dynamism to promote reproductive medicine and its development, Rihachi Iizuka.

PubMed

Sueoka, K

2001-12-01

Rihachi Iizuka has contributed strong leadership for the remarkable development of reproductive medicine which has undergone a complete transformation in the previous half century. The Keio University Hospital introduced artificial insemination as the first assisted reproductive technology in Japan. As it follows, lizuka and his colleagues first reported the live birth of a female infant in August 1949 after heterologous insemination: AID. Iizuka and his colleagues were also among the first to successfully inseminate a woman with sperm that had been frozen. He developed the new cryopreservation medium for human semen called "KS Cryo-medium". He also developed semen preparation methods of washing and concentrating sperm counts by centrifugation with Percoll (colloidal silica derivative) solution for oligozoospermic patients. These methods are broadly used in the clinical field. Furthermore, he developed the X-, Y-bearing sperm preseparation method using Percoll which is the so-called "gender selection" procedure for preventing X-linked genetic disorders. The most striking assisted reproductive technology was in vitro fertilization first carried out in Britain. Prior to the clinical application in Japan, the Japan Society of Fertilization and Implantation was established as the main organ for the exchange of official scientific information by lizuka in 1982. As rapid development and spreading of in vitro fertilization and its implicated technologies, lizuka and his colleague of the department had the first success of offspring following embryo freezing and thawing in Japan which was performed at the Tokyo Dental College Ichikawa General Hospital. Already the numbers of offspring following in vitro fertilization treatment has risen to approximately 1% of births in Japan. Rihachi lizuka still undertakes the responsibility for reproductive medicine as he has done so far.

Assessment of human epidermal model LabCyte EPI-MODEL for in vitro skin irritation testing according to European Centre for the Validation of Alternative Methods (ECVAM)-validated protocol.

PubMed

Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-Ichiro

2009-06-01

A validation study of an in vitro skin irritation testing method using a reconstructed human skin model has been conducted by the European Centre for the Validation of Alternative Methods (ECVAM), and a protocol using EpiSkin (SkinEthic, France) has been approved. The structural and performance criteria of skin models for testing are defined in the ECVAM Performance Standards announced along with the approval. We have performed several evaluations of the new reconstructed human epidermal model LabCyte EPI-MODEL, and confirmed that it is applicable to skin irritation testing as defined in the ECVAM Performance Standards. We selected 19 materials (nine irritants and ten non-irritants) available in Japan as test chemicals among the 20 reference chemicals described in the ECVAM Performance Standard. A test chemical was applied to the surface of the LabCyte EPI-MODEL for 15 min, after which it was completely removed and the model then post-incubated for 42 hr. Cell v iability was measured by MTT assay and skin irritancy of the test chemical evaluated. In addition, interleukin-1 alpha (IL-1alpha) concentration in the culture supernatant after post-incubation was measured to provide a complementary evaluation of skin irritation. Evaluation of the 19 test chemicals resulted in 79% accuracy, 78% sensitivity and 80% specificity, confirming that the in vitro skin irritancy of the LabCyte EPI-MODEL correlates highly with in vivo skin irritation. These results suggest that LabCyte EPI-MODEL is applicable to the skin irritation testing protocol set out in the ECVAM Performance Standards.

Morphometric assessment of in vitro matured dromedary camel oocytes determines the developmental competence after parthenogenetic activation.

PubMed

Saadeldin, Islam M; Swelum, Ayman Abdel-Aziz; Yaqoob, Syed Hilal; Alowaimer, Abdullah Nasser

2017-06-01

The aim of the current study was to improve the selection method of camel oocytes after in vitro maturation by reducing exclusion criteria that were based only on the presence of the first polar body. A combined nuclear and morphometric assessment of camel oocytes after in vitro maturation was included to perform a judgment. The nuclear status of the oocytes, including the presence of the first polar body, meiosis I stage, and lack of nuclear materials, was investigated. The morphometric criteria that comprised the dimensions of each oocyte were as follows: diameter of the whole oocyte, including the zona pellucida (ZPO), zona pellucida thickness (ZPT), ooplasm diameter (OD), the perivitelline space (PVS) area, and PVS diameter. Among the oocytes with different nuclear status, there were no differences in ZPO and ZPT. However, oocytes with no nuclear material showed a significant reduction in OD (110.19 ± 1.4 μm) and a significant increase in PVS area (2139 ± 324.6 μm 2 ) and PVS diameter (13.9 ± 1.96 μm) when compared with oocytes in the meiosis I stage (117.41 ± 2.85 μm, 1287.4 ± 123.4 μm 2 , and 8.56 ± 0.65 μm, respectively). To simplify the selection, the major difference between meiosis I and degenerated oocytes was the diameter of the PVS, which was greater than the ZPT in degenerated oocytes. Therefore, three groups were morphologically differentiated into oocytes with polar bodies (PB1), meiosis I (MI) oocytes, and degenerated oocytes. MI oocytes were able to extrude the polar body after activation but were not able to develop into blastocysts. In contrast, MI oocytes were able to develop into blastocysts after a biphasic activation protocol in which the oocytes were electrically activated and treated with ionomycin after 2 h. In conclusion, the results obtained by the morphometric assessment allowed us to develop a simple and objective classification system for in vitro matured dromedary camel oocytes, which will lead to accurate oocyte selection for the support of subsequent embryonic development. Copyright © 2017 Elsevier Inc. All rights reserved.

Nanotoxicology: in Vitro-in Vivo Dosimetry:Gangwal et al. Respond

EPA Science Inventory

We appreciate the letter from Oberdörster commenting on the importance of careful selection of in vitro doses for nanomaterial (NM) toxicity testing and his assessment of our article (Gangwal et al. 2011). Because the objective of our study was to use limited data on potential hu...

Screening larch in vitro for resistance to Mycosphaerella laricina

Treesearch

M.E. Ostry; Paula M. Pijut; D.D. Skilling

1991-01-01

Needle blight of larch caused by Mycosphaerella laricina seriously limits the productivity of susceptible trees in the north central and northeastern United States. Adventitious shoots, derived from cotyledon tissue culture, of selected European larch (Larix decidua) and a hybrid larch were inoculated in vitro with three isolates...

Quantifying statistical relationships between commonly used in vitro models for estimating lead bioaccessibility.

PubMed

Yan, Kaihong; Dong, Zhaomin; Liu, Yanju; Naidu, Ravi

2016-04-01

Bioaccessibility to assess potential risks resulting from exposure to Pb-contaminated soils is commonly estimated using various in vitro methods. However, existing in vitro methods yield different results depending on the composition of the extractant as well as the contaminated soils. For this reason, the relationships between the five commonly used in vitro methods, the Relative Bioavailability Leaching Procedure (RBALP), the unified BioAccessibility Research Group Europe (BARGE) method (UBM), the Solubility Bioaccessibility Research Consortium assay (SBRC), a Physiologically Based Extraction Test (PBET), and the in vitro Digestion Model (RIVM) were quantified statistically using 10 soils from long-term Pb-contaminated mining and smelter sites located in Western Australia and South Australia. For all 10 soils, the measured Pb bioaccessibility regarding all in vitro methods varied from 1.9 to 106% for gastric phase, which is higher than that for intestinal phase: 0.2 ∼ 78.6%. The variations in Pb bioaccessibility depend on the in vitro models being used, suggesting that the method chosen for bioaccessibility assessment must be validated against in vivo studies prior to use for predicting risk. Regression studies between RBALP and SRBC, RBALP and RIVM (0.06) (0.06 g of soil in each tube, S:L ratios for gastric phase and intestinal phase are 1:375 and 1:958, respectively) showed that Pb bioaccessibility based on the three methods were comparable. Meanwhile, the slopes between RBALP and UBM, RBALP and RIVM (0.6) (0.6 g soil in each tube, S:L ratios for gastric phase and intestinal phase are 1:37.5 and 1:96, respectively) were 1.21 and 1.02, respectively. The findings presented in this study could help standardize in vitro bioaccessibility measurements and provide a scientific basis for further relating Pb bioavailability and soil properties.

Potential countersample materials for in vitro simulation wear testing.

PubMed

Shortall, Adrian C; Hu, Xiao Q; Marquis, Peter M

2002-05-01

Any laboratory investigation of the wear resistance of dental materials needs to consider oral conditions so that in vitro wear results can be correlated with in vivo findings. The choice of the countersample is a critical factor in establishing the pattern of tribological wear and in achieving an efficient in vitro wear testing system. This research investigated the wear behavior and surface characteristics associated with three candidate countersample materials used for in vitro wear testing in order to identify a possible suitable substitute for human dental enamel. Three candidate materials, stainless steel, steatite and dental porcelain were evaluated and compared to human enamel. A variety of factors including hardness, wear surface evolution and frictional coefficients were considered, relative to the tribology of the in vivo situation. The results suggested that the dental porcelain investigated bore the closest similarity to human enamel of the materials investigated. Assessment of potential countersample materials should be based on the essential tribological simulation supported by investigations of mechanical, chemical and structural properties. The selected dental porcelain had the best simulating ability among the three selected countersample materials and this class of material may be considered as a possible countersample material for in vitro wear test purposes. Further studies are required, employing a wider range of dental ceramics, in order to optimise the choice of countersample material for standardized in vitro wear testing.

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Relative Bioavailability and Bioaccessability and Speciation of ...

EPA Pesticide Factsheets

Background: Assessment of soil arsenic (As) bioavailability may profoundly affect the extent of remediation required at contaminated sites by improving human exposure estimates. Because small adjustments in soil As bioavailability estimates can significantly alter risk assessments and remediation goals, convenient, rapid, reliable, and inexpensive tools are needed to determine soil As bioavailability. Objectives: We evaluated inexpensive methods for assessing As bioavailability in soil as a means to improve human exposure estimates and potentially reduce remediation costs. Methods: Nine soils from residential sites affected by mining or smelting activity and two National Institute of Standards and Technology standard reference materials were evaluated for As bioavailability, bioaccessibility, and speciation. Arsenic bioavailability was determined using an in vivo mouse model, and As bioaccessibility was determined using the Solubility/Bioavailability Research Consortium in vitro assay. Arsenic speciation in soil and selected soil physicochemical properties were also evaluated to determine whether these parameters could be used as predictors of As bio¬availability and bioaccessibility. Results: In the mouse assay, we compared bioavailabilities of As in soils with that for sodium arsenate. Relative bioavailabilities (RBAs) of soil As ranged from 11% to 53% (mean, 33%). In vitro soil As bioaccessibility values were strongly correlated with soil As RBAs (R

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.

A fibrin-supported myocardial organ culture for isolation of cardiac stem cells via the recapitulation of cardiac homeostasis.

PubMed

Kim, Jong-Tae; Chung, Hye Jin; Seo, Ji-Yeon; Yang, Young-Il; Choi, Min-Young; Kim, Hyeong-In; Yang, Tae-Hyun; Lee, Won-Jin; Youn, Young Chul; Kim, Hye Jung; Kim, Yeon Mee; Lee, Hyukjin; Jang, Yang-Soo; Lee, Seung-Jin

2015-04-01

There is great interest in the development of cardiac stem cells (CSCs) cell-based therapeutics; thus, clinical translation requires an efficient method for attaining therapeutic quantities of these cells. Furthermore, an in vitro model to investigate the mechanisms regulating the cardiac homeostasis is crucial. We sought to develop a simple myocardial culture method for enabling both the recapitulation of myocardial homeostasis and the simultaneous isolation of CSCs. The intact myocardial fragments were encapsulated 3-dimensionally into the fibrin and cultured under dynamic conditions. The fibrin provided secure physical support and substratum to the myocardium, which mediated integrin-mediated cell signaling that allowed in situ renewal, outgrowth and cardiomyogenic differentiation of CSCs, mimicking myocardial homeostasis. Since our culture maintained the myocardial CSCs niches, it was possible to define the identity of in vitro renewed CSCs that situated in the interstitium between cardiomyocytes and microvessels. Lastly, the use of matrix-restricted fibrinolysis enabled the selective isolation of outgrown CSCs that retained the clonogenicity, long-term growth competency and cardiovascular commitment potential. Collectively, this myocardial culture might be used as an alternative tool for studying cardiac biology and developing cell-based therapeutics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Association of two single-isomer anionic CD in NACE for the chiral and achiral separation of fenbendazole, its sulphoxide and sulphone metabolites: application to their determination after in vitro metabolism.

PubMed

Rousseau, Anne; Gillotin, Florian; Chiap, Patrice; Crommen, Jacques; Fillet, Marianne; Servais, Anne-Catherine

2010-05-01

A NACE method was developed for the separation of fenbendazole (FBZ), a prochiral drug giving rise to chiral (oxfendazole or OFZ) and nonchiral (FBZ sulphone or FBZSO(2)) metabolites. First, the effect of the nature and the concentration of CD as well as that of the acidic BGE on the enantiomeric separation of OFZ were studied. OFZ enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 0.5 M TFA in methanol containing 10 mM heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD and 10 mM heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD. Moreover, the NACE method was found to be particularly well suited to the simultaneous determination of FBZ, OFZ enantiomers, and FBZSO(2). Thiabendazole was selected as an internal standard. The CD-NACE potential was then evaluated for in vitro metabolism studies using FBZ as a model case. The OFZ enantiomers and FBZSO(2) could be detected after incubation of FBZ in the phenobarbital-induced male rat liver microsomes systems.

Phenotypic, Functional, and Safety Control at Preimplantation Phase of MSC-Based Therapy.

PubMed

Lech, Wioletta; Figiel-Dabrowska, Anna; Sarnowska, Anna; Drela, Katarzyna; Obtulowicz, Patrycja; Noszczyk, Bartlomiej Henryk; Buzanska, Leonora; Domanska-Janik, Krystyna

2016-01-01

Mesenchymal stem cells (MSC) exhibit enormous heterogeneity which can modify their regenerative properties and therefore influence therapeutic effectiveness as well as safety of these cells transplantation. In addition the high phenotypic plasticity of MSC population makes it enormously sensitive to any changes in environmental properties including fluctuation in oxygen concentration. We have shown here that lowering oxygen level far below air atmosphere has a beneficial impact on various parameters characteristic for umbilical cord Wharton Jelly- (WJ-) MSC and adipose tissue- (AD-) derived MSC cultures. This includes their cellular composition, rate of proliferation, and maintenance of stemness properties together with commitment to cell differentiation toward mesodermal and neural lineages. In addition, the culture genomic stability increased significantly during long-term cell passaging and eventually protected cells against spontaneous transformation. Also by comparing of two routinely used methods of MSCs isolation (mechanical versus enzymatic) we have found substantial divergence arising between cell culture properties increasing along the time of cultivation in vitro. Thus, in this paper we highlight the urgent necessity to develop the more sensitive and selective methods for prediction and control cells fate and functioning during the time of growth in vitro.

Developing High-Affinity Protein Capture Agents and Nanotechnology-Based Platforms for In Vitro Diagnostics

NASA Astrophysics Data System (ADS)

Rohde, Rosemary Dyane

In this thesis, I describe projects that were aimed at improving ways to capture proteins for clinical diagnostics. Nanoelectronic sensors, such as silicon nanowires (SiNWs), can provide label-free quantitative measurements of protein biomarkers in real time. One technical challenge for SiNWs is to develop chemistry that can be applied for selectively encoding the nanowire surfaces with capture agents, thus making them sensors that have selectivity for specific proteins. Furthermore, because of the nature of how the sensor works, it is desirable to achieve this spatially selective chemical functionalization without having the silicon undergo oxidation. This method is described here and provides a general platform that can incorporate organic and biological molecules on Si (111) with minimal oxidation of the silicon surface. The development of these devices is, in part, driven by early diagnosis, treatment, monitoring, and personalized medicine---all of which are increasingly requiring quantitative, rapid, and multiparameter measurements. To begin achieving this goal, a large number of protein biomarkers need to be captured and quantitatively measured to create a diagnostic panel. One of the greatest challenges towards making protein-biomarker-based in vitro diagnostics inexpensive involves developing capture agents to detect the proteins. A major thrust of this thesis is to develop multi-valent, high-affinity and high-selectivity protein capture agents using in situ click chemistry. In situ click chemistry is a tool that utilizes the protein itself to catalyze the formation of a biligand from individual azide and alkyne ligands that are co-localized. Large one-bead one-compound (OBOC) libraries of peptides are used to form the body of these ligands, also providing high chemical diversity with minimal synthetic effort. This process can be repeated to identify a triligand, tetraligand, and so forth. Moreover, the resulting multiligand protein capture agents can be produced in gram-scale quantities with designed control over chemical and biochemical stability and water solubility. This is a general and robust method for inexpensive, high-throughput capture agent discovery that can be utilized to capture the relevant biomarker proteins for blood protein diagnostics.

Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection.

PubMed Central

Tang, J; Breaker, R R

1997-01-01

We have designed a self-cleaving ribozyme construct that is rendered inactive during preparative in vitro transcription by allosteric interactions with ATP. This allosteric ribozyme was constructed by joining a hammerhead domain to an ATP-binding RNA aptamer, thereby creating a ribozyme whose catalytic rate can be controlled by ATP. Upon purification by PAGE, the engineered ribozyme undergoes rapid self-cleavage when incubated in the absence of ATP. This strategy of "allosteric delay" was used to prepare intact hammerhead ribozymes that would otherwise self-destruct during transcription. Using a similar strategy, we have prepared a combinatorial pool of RNA in order to assess the catalytic fitness of ribozymes that carry the natural consensus sequence for the hammerhead. Using in vitro selection, this comprehensive RNA pool was screened for sequence variants of the hammerhead ribozyme that also display catalytic activity. We find that sequences that comprise the core of naturally occurring hammerhead dominate the population of selected RNAs, indicating that the natural consensus sequence of this ribozyme is optimal for catalytic function. PMID:9257650

LANTCET: laser nanotechnology for screening and treating tumors ex vivo and in vivo

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri O.; Lukianova-Hleb, Ekaterina Y.; Zhdanok, Sergei A.; Hafner, Jason H.; Rostro, Betty C.; Scully, Peter; Konopleva, Marina; Andreeff, Michael; Li, Chun; Hanna, Ehab Y.; Myers, Jeffrey N.; Oraevsky, Alexander A.

2007-06-01

LANTCET (laser-activated nano-thermolysis as cell elimination technology) was developed for selective detection and destruction of individual tumor cells through generation of photothermal bubbles around clusters of light absorbing gold nanoparticles (nanorods and nanoshells) that are selectively formed in target tumor cells. We have applied bare nanoparticles and their conjugates with cell-specific vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia) and C225 (specific for carcinoma cells that express epidermal growth factor -EGF). Clusters were formed by using vector-receptor interactions with further clusterization of nanoparticles due to endocytosis. Formation of clusters was verified directly with optical resonance scattering microscopy and microspectroscopy. LANTCET method was tested in vitro for living cell samples with: (1) model myeloid K562 cells (CD33 positive), (2) primary human bone marrow CD33-positive blast cells from patients with the diagnosis of acute myeloid leukemia, (3) monolayers of living EGF-positive carcinoma cells (Hep-2C), (4) human lymphocytes and red blood cells as normal cells. The LANTCET method was also tested in vivo using rats with experimental polymorphic sarcoma. Photothermal bubbles were generated and detected in vitro with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser. We have found that cluster formation caused an almost 100-fold decrease in the bubble generation threshold of laser pulse fluence in tumor cells compared to the bubble generation threshold for normal cells. The animal tumor that was treated with a single laser pulse showed a necrotic area of diameter close to the pump laser beam diameter and a depth of 1-2 mm. Cell level selectivity of tumor damage with single laser pulse was demonstrated. Combining lightscattering imaging with bubble imaging, we introduced a new image-guided mode of the LANTCET operation for screening and treatment of tumors ex vivo and in vivo.

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

EPA Science Inventory

Chemical biotransformation represents the single largest source of uncertainty in chemical bioaccumulation assessments for fish. In vitro methods employing isolated hepatocytes and liver subcellular fractions (S9) can be used to estimate whole-body rates of chemical metabolism, ...

A comparative study of additive and subtractive manufacturing for dental restorations.

PubMed

Bae, Eun-Jeong; Jeong, Il-Do; Kim, Woong-Chul; Kim, Ji-Hwan

2017-08-01

Digital systems have recently found widespread application in the fabrication of dental restorations. For the clinical assessment of dental restorations fabricated digitally, it is necessary to evaluate their accuracy. However, studies of the accuracy of inlay restorations fabricated with additive manufacturing are lacking. The purpose of this in vitro study was to evaluate and compare the accuracy of inlay restorations fabricated by using recently introduced additive manufacturing with the accuracy of subtractive methods. The inlay (distal occlusal cavity) shape was fabricated using 3-dimensional image (reference data) software. Specimens were fabricated using 4 different methods (each n=10, total N=40), including 2 additive manufacturing methods, stereolithography apparatus and selective laser sintering; and 2 subtractive methods, wax and zirconia milling. Fabricated specimens were scanned using a dental scanner and then compared by overlapping reference data. The results were statistically analyzed using a 1-way analysis of variance (α=.05). Additionally, the surface morphology of 1 randomly (the first of each specimen) selected specimen from each group was evaluated using a digital microscope. The results of the overlap analysis of the dental restorations indicated that the root mean square (RMS) deviation observed in the restorations fabricated using the additive manufacturing methods were significantly different from those fabricated using the subtractive methods (P<.05). However, no significant differences were found between restorations fabricated using stereolithography apparatus and selective laser sintering, the additive manufacturing methods (P=.466). Similarly, no significant differences were found between wax and zirconia, the subtractive methods (P=.986). The observed RMS values were 106 μm for stereolithography apparatus, 113 μm for selective laser sintering, 116 μm for wax, and 119 μm for zirconia. Microscopic evaluation of the surface revealed a fine linear gap between the layers of restorations fabricated using stereolithography apparatus and a grooved hole with inconsistent weak scratches when fabricated using selective laser sintering. In the wax and zirconia restorations, possible traces of milling bur passes were observed. The results indicate that the accuracy of dental restorations fabricated using the additive manufacturing methods is higher than that of subtractive methods. Therefore, additive manufacturing methods are a viable alternative to subtractive methods. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism.

PubMed

López-Arredondo, Damar L; Herrera-Estrella, Luis

2013-05-01

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large-scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods. © 2013 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Glioma Selectivity of Magnetically Targeted Nanoparticles: A Role of Abnormal Tumor Hydrodynamics

PubMed Central

Chertok, Beata; David, Allan E.; Huang, Yongzhuo; Yang, Victor C.

2007-01-01

Magnetic targeting is a promising strategy for achieving localized drug delivery. Application of this strategy to treat brain tumors, however, is complicated by their deep intracranial location, since magnetic field density cannot be focused at a distance from an externally applied magnet. This study intended to examine whether, with magnetic targeting, pathological alteration in brain tumor flow dynamics could be of value in discriminating the diseased site from healthy brain. To address this question, the capture of magnetic nanoparticles was first assessed in vitro using a simple flow system under theoretically estimated glioma and normal brain flow conditions. Secondly, accumulation of nanoparticles via magnetic targeting was evaluated in vivo using 9L-glioma bearing rats. In vitro results that predicted a 7.6-fold increase in nanoparticle capture at glioma-versus contralateral brain-relevant flow rates were relatively consistent with the 9.6-fold glioma selectivity of nanoparticle accumulation over the contralateral brain observed in vivo. Based on these finding, the in vitro ratio of nanoparticle capture can be viewed as a plausible indicator of in vivo glioma selectivity. Overall, it can be concluded that the decreased blood flow rate in glioma, reflecting tumor vascular abnormalities, is an important contributor to glioma-selective nanoparticle accumulation with magnetic targeting. PMID:17628157

Glioma selectivity of magnetically targeted nanoparticles: a role of abnormal tumor hydrodynamics.

PubMed

Chertok, Beata; David, Allan E; Huang, Yongzhuo; Yang, Victor C

2007-10-08

Magnetic targeting is a promising strategy for achieving localized drug delivery. Application of this strategy to treat brain tumors, however, is complicated by their deep intracranial location, since magnetic field density cannot be focused at a distance from an externally applied magnet. This study intended to examine whether, with magnetic targeting, pathological alteration in brain tumor flow dynamics could be of value in discriminating the diseased site from healthy brain. To address this question, the capture of magnetic nanoparticles was first assessed in vitro using a simple flow system under theoretically estimated glioma and normal brain flow conditions. Secondly, accumulation of nanoparticles via magnetic targeting was evaluated in vivo using 9L-glioma bearing rats. In vitro results that predicted a 7.6-fold increase in nanoparticle capture at glioma- versus contralateral brain-relevant flow rates were relatively consistent with the 9.6-fold glioma selectivity of nanoparticle accumulation over the contralateral brain observed in vivo. Based on these finding, the in vitro ratio of nanoparticle capture can be viewed as a plausible indicator of in vivo glioma selectivity. Overall, it can be concluded that the decreased blood flow rate in glioma, reflecting tumor vascular abnormalities, is an important contributor to glioma-selective nanoparticle accumulation with magnetic targeting.

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme.

PubMed

da Silva, Maria Cristina Mattar; Del Sarto, Rafael Perseghini; Lucena, Wagner Alexandre; Rigden, Daniel John; Teixeira, Fabíola Rodrigues; Bezerra, Caroline de Andrade; Albuquerque, Erika Valéria Saliba; Grossi-de-Sa, Maria Fatima

2013-09-20

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸ α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT-751

PubMed Central

Innocenti, Federico; Ramírez, Jacqueline; Obel, Jennifer; Xiong, Julia; Mirkov, Snezana; Chiu, Yi-Lin; Katz, David A.; Carr, Robert A.; Zhang, Wei; Das, Soma; Adjei, Araba; Moyer, Ann M.; Chen, Pei Xian; Krivoshik, Andrew; Medina, Diane; Gordon, Gary B.; Ratain, Mark J.; Sahelijo, Leonardo; Weinshilboum, Richard M.; Fleming, Gini F.; Bhathena, Anahita

2013-01-01

Objective ABT-751, a novel orally available antitubulin agent, is mainly eliminated as inactive glucuronide (ABT-751G) and sulfate (ABT-751S) conjugates. We performed a pharmacogenetic investigation of ABT-751 pharmacokinetics using in-vitro data to guide the selection of genes for genotyping in a phase I trial of ABT-751. Methods UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes were screened for ABT-751 metabolite formation in vitro. Forty-seven cancer patients treated with ABT-751 were genotyped for 21 variants in these genes. Results UGT1A1, UGT1A4, UGT1A8, UGT2B7, and SULT1A1 were found to be involved in the formation of inactive ABT-751 glucuronide (ABT-751G) and sulfate (ABT-751S). SULT1A1 copy number (> 2) was associated with an average 34% increase in ABT-751 clearance (P= 0.044), an 18% reduction in ABT-751 AUC (P = 0.045), and a 50% increase in sulfation metabolic ratios (P=0.025). UGT1A8 rs6431558 was associated with a 28% increase in glucuronidation metabolic ratios (P =0.022), and UGT1A4*2 was associated with a 65% decrease in ABT-751 Ctrough (P = 0.009). Conclusion These results might represent the first example of a clinical pharmacokinetic effect of the SULT1A1 copy number variant on the clearance of a SULT1A1 substrate. A-priori selection of candidate genes guided by in-vitro metabolic screening enhanced our ability to identify genetic determinants of interpatient pharmacokinetic variability. PMID:23670235

Toxicity Assessment of Wild Mushrooms from the Western Ghats, India: An in Vitro and Sub-Acute in Vivo Study

PubMed Central

Sai Latha, S.; Naveen, S.; Pradeep, C. K.; Sivaraj, C.; Dinesh, M. G.; Anilakumar, K. R.

2018-01-01

Background: Poisoning by different kinds of toxic mushrooms is unfortunately becoming an increasingly important medical problem, evident from the growing number of reports worldwide since the 1950s. Mycetism being a health concern, deserves scientific attention. In this perspective, the present study aims to assess the potential effects of ingesting the selected wild mushrooms from regions of the Western Ghats, India. Methods: The preliminary cytotoxicity of the selected mushrooms was studied in vitro on the intestinal NCM460 and the Chang's liver cell lines on the basis of cell viability. Further, the hepatotoxicity was assessed by measuring biologically relevant endpoints such as membrane integrity, mitochondrial stress and oxidative status. A 28 day sub-acute toxicity study was carried out by orally administering the mushroom extracts to mice at 250 and 500 mg/kg body weight. The hematological and serum analysis as well as histological examinations were carried out to evaluate their in vivo toxicity. GC-MS analysis of the mushrooms facilitated the identification of their volatile chemical profile. Result: The in vitro intestinal cytotoxicity exhibited by these wild mushrooms in comparison to the edible mushroom indicated their potential gastrointestinal toxicity. The pathological findings in small intestine on exposure to Chlorophyllum molybdites and Agaricus endoxanthus also validates the speculations about their intestinal toxicity. The toxic insult to the hepatocytes due to Amanita angustilamellata, Entoloma crassum, and Clarkeinda trachodes was predictive of the observed in vivo hepatotoxicity which was also accompanied by renal toxicity at the higher dose of 500 mg/kg bwt. Conclusion: The potential toxicity exhibited by these representative mushrooms from the wild warrants caution about their consumption. The present work could also have broader implications for global mycetism. PMID:29487528

Oncolytic Adenoviruses Targeted to Human Papilloma Virus-Positive Head and Neck Squamous Cell Carcinomas

PubMed Central

LaRocca, Christopher J.; Han, Joohee; Oliveira, Amanda R.; Davydova, Julia; Herzberg, Mark; Gopalakrishnan, Rajaram; Yamamoto, Masato

2016-01-01

Objectives In recent years, the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has markedly increased. Our aim was to design a novel therapeutic agent through the use of conditionally replicative adenoviruses (CRAds) that are targeted to the HPV E6 and E7 oncoproteins. Methods Each adenovirus included small deletion(s) in the E1a region of the genome (Δ24 or CB016) intended to allow for selective replication in HPV-positive cells. In vitro assays were performed to analyze the transduction efficiency of the vectors and the cell viability following viral infection. Then, the UPCI SCC 090 cell line (HPV-positive) was used to establish subcutaneous tumors in the flanks of nude mice. The tumors were then treated with either one dose of the virus or four doses (injected every fourth day). Results The transduction analysis with luciferase-expressing viruses demonstrated that the 5/3 fiber modification maximized virus infectivity. In vitro, both viruses (5/3Δ24 and 5/3CB016) demonstrated profound oncolytic effects. The 5/3CB016 virus was selective for only HPV-positive HNSCC cells, whereas the 5/3Δ24 virus killed HNSCC cells regardless of HPV status. In vivo, single injections of both viruses demonstrated anti-tumor effects until only 6–8 days following viral inoculation. However, after four viral injections, there was statistically significant reduction in tumor growth when compared to the control group (p<0.05). Conclusion CRAds targeted to HPV-positive HNSCCs demonstrated excellent in vitro and in vivo therapeutic effects, and they have the potential to be clinically translated as a novel treatment modality for this emerging disease. PMID:27086483

Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli--an in vitro study.

PubMed

Hasslöf, Pamela; Hedberg, Maria; Twetman, Svante; Stecksén-Blicks, Christina

2010-07-02

Probiotic bacteria are suggested to play a role in the maintenance of oral health. Such health promoting bacteria are added to different commercial probiotic products. The aim of the study was to investigate the ability of a selection of lactobacilli strains, used in commercially available probiotic products, to inhibit growth of oral mutans streptococci and C. albicans in vitro. Eight probiotic lactobacilli strains were tested for growth inhibition on three reference strains and two clinical isolates of mutans streptococci as well as two reference strains and three clinical isolates of Candida albicans with an agar overlay method. At concentrations ranging from 109 to 105 CFU/ml, all lactobacilli strains inhibited the growth of the mutans streptococci completely with the exception of L. acidophilus La5 that executed only a slight inhibition of some strains at concentrations corresponding to 107 and 105 CFU/ml. At the lowest cell concentration (103 CFU/ml), only L. plantarum 299v and L. plantarum 931 displayed a total growth inhibition while a slight inhibition was seen for all five mutans streptococci strains by L. rhamnosus LB21, L. paracasei F19, L. reuteri PTA 5289 and L. reuteri ATCC 55730. All the tested lactobacilli strains reduced candida growth but the effect was generally weaker than for mutans streptococci. The two L. plantarum strains and L. reuteri ATCC 55730 displayed the strongest inhibition on Candida albicans. No significant differences were observed between the reference strains and the clinical isolates. The selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and Candida albicans in vitro.

Comparison of in vitro digestive fluid extraction and traditional in vivo approaches as measures of polycyclic aromatic hydrocarbon bioavailability from sediments

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

Weston, D.P.; Mayer, L.M.

1998-05-01

The bioavailability of particle-associated contaminants was measured by a new approach that employs the digestive fluid of deposit feeders to solubilize contaminants in vitro. The proportion of contaminant solubilized by digestive fluid of the polychaete Arenicola brasiliensis was considered a measure of bioavailability and was contrasted with other, more traditional measures (i.e., uptake clearance, bioaccumulation factor, and absorption efficiency). There was generally good agreement among the four methods on the relative bioavailability of benzo[a]pyrene from six sandy sediments. Measures of phenanthrene bioavailability did not show strong correlations due to both a more limited data set and perhaps greater importance ofmore » uptake from the dissolved phase. The bioavailability of spiked polycyclic aromatic hydrocarbons (PAHs) differed from that of equivalent in situ-contaminated PAH but not in a predictable and consistent manner. By direct measurement of PAH content of recently ingested sediments collected from the foregut the authors were able to quantify the importance of particle-selective feeding in increasing PAH content of ingested material relative to the bulk, ambient sediments. In most instances, the effect of selective feeding by A. brasiliensis was minimal, increasing PAH content of ingested material <20% above the ambient sediments. Absorption efficiencies of PAH during gut passage were determined by direct measurement of PAH concentration in sediments at various points along the digestive tract. Overall digestive absorption efficiencies were similar to the extent of in vitro solubilization by digestive fluids from the same sediments. These data suggest that extent of solubilization of sediment-bound contaminants during gut passage is a critical constraint on uptake and that absorption efficiency, with respect to the solubilized fraction, approaches 100%.« less

Taste masking of ondansetron hydrochloride by polymer carrier system and formulation of rapid-disintegrating tablets.

PubMed

Khan, Shagufta; Kataria, Prashant; Nakhat, Premchand; Yeole, Pramod

2007-06-22

The purpose of this research was to mask the intensely bitter taste of ondansetron HCl and to formulate a rapid-disintegrating tablet (RDT) of the taste-masked drug. Taste masking was done by complexing ondansetron HCl with aminoalkyl methacrylate copolymer (Eudragit EPO) in different ratios by the precipitation method. Drug-polymer complexes (DPCs) were tested for drug content, in vitro taste in simulated salivary fluid (SSF) of pH 6.2, and molecular property. Complex that did not release drug in SSF was considered taste-masked and selected for formulation RDTs. The complex with drug-polymer ratio of 8:2 did not show drug release in SSF; therefore, it was selected. The properties of tablets such as tensile strength, wetting time, water absorption ratio, in vitro disintegration time, and disintegration in the oral cavity were investigated to elucidate the wetting and disintegration characteristics of tablets. Polyplasdone XL-10 7% wt/wt gave the minimum disintegration time. Tablets of batch F4 containing spray-dried mannitol and microcrystalline cellulose in the ratio 1:1 and 7% wt/wt Polyplasdone XL-10 showed faster disintegration, within 12.5 seconds, than the marketed tablet (112 seconds). Good correlation between in vitro disintegration behavior and in the oral cavity was recognized. Taste evaluation of RDT in human volunteers revealed considerable taste masking with the degree of bitterness below threshold value (0.5) ultimately reaching to 0 within 15 minutes, whereas ondansetron HCl was rated intensely bitter with a score of 3 for 10 minutes. Tablets of batch F4 also revealed rapid drug release (t(90), 60 seconds) in SGF compared with marketed formulation (t(90), 240 seconds; P < .01). Thus, results conclusively demonstrated successful masking of taste and rapid disintegration of the formulated tablets in the oral cavity.

Selective factors governing in vitro β-carotene bioaccessibility: negative influence of low filtration cutoffs and alterations by emulsifiers and food matrices.

PubMed

Corte-Real, Joana; Richling, Elke; Hoffmann, Lucien; Bohn, Torsten

2014-12-01

Because of their putative health benefits, the biological fate of carotenoids after digestion has been met with much interest, and ex vivo methods using carotenoid standards to study their digestion and further metabolism have been developed. In the absence of a complex food matrix, that is, when studying isolated carotenoids, protocol conditions of gastrointestinal digestion models have to be adjusted. In this investigation, we hypothesized that certain selected factors would significantly influence the bioaccessibility of β-carotene in vitro. The factors considered included (i) type of lipid matrix employed (milk, cream, or oil), (ii) presence/absence of emulsifiers (e.g. lecithin and taurocholate), (iii) addition of a gastric lipase, and (iv) final filtration (20 or 200 nm) of the digesta. Adding an emulsifier mixture (10 mg lecithin + 50 mg monoolein + 5 mg oleic acid) enhanced β-carotene bioaccessibility 3 times (P < 0.001), whereas additional taurocholate and the presence/absence of gastric lipase added before intestinal digestion had no significant effect. β-Carotene bioaccessibility was superior with oil than with milk (18.8% ± 0.7% and 6.1% ± 0.7%, respectively; P = 0.03), especially after filtration, thus suggesting incomplete micelle formation after addition of milk. Filtration through 20 nm filters reduced carotenoid concentration in the aqueous fraction (from 7.1% ± 0.2% to 5.5% ± 0.2% in samples digested with canola oil, P < 0.001), indicating that not all formed micelles compared in size with those normally formed in vivo. When studying carotenoid standards during in vitro digestion, care should be taken to separate mixed micelles by filtration, and the choice of emulsifier and matrix should be considered. Copyright © 2014 Elsevier Inc. All rights reserved.

The Effect of Nizatidine, a MATE2K Selective Inhibitor, on the Pharmacokinetics and Pharmacodynamics of Metformin in Healthy Volunteers

PubMed Central

Morrissey, Kari M.; Stocker, Sophie L.; Chen, Eugene C.; Castro, Richard A.; Brett, Claire M.; Giacomini, Kathleen M.

2015-01-01

Background and Objectives In the proximal tubule, basic drugs are transported from the renal cells to the tubule lumen through the concerted action of the H+/organic cation antiporters, multidrug and toxin extrusion 1 (MATE1) and 2K (MATE2K). Dual inhibitors of the MATE transporters have been shown to have a clinically relevant effect on the pharmacokinetics of concomitantly administered basic drugs. However, the clinical impact of selective renal organic cation transport inhibition on the pharmacokinetics and pharmacodynamics of basic drugs, such as metformin, is unknown. This study sought to identify a selective MATE2K inhibitor in vitro and to determine its clinical impact on the pharmacokinetics and pharmacodynamics of metformin in healthy subjects. Methods A strategic cell-based screen of 71 U.S. Food and Drug Administration (FDA)-approved medications was conducted to identify selective inhibitors of renal organic cation transporters that are capable of inhibiting at clinically relevant concentrations. From this screen, nizatidine was identified and predicted to be a clinically potent and selective inhibitor of MATE2K-mediated transport. The effect of nizatidine on the pharmacokinetics and pharmacodynamics of metformin was evaluated in 12 healthy volunteers in an open-label, randomized, two-phase crossover drug-drug interaction (DDI) study. Results In healthy volunteers, the MATE2K-selective inhibitor, nizatidine, significantly increased the apparent volume of distribution, half-life and hypoglycemic activity of metformin. However, despite achieving unbound maximum concentrations greater than the in vitro inhibition potency (IC50) of MATE2K-mediated transport, nizatidine did not affect the renal clearance or net secretory clearance of metformin. Conclusion This study demonstrates that a selective inhibition of MATE2K by nizatidine, affected the apparent volume of distribution, tissue levels and peripheral effects of metformin. However, nizatidine did not alter systemic concentrations or the renal clearance of metformin, suggesting that specific MATE2K inhibition may not be sufficient to cause renal DDIs with basic drugs. PMID:26507723

Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme

PubMed Central

Rublack, Nico

2014-01-01

Summary Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated. PMID:25246949

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

In Vitro Selection of pH-Activated DNA Nanostructures.

PubMed

Fong, Faye Yi; Oh, Seung Soo; Hawker, Craig J; Soh, H Tom

2016-12-05

We report the first in vitro selection of DNA nanostructures that switch their conformation when triggered by change in pH. Previously, most pH-active nanostructures were designed using known pH-active motifs, such as the i-motif or the triplex structure. In contrast, we performed de novo selections starting from a random library and generated nanostructures that can sequester and release Mipomersen, a clinically approved antisense DNA drug, in response to pH change. We demonstrate extraordinary pH-selectivity, releasing up to 714-fold more Mipomersen at pH 5.2 compared to pH 7.5. Interestingly, none of our nanostructures showed significant sequence similarity to known pH-sensitive motifs, suggesting that they may operate via novel structure-switching mechanisms. We believe our selection scheme is general and could be adopted for generating DNA nanostructures for many applications including drug delivery, sensors and pH-active surfaces. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Experimental Implementation of Chemical Subtraction

PubMed Central

Chen, Shao-Nong; Turner, Allison; Jaki, Birgit U.; Nikolic, Dejan; van Breemen, Richard B.; Friesen, J. Brent; Pauli, Guido F.

2008-01-01

A preparative analytical method was developed to selectively remove (“chemically subtract”) a single compound from a complex mixture, such as a natural extract or fraction, in a single step. The proof of concept is demonstrated by the removal of pure benzoic acid (BA) from cranberry (Vaccinium macrocarpon Ait.) juice fractions that exhibit anti-adhesive effects vs. uropathogenic E. coli. Chemical subtraction of BA, representing a major constituent of the fractions, eliminates the potential in vitro interference of the bacteriostatic effect of BA on the E. coli anti-adherence action measured in bioassays. Upon BA removal, the anti-adherent activity of the fraction was fully retained, 36% inhibition of adherence in the parent fraction at 100 ug/mL increased to 58% in the BA-free active fraction. The method employs countercurrent chromatography (CCC) and operates loss-free for both the subtracted and the retained portions as only liquid-liquid partitioning is involved. While the high purity (97.47% by quantitative 1H NMR) of the subtracted BA confirms the selectivity of the method, one minor impurity was determined to be scopoletin by HR-ESI-MS and (q)HNMR and represents the first coumarin reported from cranberries. A general concept for the selective removal of phytoconstituents by CCC is presented, which has potential broad applicability in the biological evaluation of medicinal plant extracts and complex pharmaceutical preparations. PMID:18234463

Radiogenetic therapy: strategies to overcome tumor resistance.

PubMed

Marples, B; Greco, O; Joiner, M C; Scott, S D

2003-01-01

The aim of cancer gene therapy is to selectively kill malignant cells at the tumor site, by exploiting traits specific to cancer cells and/or solid tumors. Strategies that take advantage of biological features common to different tumor types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia, the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumor volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene (e.g., herpes simplex virus thymidine kinase) to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug (e.g., ganciclovir). The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumor targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

[Analysis on microdialysis probe recovery of baicalin in vitro and in vivo based on LC-MS/MS].

PubMed

Chen, Teng-Fei; Liu, Jian-Xun; Zhang, Ying; Lin, Li; Song, Wen-Ting; Yao, Ming-Jiang

2017-06-01

To further study the brain behavior and the pharmacokinetics of baicalin in intercellular fluid of brain, and study the recovery rate and stability of brain and blood microdialysis probe of baicalin in vitro and in vivo. The concentration of baicalin in brain and blood microdialysates was determined by LC-MS/MS and the probe recovery for baicalin was calculated. The effects of different flow rates (0.50, 1.0, 1.5, 2.0，3.0 μL•min⁻¹) on recovery in vitro were determined by incremental method and decrement method. The effects of different drug concentrations (50.00, 200.0, 500.0, 1 000 μg•L⁻¹) and using times (0, 1, 2) on recovery in vitro were determined by incremental method. The probe recovery stability and effect of flow rate on recovery in vivo were determined by decrement method, and its results were compared with those in in vitro trial. The in vitro recovery of brain and blood probe of baicalin was decreased with the increase of flow rate under the same concentration; and at the same flow rate, different concentrations of baicalin had little influence on the recovery. The probe which had been used for 2 times showed no obvious change in probe recovery by syringe with 2% heparin sodium and ultrapure water successively. In vitro recovery rates obtained by incremental method and decrement method were approximately equal under the same condition, and the in vivo recovery determined by decrement method was similar with the in vitro results and they were showed a good stability within 10 h. The results showed that decrement method can be used for pharmacokinetic study of baicalin, and can be used to study probe recovery in vivo at the same time. Copyright© by the Chinese Pharmaceutical Association.

Role of In Vitro Release Methods in Liposomal Formulation Development: Challenges and Regulatory Perspective.

PubMed

Solomon, Deepak; Gupta, Nilesh; Mulla, Nihal S; Shukla, Snehal; Guerrero, Yadir A; Gupta, Vivek

2017-11-01

In the past few years, measurement of drug release from pharmaceutical dosage forms has been a focus of extensive research because the release profile obtained in vitro can give an indication of the drug's performance in vivo. Currently, there are no compendial in vitro release methods designed for liposomes owing to a range of experimental challenges, which has created a major hurdle for both development and regulatory acceptance of liposome-based drug products. In this paper, we review the current techniques that are most often used to assess in vitro drug release from liposomal products; these include the membrane diffusion techniques (dialysis, reverse dialysis, fractional dialysis, and microdialysis), the sample-and-separate approach, the in situ method, the continuous flow, and the modified United States Pharmacopeia methods (USP I and USP IV). We discuss the principles behind each of the methods and the criteria that assist in choosing the most appropriate method for studying drug release from a liposomal formulation. Also, we have included information concerning the current regulatory requirements for liposomal drug products in the United States and in Europe. In light of increasing costs of preclinical and clinical trials, applying a reliable in vitro release method could serve as a proxy to expensive in vivo bioavailability studies. Graphical Abstract Appropriate in-vitro drug release test from liposomal products is important to predict the in-vivo performance.

Biotechnological approaches to enhance salidroside, rosin and its derivatives production in selected Rhodiola spp. in vitro cultures.

PubMed

Grech-Baran, Marta; Sykłowska-Baranek, Katarzyna; Pietrosiuk, Agnieszka

Rhodiola (Crassulaceae) an arctic-alpine plant, is extensively used in traditional folk medicine in Asian and European countries. A number of investigations have demonstrated that Rhodiola preparations exhibit adaptogenic, neuroprotective, anti-tumour, cardioprotective, and anti-depressant effects. The main compounds responsible for these activities are believed to be salidroside, rosin and its derivatives which became the target of biotechnological investigations. This review summarizes the results of the diverse biotechnological approaches undertaken to enhance the production of salidroside, rosin and its derivatives in callus, cell suspension and organ in vitro cultures of selected Rhodiola species.

Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity.

PubMed

Mulligan, M E; Hawley, D K; Entriken, R; McClure, W R

1984-01-11

We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

[Evaluation on a fast weight reduction model in vitro].

PubMed

Li, Songtao; Li, Ying; Wen, Ying; Sun, Changhao

2010-03-01

To establish a fast and effective model in vitro for screening weight-reducing drugs and taking preliminary evaluation of the model. Mature adipocytes of SD rat induced by oleic acid were used to establish a obesity model in vitro. Isoprel, genistein, caffeine were selected as positive agents and curcumine as negative agent to evaluate the obesity model. Lipolysis of adipocytes was stimulated significantly by isoprel, genistein and caffeine rather than curcumine. This model could be used efficiently for screening weight-losing drugs.

In Vitro Pharmacodynamics of AZD5206 against Staphylococcus aureus

PubMed Central

Chang, Kai-Tai; Yang, Zhen; Newman, Joseph; Hu, Ming

2013-01-01

AZD5206 is a novel antimicrobial agent with potent in vitro activity against Staphylococcus aureus. We evaluated the in vitro pharmacodynamics of AZD5206 against a standard wild-type methicillin-susceptible strain (ATCC 29213) and a clinical strain of methicillin-resistant S. aureus (SA62). Overall, bacterial killing against a low baseline inoculum was more remarkable. Low dosing exposures and/or high baseline inoculum resulted in early reduction in bacterial burden, followed by regrowth and selective amplification of the resistant population. PMID:23229481

Structure-activity relationships of selected phenazines against Mycobacterium leprae in vitro.

PubMed Central

Franzblau, S G; O'Sullivan, J F

1988-01-01

Structure-activity relationships of phenazines against Mycobacterium leprae were investigated by using an in vitro radiorespirometric assay. In general, activity in ascending order was observed in compounds containing no chlorine atoms, a monochlorinated phenazine nucleus, and chlorines in the para positions of both the anilino and phenyl rings. The most active compounds contained a 2,2,6,6-tetramethylpiperidine substitution at the imino nitrogen. Most of these chlorinated phenazines were considerably more active in vitro than clofazimine (B663). PMID:3056241

On Selecting a Minimal Set of In Vitro Assays to Reliably Determine Estrogen Agonist Activity

EPA Science Inventory

The US EPA is charged with screening chemicals for their ability to be endocrine disruptors through interaction with the estrogen, androgen and thyroid axes. The agency is starting to explore the use of high-throughput in vitro assays to use in the Endocrine Disruptor Screening P...

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.

Comparison of in situ and in vitro techniques for measuring ruminal degradation of animal by-product proteins.

PubMed

England, M L; Broderick, G A; Shaver, R D; Combs, D K

1997-11-01

Ruminally undegraded protein (RUP) values of blood meal (n = 2), hydrolyzed feather meal (n = 2), fish meal (n = 2), meat and bone meal, and soybean meal were estimated using an in situ method, an inhibitor in vitro method, and an inhibitor in vitro technique applying Michaelis-Menten saturation kinetics. Degradation rates for in situ and inhibitor in vitro methods were calculated by regression of the natural log of the proportion of crude protein (CP) remaining undegraded versus time. Nonlinear regression analysis of the integrated Michaelis-Menten equation was used to determine maximum velocity, the Michaelis constant, and degradation rate (the ratio of maximum velocity to the Michaelis constant). A ruminal passage rate of 0.06/h was assumed in the calculation of RUP. The in situ and inhibitor in vitro techniques yielded similar estimates of ruminal degradation. Mean RUP estimated for soybean meal, blood meal, hydrolyzed feather meal, fish meal, and meat and bone meal were, respectively, 28.6, 86.0, 77.4, 52.9, and 52.6% of CP by the in situ method and 26.4, 86.1, 76.0, 59.6, and 49.5% of CP by the inhibitor in vitro technique. The Michaelis-Menten inhibitor in vitro technique yielded more rapid CP degradation rates and decreased estimates of RUP. The inhibitor in vitro method required less time and labor than did the other two techniques to estimate the RUP values of animal by-product proteins. Results from in vitro incubations with pepsin.HCl suggested that low postruminal digestibility of hydrolyzed feather meal may impair its value as a source of RUP.

Simultaneous generation of aptamers to multiple gamma-carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection.

PubMed

Layzer, Juliana M; Sullenger, Bruce A

2007-01-01

By using the in vitro selection method SELEX against the complex mixture of GLA proteins and utilizing methods to deconvolute the resulting ligands, we were able to successfully generate 2'-ribo purine, 2'-fluoro pyrimidine aptamers to various individual targets in the GLA protein proteome that ranged in concentration from 10 nM to 1.4 microM in plasma. Perhaps not unexpectedly, the majority of the aptamers isolated following SELEX bind the most abundant protein in the mixture, prothrombin (FII), with high affinity. We show that by deselecting the dominant prothrombin aptamer the selection can be redirected. By using this DeSELEX approach, we were able to shift the selection toward other sequences and to less abundant protein targets and obtained an aptamer to Factor IX (FIX). We also demonstrate that by using an RNA library that is focused around a proteome, purified protein targets can then be used to rapidly generate aptamers to the protein targets that are rare in the initial mixture such as Factor VII (FVII) and Factor X (FX). Moreover, for all four proteins targeted (FII, FVII, FIX, and FX), aptamers were identified that could inhibit the individual protein's activitity in coagulation assays. Thus, by applying the concepts of DeSELEX and focused library selection, aptamers specific for any protein in a particular proteome can theoretically be generated, even when the proteins in the mixture are present at very different concentrations.

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against the Pesticide Fipronil and Sensitive Detection in River Water

PubMed Central

Sooter, Letha J.

2017-01-01

Fipronil is a commonly used insecticide that has been shown to have environmental and human health risks. The current standard methods of detection for fipronil and its metabolites, such as GC-MS, are time consuming and labor intensive. In this study, a variant of systematic evolution of ligands by exponential enrichment (SELEX), was utilized to identify the first single-stranded DNA (ssDNA) molecular recognition element (MRE) that binds to fipronil with high affinity (Kd = 48 ± 8 nM). The selected MRE displayed low cross binding activity on various environmentally relevant, structurally unrelated herbicides and pesticides, in addition to broad-spectrum binding activity on major metabolites of fipronil and a structurally similar pesticide in prepared river samples. Additionally, a proof-of-principle fluorescent detection assay was developed by using the selected ssDNA MRE as a signal-reporting element, with a limit of detection of 105 nM in a prepared river water sample. PMID:29283416

Bispecific small molecule-antibody conjugate targeting prostate cancer.

PubMed

Kim, Chan Hyuk; Axup, Jun Y; Lawson, Brian R; Yun, Hwayoung; Tardif, Virginie; Choi, Sei Hyun; Zhou, Quan; Dubrovska, Anna; Biroc, Sandra L; Marsden, Robin; Pinstaff, Jason; Smider, Vaughn V; Schultz, Peter G

2013-10-29

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

Functionalized Gold Nanoparticles for the Detection of C-Reactive Protein

PubMed Central

António, Maria

2018-01-01

C-reactive protein (CRP) is a very important biomarker of infection and inflammation for a number of diseases. Routine CRP measurements with high sensitivity and reliability are highly relevant to the assessment of states of inflammation and the efficacy of treatment intervention, and require the development of very sensitive, selective, fast, robust and reproducible assays. Gold nanoparticles (Au NPs) are distinguished for their unique electrical and optical properties and the ability to conjugate with biomolecules. Au NP-based probes have attracted considerable attention in the last decade in the analysis of biological samples due to their simplicity, high sensitivity and selectivity. Thus, this article aims to be a critical and constructive analysis of the literature of the last three years regarding the advances made in the development of bioanalytical assays based on gold nanoparticles for the in vitro detection and quantification of C-reactive protein from biological samples. Current methods for Au NP synthesis and the strategies for surface modification aiming at selectivity towards CRP are highlighted. PMID:29597295

Highly Sensitive and Selective Immuno-capture/Electrochemical Assay of Acetylcholinesterase Activity in Red Blood Cells: A Biomarker of Exposure to Organophosphorus Pesticides and Nerve Agents

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

Chen, Aiqiong; Du, Dan; Lin, Yuehe

Acetylcholinesterase (AChE) enzyme activity in red blood cells (RBCs) is a useful biomarker for biomonitoring of exposures to organophosphorus (OP) pesticides and chemical nerve agents. In this paper, we reported a new method for AChE activity assay based on selective immuno-capture of AChE from biological samples followed by enzyme activity assay of captured AChE using a disposable electrochemical sensor. The electrochemical sensor is based on multiwalled carbon nanotubes-gold nanocomposites (MWCNTs-Au) modified screen printed carbon electrode (SPCE). Upon the completion of immunoreaction, the target AChE (including active and inhibited) is captured onto the electrode surface and followed by an electrochemical detectionmore » of enzymatic activity in the presence of acetylthiocholine. A linear response is obtained over standard AChE concentration range from 0.1 to 10 nM. To demonstrate the capability of this new biomonitoring method, AChE solutions dosed with different concentration of paraoxon were used to validate the new AChE assay method. AChE inhibition in OP dosed solutions was proportional to its concentration from 0.2 to 50 nM. The new AChE activity assay method for biomonitoring of OP exposure was further validated with in-vitro paraoxon-dosed RBC samples. The established electrochemical sensing platform for AChE activity assay not only avoids the problem of overlapping substrate specificity with esterases by using selective antibody, but also eliminates potential interference from other electroactive species in biological samples. It offers a new approach for sensitive, selective, and rapid AChE activity assay for biomonitoring of exposures to OPs.« less

COMPARISON OF IN VIVO AND IN VITRO CHEMICAL SCREENING ASSAYS FOR ESTROGEN-RESPONSIVE PROTEIN BIOMARKER EXPRESSION IN THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)

EPA Science Inventory

Animal welfare concerns and the high cost associated with in vivo chemical screening methods have prompted efforts to develop highly specific and sensitive in vitro techniques to evaluate chemicals for endocrine activity. Diagnostic biomarkers developed using in vitro methods sho...

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish

USDA-ARS?s Scientific Manuscript database

Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate, and D-mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that,...

In vitro evidence for efficacy in food intolerance for the multispecies probiotic formulation Ecologic® Tolerance (Syngut™).

PubMed

Besseling-van der Vaart, I; Heath, M D; Guagnini, F; Kramer, M F

2016-02-01

The beneficial effects of probiotics are currently the subject of extensive studies in health and medical research. The aim of this research was to specifically design a new probiotic formulation for supplementation in people suffering from food intolerance. The selection of strains was focussed on the capacity to influence mechanisms of action that are important in development of food intolerance with the following parameters measure: in vitro capacity to produce β-galactosidase, in vitro strengthening of the epithelial barrier, in vitro stimulation of cytokines produced by regulatory T cells, in addition to assessing fundamental quality criteria (stability, gastrointestinal (GI)-survival, multispecies concept, allergen-free). Ecologic®Tolerance/Syngut™ was subsequently developed consisting of a multispecies concept using 4 different probiotic strains (Bifidobacterium lactis W51, Lactobacillus acidophilus W22, Lactobacillus plantarum W21 and Lactococcus lactis W19). Each of these strains demonstrated ability to survive the GI-tract and strain specific effects in producing β-galactosidase, strengthening the gut barrier function after immunological-induced stress and inhibiting Th2 cytokines (IL-4, IL-5 and IL-13 (≥50%), in addition to stimulating interleukin-10 levels; thus, providing in vitro evidence for the efficacy of the selected strains to provide beneficial effects in patients suffering from food intolerance.

Screening and Characterization of Lactic Acid Bacteria Strains with Anti-inflammatory Activities through in vitro and Caenorhabditis elegans Model Testing

PubMed Central

Park, Mi Ri; Kim, Younghoon; Lee, Myung-Ki

2015-01-01

The present study was conducted to screen candidate probiotic strains for anti-inflammatory activity. Initially, a nitric oxide (NO) assay was used to test selected candidate probiotic strains for anti-inflammatory activity in cultures of the murine macrophage cell line, RAW 264.7. Then, the in vitro probiotic properties of the strains, including bile tolerance, acid resistance, and growth in skim milk media, were investigated. We also performed an in vitro hydrophobicity test and an intestinal adhesion assay using Caenorhabditis elegans as a surrogate in vivo model. From our screening, we obtained 4 probiotic candidate lactic acid bacteria (LAB) strains based on their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell cultures and the results of the in vitro and in vivo probiotic property assessments. Molecular characterization using 16S rDNA sequencing analysis identified the 4 LAB strains as Lactobacillus plantarum. The selected L. plantarum strains (CAU1054, CAU1055, CAU1064, and CAU1106) were found to possess desirable in vitro and in vivo probiotic properties, and these strains are good candidates for further investigations in animal models and human clinical studies to elucidate the mechanisms underlying their anti-inflammatory activities. PMID:26761805

The Consequences of Sex Selection

ERIC Educational Resources Information Center

Rothman, Barbara Katz

2006-01-01

A group of researchers at Baylor College of Medicine in Houston are set to do a long-term study of families that would permit to select the sex of their babies through genetic testing before implanting the embryo in the mother. Technologies such as in vitro fertilization involved in selecting a baby's sex has societal and psychological…

Effect of micro-environment modification and polymer type on the in-vitro dissolution behavior and in-vivo performance of amorphous solid dispersions.

PubMed

Sun, Weiwei; Pan, Baoliang

2017-06-15

This study investigates the effects of micro-environment modification and polymer type on the in-vitro dissolution behavior and in-vivo performance of micro-environment pH modifying solid dispersions (pH M -SD) for the poorly water-soluble model drug Toltrazuril (TOL). Various pH M -SDs were prepared using Ca(OH) 2 as a pH-modifier in hydrophilic polymers, including polyethylene glycol 6000 (PEG6000), polyvinylpyrrolidone k30 (PVPk30) and hydroxypropyl methylcellulose (HPMC). Based on the results of physicochemical characterizations and in-vitro dissolution testing, the representative ternary (Ca(OH) 2 :TOL:PEG6000/HPMC/PVPk30=1:8:24, w/w/w) and binary (TOL:PVPk30=1:3, w/w) solid dispersions were selected and optimized to perform in-vivo pharmacokinetic study. The micro-environment pH modification improved the in-vitro water-solubility and in-vivo bioavailability of parent drug TOL. Furthermore, the addition of alkalizers not only enhanced the release and absorption of prototype drug, but also promoted the generation of active metabolites, including toltrazuril sulfoxide (TOLSO) and toltrazuril sulfone (TOLSO 2 ). The in-vitro dissolution profiles and in-vivo absorption, distribution and metabolism behaviors of the pH M -SDs varied with polymer type. Moreover, in-vivo bioavailability of three active pharmaceutical ingredients increased with an increase in in-vitro dissolution rates of the drug from the pH M -SDs prepared with various polymers. Therefore, a non-sink in-vitro dissolution method can be used to predict the in-vivo performance of pH M -SDs formulated with various polymers with trend consistency. In-vitro and in-vivo screening procedures revealed that the pH M -SD composed of Ca(OH) 2 , TOL and PVPk30 at a weight ratio of 1:8:24, of which the safety was adequately proved via histopathological examination, may be a promising candidate for providing better clinical outcomes. Copyright © 2017. Published by Elsevier B.V.

In vitro methods for hazard assessment of industrial chemicals – opportunities and challenges

PubMed Central

Wong, Chin Lin; Ghassabian, Sussan; Smith, Maree T.; Lam, Ai-Leen

2015-01-01

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay is the ‘method of choice’ for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement, and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals. PMID:25999858

In vitro methods for hazard assessment of industrial chemicals - opportunities and challenges.

PubMed

Wong, Chin Lin; Ghassabian, Sussan; Smith, Maree T; Lam, Ai-Leen

2015-01-01

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay is the 'method of choice' for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement, and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals.

Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

PubMed Central

Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

2015-01-01

Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

Organogenesis from transformed tomato explants.

PubMed

Frary, Anne; Van Eck, Joyce

2005-01-01

Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants. Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development. In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer. The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation. After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin. Regenerated shoots are then rooted on a separate selective medium. This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10-15%.

Reverse breeding: a novel breeding approach based on engineered meiosis.

PubMed

Dirks, Rob; van Dun, Kees; de Snoo, C Bastiaan; van den Berg, Mark; Lelivelt, Cilia L C; Voermans, William; Woudenberg, Leo; de Wit, Jack P C; Reinink, Kees; Schut, Johan W; van der Zeeuw, Eveline; Vogelaar, Aat; Freymark, Gerald; Gutteling, Evert W; Keppel, Marina N; van Drongelen, Paul; Kieny, Matthieu; Ellul, Philippe; Touraev, Alisher; Ma, Hong; de Jong, Hans; Wijnker, Erik

2009-12-01

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.

Reverse breeding: a novel breeding approach based on engineered meiosis

PubMed Central

Dirks, Rob; van Dun, Kees; de Snoo, C Bastiaan; van den Berg, Mark; Lelivelt, Cilia L C; Voermans, William; Woudenberg, Leo; de Wit, Jack P C; Reinink, Kees; Schut, Johan W; van der Zeeuw, Eveline; Vogelaar, Aat; Freymark, Gerald; Gutteling, Evert W; Keppel, Marina N; van Drongelen, Paul; Kieny, Matthieu; Ellul, Philippe; Touraev, Alisher; Ma, Hong; de Jong, Hans; Wijnker, Erik

2009-01-01

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome. PMID:19811618

The Global Error Assessment (GEA) model for the selection of differentially expressed genes in microarray data.

PubMed

Mansourian, Robert; Mutch, David M; Antille, Nicolas; Aubert, Jerome; Fogel, Paul; Le Goff, Jean-Marc; Moulin, Julie; Petrov, Anton; Rytz, Andreas; Voegel, Johannes J; Roberts, Matthew-Alan

2004-11-01

Microarray technology has become a powerful research tool in many fields of study; however, the cost of microarrays often results in the use of a low number of replicates (k). Under circumstances where k is low, it becomes difficult to perform standard statistical tests to extract the most biologically significant experimental results. Other more advanced statistical tests have been developed; however, their use and interpretation often remain difficult to implement in routine biological research. The present work outlines a method that achieves sufficient statistical power for selecting differentially expressed genes under conditions of low k, while remaining as an intuitive and computationally efficient procedure. The present study describes a Global Error Assessment (GEA) methodology to select differentially expressed genes in microarray datasets, and was developed using an in vitro experiment that compared control and interferon-gamma treated skin cells. In this experiment, up to nine replicates were used to confidently estimate error, thereby enabling methods of different statistical power to be compared. Gene expression results of a similar absolute expression are binned, so as to enable a highly accurate local estimate of the mean squared error within conditions. The model then relates variability of gene expression in each bin to absolute expression levels and uses this in a test derived from the classical ANOVA. The GEA selection method is compared with both the classical and permutational ANOVA tests, and demonstrates an increased stability, robustness and confidence in gene selection. A subset of the selected genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). All these results suggest that GEA methodology is (i) suitable for selection of differentially expressed genes in microarray data, (ii) intuitive and computationally efficient and (iii) especially advantageous under conditions of low k. The GEA code for R software is freely available upon request to authors.

Emulsion of Chloramphenicol: an Overwhelming Approach for Ocular Delivery.

PubMed

Ashara, Kalpesh C; Shah, Ketan V

2017-03-01

Ophthalmic formulations of chloramphenicol have poor bioavailability of chloramphenicol in the ocular cavity. The present study aimed at exploring the impact of different oil mixtures in the form of emulsion on the permeability of chloramphenicol after ocular application. Selection of oil mixture and ratio of the components was made by an equilibrium solubility method. An emulsifier was chosen according to its emulsification properties. A constrained simplex centroid design was used for the assessment of the emulsion development. Emulsions were evaluated for physicochemical properties; zone of inhibition, in-vitro diffusion and ex-vivo local accumulation of chloramphenicol. Validation of the design using check-point batch and reduced polynomial equations were also developed. Optimization of the emulsion was developed by software Design® expert 6.0.8. Assessment of the osmolarity, ocular irritation, sterility testing and isotonicity of optimized batch were also made. Parker Neem®, olive and peppermint oils were selected as an oil phase in the ratio 63.64:20.2:16.16. PEG-400 was selected as an emulsifier according to a pseudo-ternary phase diagram. Constrained simplex-centroid design was applied in the range of 25-39% water, 55-69% PEG-400, 5-19% optimized oil mixture, and 1% chloramphenicol. Unpaired Student's t-test showed for in-vitro and ex-vivo studies that there was a significant difference between the optimized batch of emulsion and Chloramphenicol eye caps (a commercial product) according to both were equally safe. The optimized batch of an emulsion of chloramphenicol was found to be as safe as and more effective than Chloramphenicol eye caps.

Mutation Analysis in Cultured Cells of Transgenic Rodents

PubMed Central

Zheng, Albert; Bates, Steven E.; Tommasi, Stella

2018-01-01

To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable. PMID:29337872

Fermentation quality and in vitro methane production of sorghum silage prepared with cellulase and lactic acid bacteria

PubMed Central

Khota, Waroon; Pholsen, Suradej; Higgs, David; Cai, Yimin

2017-01-01

Objective The effects of lactic acid bacteria (LAB) and cellulase enzyme on fermentation quality, microorganism population, chemical composition and in vitro gas production of sorghum silages were studied. Methods Commercial inoculant Lactobacillus plantarum Chikuso 1 (CH), local selected strain Lactobacillus casei (L. casei) TH 14 and Acremonium cellulase (AC) were used as additives in sorghum silage preparation. Results Prior to ensiling Sorghum contained 104 LAB and 106 cfu/g fresh matter coliform bacteria. The chemical compositions of sorghum was 26.6% dry matter (DM), 5.2% crude protein (CP), and 69.7% DM for neutral detergent fiber. At 30 days of fermentation after ensiling, the LAB counts increased to a dominant population; the coliform bacteria and molds decreased to below detectable level. All sorghum silages were good quality with a low pH (<3.5) and high lactic acid content (>66.9 g/kg DM). When silage was inoculated with TH14, the pH value was significantly (p<0.05) lower and the CP content significantly (p<0.05) higher compared to control, CH and AC-treatments. The ratio of in vitro methane production to total gas production and DM in TH 14 and TH 14+AC treatments were significantly (p<0.05) reduced compared with other treatments while in vitro dry matter digestibility and gas production did not differ among treatments. Conclusion The results confirmed that L. casei TH14 could improve sorghum silage fermentation, inhibit protein degradation and decrease methane production. PMID:28728399

Establishment of a biological reference preparation for hepatitis A vaccine (inactivated, non-adsorbed).

PubMed

Stalder, J; Costanzo, A; Daas, A; Rautmann, G; Buchheit, K-H

2010-04-01

A reference standard calibrated in International Units (IU) is needed for the in vitro potency assay of hepatitis A vaccines prepared by formalin-inactivation of purified hepatitis A virus grown in cell cultures. Thus, a project was launched by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish one or more non-adsorbed inactivated hepatitis A vaccine reference preparation(s) as working standard(s), calibrated against the 1st International Standard (IS), for the in vitro potency assay (ELISA) of all vaccines present on the European market. Four non-adsorbed liquid preparations of formalin-inactivated hepatitis A antigen with a known antigen content were obtained from 3 manufacturers as candidate Biological Reference Preparations (BRPs). Thirteen laboratories participated in the collaborative study. They were asked to use an in vitro ELISA method adapted from a commercially available kit for the detection of antibodies to hepatitis A virus. In-house validated assays were to be run in parallel, where available. Some participants also included commercially available hepatitis A vaccines in the assays, after appropriate desorption. During the collaborative study, several participants using the standard method were faced with problems with some of the most recent lots of the test kits. Due to these problems, the standard method did not perform satisfactorily and a high number of assays were invalid, whereas the in-house methods appeared to perform better. Despite this, the overall mean results of the valid assays using both methods were in agreement. Nonetheless, it was decided to base the assignment of the potency values on the in-house methods only. The results showed that all candidate BRPs were suitable for the intended purpose. However, based on availability of the material and on the results of end-product testing, 2 candidate reference preparations, Samples C and D, were selected. Both were from the same batch but filled on different days; no statistically significant difference in potency was observed. They were thus combined in 1 single batch. The candidate preparation (Sample C/D) was adopted at the June 2009 session of the European Pharmacopoeia (Ph. Eur.) Commission as the Ph. Eur. BRP batch 1 for hepatitis A vaccine (inactivated, non-adsorbed), with an assigned potency of 12 IU/ml for in vitro antigen content assays. Accelerated degradation studies have been initiated. The preliminary data show that the BRP is stable at the recommended storage temperature (< -50 degrees C). The BRP will be monitored at regular intervals throughout its lifetime.

Application of boronated anti-CEA immunoliposome to tumour cell growth inhibition in in vitro boron neutron capture therapy model.

PubMed Central

Yanagië, H.; Tomita, T.; Kobayashi, H.; Fujii, Y.; Takahashi, T.; Hasumi, K.; Nariuchi, H.; Sekiguchi, M.

1991-01-01

An immunoliposome containing a 10B-compound has been examined as a selective drug delivery system in boron neutron-capture therapy. Liposomes, conjugated with monoclonal antibodies specific for carcinoembryonic antigen (CEA) were shown to bind selectively to cells bearing CEA on their surface. The immunoliposomes attached to tumour cells suppressed growth in vitro upon thermal neutron irradiation and suppression was dependent upon the concentration of the 10B-compound in the liposomes and on the density of antibody conjugated to the liposomes. The results suggest that immunoliposomes containing the 10B-compound could act as a selective and efficient carrier of 10B atoms to target tumour cells in boron neutron-capture therapy. Images Figure 1 PMID:2021537

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

PubMed

Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

2016-12-01

Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

PubMed Central

Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N.

2016-01-01

ABSTRACT Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries. PMID:27715478

Ketoprofen suppository dosage forms: in vitro release and in vivo absorption studies in rabbits.

PubMed

Babar, A; Bellete, T; Plakogiannis, F M

1999-02-01

In vitro release of ketoprofen from suppository bases and in vivo absorption in rabbits were studied. Suppositories containing 50 mg of ketoprofen were prepared using theobroma oil, esterified (c10-c18) fatty acids, and polyethylene glycol 1000 bases. The displacement values of the drug were determined and found to be of the order of theobroma oil > esterified (c10-c18) fatty acids and polyethylene glycol 1000 bases. The suppository hardness data revealed that the theobroma oil base produced relatively brittle suppositories. Using the USP dissolution method, the release of ketoprofen was observed to be greatest from polyethylene glycol 1000 suppositories. With the dialysis technique, the maximum release of drug was obtained from theobroma oil suppository containing polysorbate 40 at a 6% level. Selected suppository formulations were evaluated for rectal absorption studies in rabbits. The in vivo data showed that the optimum drug absorption took place from the polyethylene glycol 1000 base and theobroma oil formulation containing 6% polysorbate 40.

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking

NASA Astrophysics Data System (ADS)

Szafraniec, Ewelina; Majzner, Katarzyna; Farhane, Zeineb; Byrne, Hugh J.; Lukawska, Malgorzata; Oszczapowicz, Irena; Chlopicki, Stefan; Baranska, Malgorzata

2016-12-01

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488 nm and 785 nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

Shearwave Dispersion Ultrasonic Vibrometry (SDUV) for measuring prostate shear stiffness and viscosity – An in vitro pilot study

PubMed Central

Urban, M.W.; Fatemi, M.; Greenleaf, J.F.

2011-01-01

This paper reports shear stiffness and viscosity “virtual biopsy” measurements of three excised non-cancerous human prostates using shearwave dispersion ultrasound vibrometry (SDUV) in vitro. Improved methods for prostate guided-biopsy are required to effectively guide needle biopsy to the suspected site. In addition, tissue stiffness measurement helps identifying a suspected site to perform biopsy because stiffness has been shown to correlate with pathology. More importantly, early detection of prostate cancer may guide minimally-invasive therapy and eliminate insidious procedures. In this work, “virtual” biopsies were taken in multiple locations in three excised prostates. Then, SDUV shear elasticity and viscosity measurements have been performed at the selected “suspicious” locations within the prostates. SDUV measurements of prostate elasticity and viscosity are generally in agreement with preliminary values reported previously in the literature. It is however important to emphasize that the obtained viscoelastic parameters values are local, and not a mean value for the whole prostate. PMID:20595086

In-vitro Drug Dissolution Studies in Medicinal Compounds.

PubMed

Bozal-Palabiyik, Burcin; Uslu, Bengi; Ozkan, Yalcin; Ozkan, Sibel A

2018-03-22

After oral administration, drug absorption from solid dosage forms depend on the release of the drug active compounds from the dosage form, the dissolution or solubilization of the drug under physiological conditions, and the permeability across the gastrointestinal tract. Dissolution testing is an essential part of designing more effective solid dosage forms in pharmaceutical industry. Moreover dissolution testing contributes to the selection of appropriate formulation excipients for improving the dosage form efficiency. This study aims to analyze in-vitro drug dissolution testing in solid dosage forms since 2010 in order to present a comprehensive outlook of recent trends. In doing that the previous studies in the literature are summarized in the form of a table to demonstrate the apparatuses used for dissolution testing, the media in which the solid dosage form is dissolved, the method preferred for analysis from dissolution media, the conditions of analyses and the results obtained. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Susceptibility of human Plasmodium knowlesi infections to anti-malarials

PubMed Central

2013-01-01

Background Evidence suggests that Plasmodium knowlesi malaria in Sarawak, Malaysian Borneo remains zoonotic, meaning anti-malarial drug resistance is unlikely to have developed in the absence of drug selection pressure. Therefore, adequate response to available anti-malarial treatments is assumed. Methods Here the ex vivo sensitivity of human P. knowlesi isolates in Malaysian Borneo were studied, using a WHO schizont maturation assay modified to accommodate the quotidian life cycle of this parasite. The in vitro sensitivities of P. knowlesi H strain adapted from a primate infection to in vitro culture (by measuring the production of Plasmodium lactate dehydrogenase) were also examined together with some assays using Plasmodium falciparum and Plasmodium vivax. Results Plasmodium knowlesi is uniformly highly sensitive to artemisinins, variably and moderately sensitive to chloroquine, and less sensitive to mefloquine. Conclusions Taken together with reports of clinical failures when P. knowlesi is treated with mefloquine, the data suggest that caution is required if using mefloquine in prevention or treatment of P. knowlesi infections, until further studies are undertaken. PMID:24245918

Studies of the in vitro anticancer, antimicrobial and antioxidant potentials of selected Yemeni medicinal plants from the island Soqotra

PubMed Central

2009-01-01

Background Recent years have witnessed that there is a revival of interest in drug discovery from medicinal plants for the maintenance of health in all parts of the world. The aim of this work was to investigate 26 plants belonging to 17 families collected from a unique place in Yemen (Soqotra Island) for their in vitro anticancer, antimicrobial and antioxidant activities. Methods The 26 plants were extracted with methanol and hot water to yield 52 extracts. Evaluation for in vitro anticancer activity was done against three human cancer cell lines (A-427, 5637 and MCF-7) by using an established microtiter plate assay based on cellular staining with crystal violet. Antimicrobial activity was tested against three Gram-positive bacteria, two Gram-negative bacteria, one yeast species and three multiresistant Staphylococcus strains by using an agar diffusion method and the determination of MIC against three Gram-positive bacteria with the broth micro-dilution assay. Antioxidant activity was investigated by measuring the scavenging activity of the DPPH radical. Moreover, a phytochemical screening of the methanolic extracts was done. Results Notable cancer cell growth inhibition was observed for extracts from Ballochia atro-virgata, Eureiandra balfourii and Hypoestes pubescens, with IC50 values ranging between 0.8 and 8.2 μg/ml. The methanol extracts of Acanthospermum hispidum, Boswellia dioscorides, Boswellia socotrana, Commiphora ornifolia and Euphorbia socotrana also showed noticeable antiproliferative potency with IC50 values < 50 μg/ml. The greatest antimicrobial activity was exhibited by extracts from Acacia pennivenia, Boswellia dioscorides, Boswellia socotrana, Commiphora ornifolia, Euclea divinorum, Euphorbia socotrana, Leucas samhaensis, Leucas virgata, Rhus thyrsiflora, and Teucrium sokotranum with inhibition zones > 15 mm and MIC values ≤ 250 μg/ml. In addition, the methanolic extracts of Acacia pennivenia, Boswellia dioscorides, Boswellia socotrana and Commiphora ornifolia showed good antioxidant potential at low concentrations (more than 80% at 50 μg/ml). Conclusion Our results show once again that medicinal plants can be promising sources of natural products with potential anticancer, antimicrobial and antioxidative activity. The results will guide the selection of some plant species for further pharmacological and phytochemical investigations. PMID:19320966

Selection signatures in four lignin genes from switchgrass populations divergently selected for in vitro dry matter digestibility

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

Chen, Shiyu; Kaeppler, Shawn M.; Vogel, Kenneth P.

Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in in vitro dry matter digestibility (IVDMD), ethanol yield, and lignin concentration. Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four locimore » in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. As a result, this study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality.« less

Selection signatures in four lignin genes from switchgrass populations divergently selected for in vitro dry matter digestibility

DOE PAGES

Chen, Shiyu; Kaeppler, Shawn M.; Vogel, Kenneth P.; ...

2016-11-28

Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in in vitro dry matter digestibility (IVDMD), ethanol yield, and lignin concentration. Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four locimore » in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. As a result, this study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality.« less

In vitro versus in vivo protein digestibility techniques for calculating PDCAAS (protein digestibility-corrected amino acid score) applied to chickpea fractions.

PubMed

Tavano, Olga Luisa; Neves, Valdir Augusto; da Silva Júnior, Sinézio Inácio

2016-11-01

Seven different in vitro methods to determine the protein digestibility for chickpea proteins were considered and also the application of these methodologies for calculating PDCAAS (protein digestibility-corrected amino acid score), seeking their correlations with the in vivo methodology. In vitro digestibility of raw and heated samples were determined using pepsin-pancreatin hydrolysis, considering soluble nitrogen via Kjeldahl (ppKJ) and hydrolysed peptide linkages using trinitrobenzenesulfonic acid and o-phthaldialdehyde. In vitro digestibility was also determined using trypsin, chymotrypsin and peptidase (3-Enz) or trypsin, chymotrypsin, peptidase and pronase solution (4-Enz). None of the correlations between in vitro and in vivo digestibilities were significant (at p<0.0500), but, strong correlations were observed between PDCAAS calculated by in vitro and in vivo results. PDCAAS-ppKJ, PDCAAS-3-Enz and PDCAAS-4-Enz presented the highest correlations with in vivo method, r=0.9316, 0.9442 and 0.9649 (p<0.0500), respectively. The use of in vitro methods for calculating PDCAAS may be promising and deserves more discussions. Copyright © 2016 Elsevier Ltd. All rights reserved.

76 FR 29752 - Nomination of In Vitro Test Methods for Detection and Quantification of Botulinum Neurotoxins and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-23

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Nomination of In Vitro Test Methods for Detection and... Evaluated by These Test Methods AGENCY: Division of National Toxicology Program (NTP), National Institute of... Methods (ICCVAM), the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods...

A novel accelerated in vitro release method to evaluate the release of thymopentin from PLGA microspheres.

PubMed

Xie, Xiangyang; Li, Zhiping; Zhang, Ling; Chi, Qiang; Yang, Yanfang; Zhang, Hui; Yang, Yang; Mei, Xingguo

2015-01-01

A novel accelerated method of good correlations with "real-time" release to evaluate in vitro thymopentin release from poly (D, L-lactide-co-glycolide) (PLGA) microsphere was developed. Thymopentin-loaded microspheres were made from three types of PLGA, and peptide release was studied in various conditions. Incomplete release of peptide (<60%) from microspheres was found in accelerated testing with two typical release media. This problem was circumvented by adding organic solvents to the release media and varying the temperature in the media heating process. Release media containing three kinds of organic solvents at 50 °C were tested, respectively, and hydro-alcoholic solution was selected for further study. After the surfactant concentration (0.06%, W/V) and ethanol concentration (20%, V/V) were fixed, a gradient heating program, consisting of three stages and each stage with a different temperature, was introduced to enhance the correlations between the short- and long-term release. After adjusting the heating time of each stage, a good correlation (R(2) = 9896, formulation 8 K; R(2) = 0.9898, formulation 13 K; R(2) = 0.9886, formulation 28 K) between accelerated and "real-time" release was obtained. By optimizing the conditions as ethanol concentration and temperature gradients, this accelerated method may be appropriate for similar peptide formulations that not well correlate with "real-time" release.

Development and validation of LC-HRMS and GC-NICI-MS methods for stereoselective determination of MDMA and its phase I and II metabolites in human urine

PubMed Central

Schwaninger, Andrea E.; Meyer, Markus R.; Huestis, Marilyn A.; Maurer, Hans H.

2013-01-01

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography–high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography–mass spectrometry with negativeion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes. PMID:21656610

Development, validation and utility of an in vitro technique for assessment of potential clinical drug-drug interactions involving P-glycoprotein.

PubMed

Keogh, John P; Kunta, Jeevan R

2006-04-01

Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug-drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug-transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [3H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC50) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [3H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A-->B] transport, [3H]-digoxin [B-->A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 microM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC50 values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC50 values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin-drug interactions mediated via Pgp.

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

In Vitro Assessment of Nanoparticle Effects on Blood Coagulation.

PubMed

Potter, Timothy M; Rodriguez, Jamie C; Neun, Barry W; Ilinskaya, Anna N; Cedrone, Edward; Dobrovolskaia, Marina A

2018-01-01

Blood clotting is a complex process which involves both cellular and biochemical components. The key cellular players in the blood clotting process are thrombocytes or platelets. Other cells, including leukocytes and endothelial cells, contribute to clotting by expressing the so-called pro-coagulant activity (PCA) complex on their surface. The biochemical component of blood clotting is represented by the plasma coagulation cascade, which includes plasma proteins also known as coagulation factors. The coordinated interaction between platelets, leukocytes, endothelial cells, and plasma coagulation factors is necessary for maintaining hemostasis and for preventing excessive bleeding. Undesirable activation of all or some of these components may lead to pathological blood coagulation and life-threatening conditions such as consumptive coagulopathy or disseminated intravascular coagulation (DIC). In contrast, unintended inhibition of the coagulation pathways may lead to hemorrhage. Thrombogenicity is the property of a test material to induce blood coagulation by affecting one or more elements of the clotting process. Anticoagulant activity refers to the property of a test material to inhibit coagulation. The tendency to cause platelet aggregation, perturb plasma coagulation, and induce leukocyte PCA can serve as an in vitro measure of a nanomaterial's likelihood to be pro- or anticoagulant in vivo. This chapter describes three procedures for in vitro analyses of platelet aggregation, plasma coagulation time, and activation of leukocyte PCA. Platelet aggregation and plasma coagulation procedures have been described earlier. The revision here includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, and updated details about assay controls. The chapter is expanded to describe a method for the leukocyte PCA analysis and case studies demonstrating the performance of these in vitro assays.

Biohybrid Membrane Systems and Bioreactors as Tools for In Vitro Drug Testing.

PubMed

Salerno, Simona; Bartolo, Loredana De

2017-01-01

In drug development, in vitro human model systems are absolutely essential prior to the clinical trials, considering the increasing number of chemical compounds in need of testing, and, keeping in mind that animals cannot predict all the adverse human health effects and reactions, due to the species-specific differences in metabolic pathways. The liver plays a central role in the clearance and biotransformation of chemicals and xenobiotics. In vitro liver model systems by using highly differentiated human cells could have a great impact in preclinical trials. Membrane biohybrid systems constituted of human hepatocytes and micro- and nano-structured membranes, represent valuable tools for studying drug metabolism and toxicity. Membranes act as an extracellular matrix for the adhesion of hepatocytes, and compartmentalise them in a well-defined physical and chemical microenvironment with high selectivity. Advanced 3-D tissue cultures are furthermore achieved by using membrane bioreactors (MBR), which ensure the continuous perfusion of cells protecting them from shear stress. MBRs with different configurations allow the culturing of cells at high density and under closely monitored high perfusion, similarly to the natural liver. These devices that promote the long-term maintenance and differentiation of primary human hepatocytes with preserved liver specific functions can be employed in drug testing for prolonged exposure to chemical compounds and for assessing repeated-dose toxicity. The use of primary human hepatocytes in MBRs is the only system providing a faster and more cost-effective method of analysis for the prediction of in vitro human drug metabolism and enzyme induction alternative and/or complementary to the animal experimentation. In this paper, in vitro models for studying drug metabolism and toxicity as advanced biohybrid membrane systems and MBRs will be reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

PubMed Central

Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

2013-01-01

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy. PMID:23626771

Photodynamic Inactivation of Acinetobacter baumannii Using Phenothiazinium Dyes: In Vitro and In Vivo Studies

PubMed Central

Ragàs, Xavier; Dai, Tianhong; Tegos, George P.; Agut, Montserrat; Nonell, Santi; Hamblin, Michael R.

2010-01-01

Background and Objective Phenothiazinium dyes have been reported to be effective photosensitizers inactivating a wide range of microorganisms in vitro after illumination with red light. However, their application in vivo has not extensively been explored. This study evaluates the bactericidal activity of phenothiazinium dyes against multidrug-resistant Acinetobacter baumannii both in vitro and in vivo. Study Design/Materials and Methods We report the investigation of toluidine blue O, methylene blue, 1,9-dimethylmethylene blue, and new methylene blue for photodynamic inactivation of multidrug-resistant A. baumannii in vitro. The most effective dye was selected to carry out in vivo studies using third-degree mouse burns infected with a bioluminescent A. baumannii strain, upon irradiation with a 652 nm noncoherent light source. The mice were imaged daily for 2 weeks to observe differences in the bioluminescence–time curve between the photodynamic therapy (PDT)-treated mice in comparison with untreated burns. Results All the dyes were effective in vitro against A. baumannii after 30 J/cm2 irradiation of 635 or 652 nm red light had been delivered, with more effective killing when the dye remained in solution. New methylene blue was the most effective of the four dyes, achieving a 3.2-log reduction of the bacterial luminescence during PDT in vivo after 360 J/cm2 and an 800 μM dye dose. Moreover, a statistically significant reduction of the area under the bioluminescence–time curve of PDT-treated mice was observed showing that the infection did not recur after PDT. Conclusions Phenothiazinium dyes, and especially new methylene blue, are potential photosensitizers for PDT to treat burns infected with multidrug-resistant A. baumannii in vivo. PMID:20583252

In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus.

PubMed

Alcolea, Pedro J; Alonso, Ana; Degayón, María A; Moreno-Paz, Mercedes; Jiménez, Maribel; Molina, Ricardo; Larraga, Vicente

2016-05-20

Leishmania infantum is the protozoan parasite responsible for zoonotic visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been reported in this area. The life cycle of the parasite is digenetic. The promastigote stage develops within the gut of phlebotomine sand flies, whereas amastigotes survive and multiply within phagolysosomes of mammalian host phagocytes. The major vector of L. infantum in Spain is Phlebotomus perniciosus. The axenic culture model of promastigotes is generally used because it is able to mimic the conditions of the natural environment (i.e. the sand fly vector gut). However, infectivity decreases with culture passages and infection of laboratory animals is frequently required. Enrichment of the stationary phase population in highly infective metacyclic promastigotes is achieved by negative selection with peanut agglutinin (PNA), which is possible only in certain Leishmania species such as L. major and L. infantum. In this study, in vitro infectivity and differential gene expression of cultured PNA-negative promastigotes (Pro-PNA(-)) and metacyclic promastigotes isolated from the sand fly anterior thoracic midgut (Pro-Pper) have been compared. In vitro infectivity is about 30 % higher in terms of rate of infected cells and number of amastigotes per infected cell in Pro-Pper than in Pro-PNA(-). This finding is in agreement with up-regulation of a leishmanolysin gene (gp63) and genes involved in biosynthesis of glycosylinositolphospholipids (GIPL), lipophosphoglycan (LPG) and proteophosphoglycan (PPG) in Pro-Pper. In addition, differences between Pro-Pper and Pro-PNA(-) in genes involved in important cellular processes (e.g. signaling and regulation of gene expression) have been found. Pro-Pper are significantly more infective than peanut lectin non-agglutinating ones. Therefore, negative selection with PNA is an appropriate method for isolating metacyclic promastigotes in stationary phase of axenic culture but it does not allow reaching the in vitro infectivity levels of Pro-Pper. Indeed, GIPL, LPG and PPG biosynthetic genes together with a gp63 gene are up-regulated in Pro-Pper and interestingly, the correlation coefficient between both transcriptomes in terms of transcript abundance is R (2) = 0.68. This means that the correlation is sufficiently high to consider that both samples are physiologically comparable (i.e. the experiment was correctly designed and performed) and sufficiently low to conclude that important differences in transcript abundance have been found. Therefore, the implications of axenic culture should be evaluated case-by-case in each experimental design even when the stationary phase population in culture is enriched in metacyclic promastigotes by negative selection with PNA.

Validation of an In Vitro Sun Protection Factor (SPF) method in blinded ring-testing.

PubMed

Pissavini, M; Tricaud, C; Wiener, G; Lauer, A; Contier, M; Kolbe, L; Trullás Cabanas, C; Boyer, F; Nollent, V; Meredith, E; Dietrich, E; Matts, P J

2018-04-20

The objective of this work was to investigate the utility of a new in vitro SPF test method in blinded ring-testing, against new ISO acceptance criteria. 24 blinded, commercial, emulsion-type, primary sunscreen products, covering the full range of labelled SPF in Europe (SPF6 - 50+), were tested by 3 test institutes using the current ISO24444:2010 In Vivo SPF Test Method and simultaneously by 3 separate test laboratories using a new candidate in vitro SPF test method, developed under the leadership of Cosmetics Europe (CE). The resulting relationship between in vitro SPF and in vivo SPF values was then compared with acceptance criteria developed recently by the International Standards (ISO) TC217 / WG7 Sun Protection Test Methods Working Group. Analysis of the mean inter-laboratory in vitro and mean inter-institute in vivo SPF values revealed a strong correlation between in vitro and in vivo values, with a Pearson correlation coefficient of r 2 =0.88 (p<0.0001), a slope of 1.01 and a non-significant intercept (-1.48; p=0.62). When these data were compared to the new ISO WG7 acceptance criteria, method bias was found to be extremely low and over 95% of the coupled data lay within the model "funnel" (defined by upper and lower confidence intervals). In conclusion, the results of blinded ring testing and comparison to new ISO WG7 acceptance criteria indicate that a new in vitro SPF test method meets (and exceeds) these minimum criteria and is an interesting candidate for possible deployment as an industry test methodology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

PubMed

Cohen-Khait, Ruth; Schreiber, Gideon

2018-04-27

Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

Monoamine Oxidase Inhibitory Activity of Ferulic Acid Amides: Curcumin-Based Design and Synthesis.

PubMed

Badavath, Vishnu N; Baysal, İpek; Uçar, Gülberk; Mondal, Susanta K; Sinha, Barij N; Jayaprakash, Venkatesan

2016-01-01

Ferulic acid has structural similarity with curcumin which is being reported for its monoamine oxidase (MAO) inhibitory activity. Based on this similarity, we designed a series of ferulic acid amides 6a-m and tested for their inhibitory activity on human MAO (hMAO) isoforms. All the compounds were found to inhibit the hMAO isoforms either selectively or non-selectively. Nine compounds (6a, 6b, 6g-m) were found to inhibit hMAO-B selectively, whereas the other four (6c-f) were found to be non-selective. There is a gradual shift from hMAO-B selectivity (6a,b) to non-selectivity (6c-f) as there is an increase in chain length at the amino terminus. In case of compounds having an aromatic nucleus at the amino terminus, increasing the carbon number between N and the aromatic ring increases the potency as well as selectivity toward hMAO-B. Compounds 6f, 6j, and 6k were subjected to membrane permeability and metabolic stability studies by in vitro assay methods. They were found to have a better pharmacokinetic profile than curcumin, ferulic acid, and selegiline. In order to understand the structural features responsible for the potency and selectivity of 6k, we carried out a molecular docking simulation study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Limitations of predicting in vivo biostability of multiphase polyurethane elastomers using temperature-accelerated degradation testing.

PubMed

Padsalgikar, Ajay; Cosgriff-Hernandez, Elizabeth; Gallagher, Genevieve; Touchet, Tyler; Iacob, Ciprian; Mellin, Lisa; Norlin-Weissenrieder, Anna; Runt, James

2015-01-01

Polyurethane biostability has been the subject of intense research since the failure of polyether polyurethane pacemaker leads in the 1980s. Accelerated in vitro testing has been used to isolate degradation mechanisms and predict clinical performance of biomaterials. However, validation that in vitro methods reproduce in vivo degradation is critical to the selection of appropriate tests. High temperature has been proposed as a method to accelerate degradation. However, correlation of such data to in vivo performance is poor for polyurethanes due to the impact of temperature on microstructure. In this study, we characterize the lack of correlation between hydrolytic degradation predicted using a high temperature aging model of a polydimethylsiloxane-based polyurethane and its in vivo performance. Most notably, the predicted molecular weight and tensile property changes from the accelerated aging study did not correlate with clinical explants subjected to human biological stresses in real time through 5 years. Further, DMTA, ATR-FTIR, and SAXS experiments on samples aged for 2 weeks in PBS indicated greater phase separation in samples aged at 85°C compared to those aged at 37°C and unaged controls. These results confirm that microstructural changes occur at high temperatures that do not occur at in vivo temperatures. In addition, water absorption studies demonstrated that water saturation levels increased significantly with temperature. This study highlights that the multiphase morphology of polyurethane precludes the use of temperature accelerated biodegradation for the prediction of clinical performance and provides critical information in designing appropriate in vitro tests for this class of materials. © 2014 Wiley Periodicals, Inc.

Rapidly disintegrating tablets containing taste masked metoclopramide hydrochloride prepared by extrusion-precipitation method.

PubMed

Randale, Shivsagar Ashok; Dabhi, Chandu Somatbhai; Tekade, Avinash Ramrao; Belgamwar, Veena Shailendra; Gattani, Surendra Ganeshlal; Surana, Sanjay Javarilal

2010-04-01

The purpose of this study was to mask the intensely bitter taste of metoclopramide HCl and to formulate a rapid disintegrating tablet (RDT) of the taste-masked drug. Taste masking was done by complexing metoclopramide HCl with aminoalkyl methacrylate copolymer (Eudragit EPO) in different ratio by the extrusion-precipitation method. Drug-polymer complexes (DPCs) were tested for drug content, in vitro taste in simulated salivary fluid (SSF) of pH 6.8, taste evaluation in oral cavity and molecular property. The complex having drug-polymer ratio of 1 : 2 shows significant taste masking, confirmed by drug release in SSF and in-vivo taste evaluation; therefore, it was selected for further study. Taste evaluation of DPCs in human volunteers revealed considerable taste masking with the degree of bitterness below threshold value (0.5) within 10 s, whereas, metoclopramide HCl was rated intensely bitter with a score of +3 for 10 s. Tablets were evaluated for various parameters like tensile strength, wetting time, water absorption ratio, in-vitro disintegration time, and disintegration in oral cavity. The effect of diluents, lubricants and sweetening agent (Xylisorb) on the disintegration time was also evaluated. Tablets of batch F3 containing mannitol and microcrystalline cellulose in the ratio 1 : 1 and 8% w/w crosspovidone showed faster disintegration (within 20 s) than the marketed formulation (180 s). Good correlation between in vitro disintegration behavior and in the oral cavity was recognized. Tablets of batch F3 also revealed rapid drug release (t(90), 90 s) in SGF compared with marketed formulation (t(90), 600 s).

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization.

PubMed

Tavares, R S; Silva, A F; Lourenço, B; Almeida-Santos, T; Sousa, A P; Ramalho-Santos, J

2013-11-01

Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ≥34.25% (p = 0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available. © 2013 American Society of Andrology and European Academy of Andrology.

The Role of Feature Selection and Statistical Weighting in Predicting In Vivo Toxicity Using In Vitro Assay and QSAR Data (SOT)

EPA Science Inventory

Our study assesses the value of both in vitro assay and quantitative structure activity relationship (QSAR) data in predicting in vivo toxicity using numerous statistical models and approaches to process the data. Our models are built on datasets of (i) 586 chemicals for which bo...

High Throughput Determination of Critical Human Dosing ...

EPA Pesticide Factsheets

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data into predicted human equivalent doses that can be linked with biologically relevant exposure scenarios. Thus, HTTK provides essential data for risk prioritization for thousands of chemicals that lack TK data. One critical HTTK parameter that can be measured in vitro is the unbound fraction of a chemical in plasma (Fub). However, for chemicals that bind strongly to plasma, Fub is below the limits of detection (LOD) for high throughput analytical chemistry, and therefore cannot be quantified. A novel method for quantifying Fub was implemented for 85 strategically selected chemicals: measurement of Fub was attempted at 10%, 30%, and 100% of physiological plasma concentrations using rapid equilibrium dialysis assays. Varying plasma concentrations instead of chemical concentrations makes high throughput analytical methodology more likely to be successful. Assays at 100% plasma concentration were unsuccessful for 34 chemicals. For 12 of these 34 chemicals, Fub could be quantified at 10% and/or 30% plasma concentrations; these results imply that the assay failure at 100% plasma concentration was caused by plasma protein binding for these chemicals. Assay failure for the remaining 22 chemicals may

Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ.

PubMed

Wang, Guang-Yu; Wang, Hu-Hu; Han, Yi-Wei; Xing, Tong; Ye, Ke-Ping; Xu, Xing-Lian; Zhou, Guang-Hong

2017-05-01

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management. Copyright © 2016 Elsevier Ltd. All rights reserved.

Suitability of [18F]altanserin and PET to determine 5-HT2A receptor availability in the rat brain: in vivo and in vitro validation of invasive and non-invasive kinetic models.

PubMed

Kroll, Tina; Elmenhorst, David; Matusch, Andreas; Wedekind, Franziska; Weisshaupt, Angela; Beer, Simone; Bauer, Andreas

2013-08-01

While the selective 5-hydroxytryptamine type 2a receptor (5-HT2AR) radiotracer [18F]altanserin is well established in humans, the present study evaluated its suitability for quantifying cerebral 5-HT2ARs with positron emission tomography (PET) in albino rats. Ten Sprague Dawley rats underwent 180 min PET scans with arterial blood sampling. Reference tissue methods were evaluated on the basis of invasive kinetic models with metabolite-corrected arterial input functions. In vivo 5-HT2AR quantification with PET was validated by in vitro autoradiographic saturation experiments in the same animals. Overall brain uptake of [18F]altanserin was reliably quantified by invasive and non-invasive models with the cerebellum as reference region shown by linear correlation of outcome parameters. Unlike in humans, no lipophilic metabolites occurred so that brain activity derived solely from parent compound. PET data correlated very well with in vitro autoradiographic data of the same animals. [18F]Altanserin PET is a reliable tool for in vivo quantification of 5-HT2AR availability in albino rats. Models based on both blood input and reference tissue describe radiotracer kinetics adequately. Low cerebral tracer uptake might, however, cause restrictions in experimental usage.

Probiotic yeasts: Anti-inflammatory potential of various non-pathogenic strains in experimental colitis in mice

PubMed Central

Foligné, Benoît; Dewulf, Joëlle; Vandekerckove, Pascal; Pignède, Georges; Pot, Bruno

2010-01-01

AIM: To evaluate the in vitro immunomodulation capacity of various non-pathogenic yeast strains and to investigate the ability of some of these food grade yeasts to prevent experimental colitis in mice. METHODS: In vitro immunomodulation was assessed by measuring cytokines [interleukin (IL)-12p70, IL-10, tumor necrosis factor and interferon γ] released by human peripheral blood mononuclear cells after 24 h stimulation with 6 live yeast strains (Saccharomyces ssp.) and with bacterial reference strains. A murine model of acute 2-4-6-trinitrobenzene sulfonic acid (TNBS)-colitis was next used to evaluate the distinct prophylactic protective capacities of three yeast strains compared with the performance of prednisolone treatment. RESULTS: The six yeast strains all showed similar non-discriminating anti-inflammatory potential when tested on immunocompetent cells in vitro. However, although they exhibited similar colonization patterns in vivo, some yeast strains showed significant anti-inflammatory activities in the TNBS-induced colitis model, whereas others had weaker or no preventive effect at all, as evidenced by colitis markers (body-weight loss, macroscopic and histological scores, myeloperoxidase activities and blood inflammatory markers). CONCLUSION: A careful selection of strains is required among the biodiversity of yeasts for specific clinical studies, including applications in inflammatory bowel disease and other therapeutic uses. PMID:20440854

Synthesis and characterization of pH-responsive nanoscale hydrogels for oral delivery of hydrophobic therapeutics.

PubMed

Puranik, Amey S; Pao, Ludovic P; White, Vanessa M; Peppas, Nicholas A

2016-11-01

pH-responsive, polyanionic nanoscale hydrogels were developed for the oral delivery of hydrophobic therapeutics, such as common chemotherapeutic agents. Nanoscale hydrogels were designed to overcome physicochemical and biological barriers associated with oral delivery of hydrophobic therapeutics such as low solubility and poor permeability due to P-glycoprotein related drug efflux. Synthesis of these nanoscale materials was achieved by a robust photoemulsion polymerization method. By varying hydrophobic monomer components, four formulations were synthesized and screened for optimal physicochemical properties and in vitro biocompatibility. All of the responsive nanoscale hydrogels were capable of undergoing a pH-dependent transition in size. Depending on the selection of the hydrophobic monomer, the sizes of the nanoparticles vary widely from 120nm to about 500nm at pH 7.4. Polymer composition was verified using Fourier transform infrared spectroscopy and 1 H-nuclear magnetic resonance spectroscopy. Polymer biocompatibility was assessed in vitro with an intestinal epithelial cell model. All formulations were found to have no appreciable cytotoxicity, defined as greater than 80% viability after polymer incubation. We demonstrate that these nanoscale hydrogels possess desirable physicochemical properties and exhibit agreeable in vitro biocompatibility for oral delivery applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Toxicity assessment of industrial chemicals and airborne contaminants: transition from in vivo to in vitro test methods: a review.

PubMed

Bakand, S; Winder, C; Khalil, C; Hayes, A

2005-12-01

Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.

A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

PubMed Central

DeGrasse, Jeffrey A.

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide. PMID:22438927

A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

PubMed

DeGrasse, Jeffrey A

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

Laser antisepsis of Phorphyromonas gingivalis in vitro with dental lasers

NASA Astrophysics Data System (ADS)

Harris, David M.

2004-05-01

It has been shown that both pulsed Nd:YAG (1064nm) and continuous diode (810nm) dental lasers kill pathogenic bacteria (laser antisepsis), but a quantitative method for determining clinical dosimetry does not exist. The purpose of this study was to develop a method to quantify the efficacy of ablation of Porphyromonas gingivalis (Pg) in vitro for two different lasers. The ablation thresholds for the two lasers were compared in the following manner. The energy density was measured as a function of distance from the output of the fiber-optic delivery system. Pg cultures were grown on blood agar plates under standard anaerobic conditions. Blood agar provides an approximation of gingival tissue for the wavelengths tested in having hemoglobin as a primary absorber. Single pulses (Nd:YAG: 100- Œs diode: 100-msec) of laser energy were delivered to Pg colonies and the energy density was increased until the appearance of a small plume was observed coincident with a laser pulse. The energy density at this point defines the ablation threshold. Ablation thresholds to a single pulse were determined for both Pg and for blood agar alone. The large difference in ablation thresholds between the pigmented pathogen and the host matrix for pulsed-Nd:YAG represented a significant therapeutic ratio and Pg was ablated without visible effect on the blood agar. Near threshold the 810-nm diode laser destroyed both the pathogen and the gel. Clinically, the pulsed Nd:YAG may selectively destroy pigmented pathogens leaving the surrounding tissue intact. The 810-nm diode laser may not demonstrate this selectivity due to its longer pulse length and greater absorption by hemoglobin.

Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola).

PubMed

Gavamukulya, Yahaya; Abou-Elella, Faten; Wamunyokoli, Fred; AEl-Shemy, Hany

2014-09-01

To determine the phytochemical composition, antioxidant and anticancer activities of ethanolic and water leaves extracts of Annona muricata (A. muricata) from the Eastern Uganda. Phytochemical screening was conducted using standard qualitative methods and a Chi-square goodness of fit test was used to assign the relative abundance of the different phytochemicals. The antioxidant activity was determined using the 2, 2-diphenyl-2-picrylhydrazyl and reducing power methods whereas the in vitro anticancer activity was determined using three different cell lines. Phytochemical screening of the extracts revealed that they were rich in secondary class metabolite compounds such as alkaloids, saponins, terpenoids, flavonoids, coumarins and lactones, anthraquinones, tannins, cardiac glycosides, phenols and phytosterols. Total phenolics in the water extract were (683.69±0.09) μg/mL gallic acid equivalents (GAE) while it was (372.92±0.15) μg/mL GAE in the ethanolic extract. The reducing power was 216.41 μg/mL in the water extract and 470.51 μg/mL GAE in the ethanolic extract. In vitro antioxidant activity IC50 was 2.0456 mg/mL and 0.9077 mg/mL for ethanolic and water leaves extracts of A. muricata respectively. The ethanolic leaves extract was found to be selectively cytotoxic in vitro to tumor cell lines (EACC, MDA and SKBR3) with IC50 values of 335.85 μg/mL, 248.77 μg/mL, 202.33 μg/mL respectively, while it had no cytotoxic effect on normal spleen cells. The data also showed that water leaves extract of A. muricata had no anticancer effect at all tested concentrations. The results showed that A. muricata was a promising new antioxidant and anticancer agent. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Tumor-specific pH-responsive peptide-modified pH-sensitive liposomes containing doxorubicin for enhancing glioma targeting and anti-tumor activity.

PubMed

Zhao, Yang; Ren, Wei; Zhong, Ting; Zhang, Shuang; Huang, Dan; Guo, Yang; Yao, Xin; Wang, Chao; Zhang, Wei-Qiang; Zhang, Xuan; Zhang, Qiang

2016-01-28

The pH environment in gliomas is acidic. Therefore, in the present research, we selected our previously reported tumor-specific pH-responsive peptide H7K(R2)2 as a targeting ligand, which could respond to the acidic pH environment in gliomas, possessing CPP characteristics. The pH-sensitive liposomes were selected as carriers which could also respond to the acidic pH environment in gliomas triggering encapsulated drug release from these pH-sensitive liposomes. The H7K(R2)2-modified pH-sensitive liposomes containing doxorubicin (DOX-PSL-H7K(R2)2) were designed and prepared in order to evaluate their potential targeting of glioma tumor cells and their anti-tumor activity in mice with glioma tumor cells. DOX-PSL-H7K(R2)2 was prepared by the thin-film hydration method followed by remote loading using an ammonium sulfate gradient method. The in vitro release of DOX from pH-sensitive liposomes was tested and the in vitro targeting characteristics of H7K(R2)2-modified liposomes regarding C6 (rat C6 glioma cells) and U87-MG (human glioblastoma cells) were evaluated. The in vivo anti-tumor activity of DOX-PSL-H7K(R2)2 was also investigated in C6 tumor-bearing mice and in U87-MG orthotopic tumor-bearing nude mice. A specific targeting effect triggered by an acidic pH was observed in our in vitro experiments in C6 and U87-MG glioma cells. The pH-triggered DOX release from the pH-sensitive liposomes under acidic conditions was also confirmed in our in vitro experiment. Anti-tumor activity of DOX-PSL-H7K(R2)2 was found in C6 tumor-bearing mice and U87-MG orthotopic tumor-bearing nude mice in in vivo experiments. The antiangiogenic activity of DOX-PSL-H7K(R2)2 was confirmed in C6 tumor-bearing mice in the in vivo experiment. These H7K(R2)2-modified pH-sensitive liposomes containing anti-tumor drugs developed in this study are a promising delivery system involving the response stimuli at the acidic pH in the glioma tumor microenvironment and are suitable for anti-tumor therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Selective Gene Transfection of Individual Cells In Vitro with Plasmonic Nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina; Samaniego, Adam P.; Wen, Jianguo; Metelitsa, Leonid; Chang, Chung-Che; Lapotko, Dmitri

2011-01-01

Gene delivery and transfection of eukaryotic cells is widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. PMID:21315120

Selective gene transfection of individual cells in vitro with plasmonic nanobubbles.

PubMed

Lukianova-Hleb, Ekaterina Y; Samaniego, Adam P; Wen, Jianguo; Metelitsa, Leonid S; Chang, Chung-Che; Lapotko, Dmitri O

2011-06-10

Gene delivery and transfection of eukaryotic cells are widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+ CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

Study of in vitro antimicrobial and antiproliferative activities of selected Saharan plants.

PubMed

Palici, Ionut F; Liktor-Busa, Erika; Zupkó, István; Touzard, Blaise; Chaieb, Mohamed; Urbán, Edit; Hohmann, Judit

2015-12-01

The aim of the present study was the evaluation of the antimicrobial and antiproliferative activities of selected Saharan species, which are applied in the traditional medicine but not studied thoroughly from chemical and pharmacological point of view. The studied plants, namely Anthyllis henoniana, Centropodia forskalii, Cornulaca monacantha, Ephedra alata var. alenda, Euphorbia guyoniana, Helianthemum confertum, Henophyton deserti, Moltkiopsis ciliata and Spartidium saharae were collected from remote areas of North Africa, especially from the Tunisian region of Sahara. After drying and applying the appropriate extraction methods, the plant extracts were tested in antimicrobial screening assay, performed on 19 Gram-positive and -negative strains of microbes. The inhibition zones produced by plant extracts were determined by disc-diffusion method. Remarkable antibacterial activities were exhibited by extracts of Ephedra alata var. alenda and Helianthemum confertum against B. subtilis, M. catarrhalis and methicillin-resistant and non-resistant S. aureus. Minimum inhibitory concentrations of these two species were also determined. Antiproliferative effects of the extracts were evaluated against 4 human adherent cell lines (HeLa, A431, A2780 and MCF7). Notable cell growth inhibition was found for extract of Helianthemum confertum and Euphorbia guyoniana. Our results provided data for selection of some plant species for further detailed pharmacological and phytochemical examinations.

Effect of gallium-arsenic laser on photosensitized periodontopathic anaerobic organisms: An in vitro study

PubMed Central

Mathur, Setu; Kothiwale, Shaila Veerappa; Nag, Buddhi Prakash; Mathur, Tanu; Bhansali, Ashoka; Khatri, Rohit Kumar

2016-01-01

Background: The mainstay of periodontal therapy is mechanical removal of subgingival plaque. There is considerable interest in supplementing it with the use of antibiotics and antiseptics. Many drawbacks are associated with these adjunctive pharmacological regimens such as development of resistance to antibiotics and disruption of microflora of the gastrointestinal tract. Hence, alternate means of killing subgingival bacteria are clearly desirable. One such method is the use of laser. Aim: This study aimed to investigate antibacterial capabilities of gallium-arsenic (Ga-As) laser on photosensitized periodontopathic organisms. The three bacteria selected for the study were Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia. Settings: The subjects for the study were selected from the patients visiting the Department of Periodontics, Karnataka Lingayat Education Society's Institute of Dental Sciences, Belgaum. Design: In vitro study design. Materials and Methods: Subgingival plaque samples collected from chronic periodontitis patients were cultured anaerobically for 72 h. Predetermined number of colonies of each bacterium was taken and was then divided into cases and control groups. Both groups were photosensitized using toluidine blue O (TBO) dye and the case groups were irradiated with Ga-As laser. Bacterial colonies were then serially diluted and were incubated for subculture. After incubation period, the number of viable bacterial count was performed. Statistical Analysis: Wilcoxon-signed rank test was carried out to determine significance of reduction on subsequent dilution within the bacterial group. Mann–Whitney U-test was performed to determine the significance of reduction between cases and control of particular bacterial group. Results: The results revealed substantial reduction in the viable bacterial count. F. nucleatum was found to be most sensitive to killing by laser irradiation followed by P. intermedia and then P. gingivalis. Further, the TBO dye per se did not have any significant bactericidal effect. Conclusion: Photodynamic Therapy may prove to be a promising method for eradicating periodontopathic bacteria in near future. PMID:28298816

Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells By The Traceable Kinase Method

PubMed Central

Chen, Xiangyu; Zhao, Xin; Abeyweera, Thushara P.; Rotenberg, Susan A.

2012-01-01

A previous report (Biochemistry 46: 2364–2370, 2007) described the application of The Traceable Kinase Method to identify substrates of PKCα in non-transformed human breast MCF-10A cells. Here, a non-radioactive variation of this method compared the phospho-protein profiles of three traceable PKC isoforms (α, δ and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N6-phenyl-ATP, and the resulting phospho-proteins were analyzed by Western blot with an antibody that recognizes the phosphorylated PKC consensus site. Phospho-protein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated effector protein Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, –δ, and −ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (mol phospho-CEP4/mol CEP4). Following knock-down with isoform-specific shRNA-encoding plasmids, phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC–α, –δ, or –ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and –δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms. PMID:22897107

Assessing the risk of pH-dependent absorption for new molecular entities: a novel in vitro dissolution test, physicochemical analysis, and risk assessment strategy.

PubMed

Mathias, Neil R; Xu, Yan; Patel, Dhaval; Grass, Michael; Caldwell, Brett; Jager, Casey; Mullin, Jim; Hansen, Luke; Crison, John; Saari, Amy; Gesenberg, Christoph; Morrison, John; Vig, Balvinder; Raghavan, Krishnaswamy

2013-11-04

Weak base therapeutic agents can show reduced absorption or large pharmacokinetic variability when coadministered with pH-modifying agents, or in achlorhydria disease states, due to reduced dissolution rate and/or solubility at high gastric pH. This is often referred to as pH-effect. The goal of this study was to understand why some drugs exhibit a stronger pH-effect than others. To study this, an API-sparing, two-stage, in vitro microdissolution test was developed to generate drug dissolution, supersaturation, and precipitation kinetic data under conditions that mimic the dynamic pH changes in the gastrointestinal tract. In vitro dissolution was assessed for a chemically diverse set of compounds under high pH and low pH, analogous to elevated and normal gastric pH conditions observed in pH-modifier cotreated and untreated subjects, respectively. Represented as a ratio between the conditions, the in vitro pH-effect correlated linearly with clinical pH-effect based on the Cmax ratio and in a non-linear relationship based on AUC ratio. Additionally, several in silico approaches that use the in vitro dissolution data were found to be reasonably predictive of the clinical pH-effect. To explore the hypothesis that physicochemical properties are predictors of clinical pH-effect, statistical correlation analyses were conducted using linear sequential feature selection and partial least-squares regression. Physicochemical parameters did not show statistically significant linear correlations to clinical pH-effect for this data set, which highlights the complexity and poorly understood nature of the interplay between parameters. Finally, a strategy is proposed for implementation early in clinical development, to systematically assess the risk of clinical pH-effect for new molecular entities that integrates physicochemical analysis and in vitro, in vivo and in silico methods.

The effect of trimethoprim on CYP2C8 mediated rosiglitazone metabolism in human liver microsomes and healthy subjects

PubMed Central

Hruska, M W; Amico, J A; Langaee, T Y; Ferrell, R E; Fitzgerald, S M; Frye, R F

2005-01-01

Aims Rosiglitazone, a thiazolidinedione antidiabetic medication used in the treatment of Type 2 diabetes mellitus, is predominantly metabolized by the cytochrome P450 (CYP) enzyme CYP2C8. The anti-infective drug trimethoprim has been shown in vitro to be a selective inhibitor of CYP2C8. The purpose of this study was to evaluate the effect of trimethoprim on the CYP2C8 mediated metabolism of rosiglitazone in vivo and in vitro. Methods The effect of trimethoprim on the metabolism of rosiglitazone in vitro was assessed in pooled human liver microsomes. The effect in vivo was determined by evaluating rosiglitazone pharmacokinetics in the presence and absence of trimethoprim. Eight healthy subjects (four men and four women) completed a randomized, cross-over study. Subjects received single dose rosiglitazone (8 mg) in the presence and absence of trimethoprim 200 mg given twice daily for 5 days. Results Trimethoprim inhibited rosiglitazone metabolism both in vitro and in vivo. Inhibition of rosiglitazone para-hydroxylation by trimethoprim in vitro was found to be competitive with apparent Ki and IC50 values of 29 µm and 54.5 µm, respectively. In the presence of trimethoprim, rosiglitazone plasma AUC was increased by 31% (P = 0.01) from 2774 ± 645 µg l−1 h to 3643 ± 1051 µg l−1 h (95% confidence interval (Cl) for difference 189, 1549), and half-life was increased by 27% (P = 0.006) from 3.3 ± 0.5 to 4.2 ± 0.8 h (95% Cl for difference 0.36, 1.5). Trimethoprim reduced the para-O-sulphate rosiglitazone/rosiglitazone and the N-desmethylrosiglitazone/rosiglitazone AUC(0–24) ratios by 22% and 38%, respectively. Conclusions These results indicate that trimethoprim is a competitive inhibitor of CYP2C8-mediated rosiglitazone metabolism in vitro and that trimethoprim administration increases plasma rosiglitazone concentrations in healthy subjects. PMID:15606443

Cell-mediated immunity in an ageing population.

PubMed Central

Girard, J P; Paychère, M; Cuevas, M; Fernandes, B

1977-01-01

Eight hundred and eighty patients hospitalized in a geriatric hospital were routinely tested with 2, 10, 30 and 100 i.u. tuberculin. Among these, fifty-four patients were selected on the basis of negative skin tests and absence of evident diseases interfering with the function of the immune apparatus. A battery of tests analysing cell-mediated immunity was applied to those fifty-four patients. It appears that elderly patients having a negative test to 100 i.u. tuberculin show very infrequent sensitization to three other thymus-dependent antigens. The capacity of this selected population to become sensitized to DNCB is poor (20%). Furthermore they exhibit a low per cent of peripheral blood T cells (36%) and a poor capacity to respond in vitro to mitogens such as PHA. Testing the in vitro response to a battery of antigens demonstrates a good correlation with the results of the skin tests. Finally the leucocytes of 25% of this selected population failed to produce LIF in vitro in the presence of PHA. These results suggest not only an absolute decrease in the population of circulating T lymphocytes in those elderly humans; but very likely, at least in some cases, a functional impairment of T cells. PMID:321161

New Coumarin Derivatives as Potent Selective COX-2 Inhibitors: Synthesis, Anti-Inflammatory, QSAR, and Molecular Modeling Studies.

PubMed

Dawood, Dina H; Batran, Rasha Z; Farghaly, Thoraya A; Khedr, Mohammed A; Abdulla, Mohamed M

2015-12-01

Two new series of coumarin derivatives incorporating thiazoline and thiazolidinone moieties were designed, synthesized, and investigated in vivo for their anti-inflammatory activities using the carrageenan-induced rat paw edema model and in vitro for their inhibitory activities against the human cyclooxygenase (COX)-1 and COX-2 isoforms. Most of the synthesized compounds demonstrated exceptionally high in vivo anti-inflammatory activity and displayed superior GI safety profiles (0-7% ulceration) as compared to indomethacin. All the bioactive compounds showed in vitro high affinity and selectivity toward the COX-2 isoenzyme, compared to the reference celecoxib with IC50 values ranging from 0.31 to 0.78 μM. The ethyl thiosemicarbazone 2b, thiazoline derivatives 3a, 3b, 5b, 6a, and 7f, and the thiazolidinone compounds 8b and 9a showed the highest in vivo and in vitro anti-inflammatory activities with remarkable COX-2 selectivity. Quantitative structure-activity relationship study (QSAR) was done and resulted in a highly predictive power R(2) (0.908). A molecular docking study revealed a relationship between the docking affinity and the biological results. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Hypoxia-Targeted Boron Neutron Capture Therapy Agent for the Treatment of Glioma

PubMed Central

Luderer, Micah John; Muz, Barbara; de la Puente, Pilar; Chavalmane, Sanmathi; Kapoor, Vaishali; Marcelo, Raymundo; Biswas, Pratim; Thotala, Dinesh; Rogers, Buck; Azab, Abdel Kareem

2016-01-01

Purpose Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells. Methods B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo. Results B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively. Conclusions B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on tumor hypoxic metabolism, and further studies are warranted to evaluate this compound and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma. PMID:27401411

A method for quantification of exportin-1 (XPO1) occupancy by Selective Inhibitor of Nuclear Export (SINE) compounds

PubMed Central

Crochiere, Marsha L.; Baloglu, Erkan; Klebanov, Boris; Donovan, Scott; del Alamo, Diego; Lee, Margaret; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef

2016-01-01

Selective Inhibitor of Nuclear Export (SINE) compounds are a family of small-molecules that inhibit nuclear export through covalent binding to cysteine 528 (Cys528) in the cargo-binding pocket of Exportin 1 (XPO1/CRM1) and promote cancer cell death. Selinexor is the lead SINE compound currently in phase I and II clinical trials for advanced solid and hematological malignancies. In an effort to understand selinexor-XPO1 interaction and to establish whether cancer cell response is a function of drug-target engagement, we developed a quantitative XPO1 occupancy assay. Biotinylated leptomycin B (b-LMB) was utilized as a tool compound to measure SINE-free XPO1. Binding to XPO1 was quantitated from SINE compound treated adherent and suspension cells in vitro, dosed ex vivo human peripheral blood mononuclear cells (PBMCs), and PBMCs from mice dosed orally with drug in vivo. Evaluation of a panel of selinexor sensitive and resistant cell lines revealed that resistance was not attributed to XPO1 occupancy by selinexor. Administration of a single dose of selinexor bound XPO1 for minimally 72 hours both in vitro and in vivo. While XPO1 inhibition directly correlates with selinexor pharmacokinetics, the biological outcome of this inhibition depends on modulation of pathways downstream of XPO1, which ultimately determines cancer cell responsiveness. PMID:26654943

Role of the immune modulator programmed cell death-1 during development and apoptosis of mouse retinal ganglion cells

PubMed Central

Chen, Ling; Sham, Caroline W.; Chan, Ann M.; Francisco, Loise M.; Wu, Yin; Mareninov, Sergey; Sharpe, Arlene H.; Freeman, Gordon J.; Yang, Xian-Jie; Braun, Jonathan; Gordon, Lynn K.

2011-01-01

PURPOSE Mammalian programmed cell death-1 (PD-1) is a membrane-associated receptor regulating the balance between T cell activation, tolerance and immunopathology, however its role in neurons has not yet been defined. We investigate the hypothesis that PD-1 signaling actively promotes retinal ganglion cell (RGC) death within the developing mouse retina. METHODS Mature retinal cell types expressing PD-1 were identified by immunofluorescence staining of vertical retina sections; developmental expression was localized by immunostaining and quantified by Western analysis. PD-1 involvement in developmental RGC survival was assessed in vitro using retina explants and in vivo using PD-1 knockout mice. PD-1 ligand gene expression was detected by RT-PCR. RESULTS PD-1 is expressed in most adult RGCs, and undergoes dynamic upregulation during the early postnatal window of retinal cell maturation and physiological programmed cell death (PCD). In vitro blockade of PD-1 signaling during this time selectively increases survival of RGCs. Furthermore, PD-1 deficient mice show a selective increase in RGC number in the neonatal retina at the peak of developmental RGC death. Lastly, throughout postnatal retina maturation, we find gene expression of both immune PD-1 ligand genes, PD-L1 and PD-L2. CONCLUSIONS These findings collectively support a novel role for a PD-1-mediated signaling pathway in developmental PCD during postnatal RGC maturation. PMID:19420345

In vitro anti-Candida activity of selective serotonin reuptake inhibitors against fluconazole-resistant strains and their activity against biofilm-forming isolates.

PubMed

Costa Silva, Rose Anny; da Silva, Cecília Rocha; de Andrade Neto, João Batista; da Silva, Anderson Ramos; Campos, Rosana Sousa; Sampaio, Letícia Serpa; do Nascimento, Francisca Bruna Stefany Aires; da Silva Gaspar, Brenda; da Cruz Fonseca, Said Gonçalves; Josino, Maria Aparecida Alexandre; Grangeiro, Thalles Barbosa; Gaspar, Danielle Macedo; de Lucena, David Freitas; de Moraes, Manoel Odorico; Cavalcanti, Bruno Coêlho; Nobre Júnior, Hélio Vitoriano

2017-06-01

Recent research has shown broad antifungal activity of the classic antidepressants selective serotonin reuptake inhibitors (SSRIs). This fact, combined with the increased cross-resistance frequency of the genre Candida regarding the main treatment today, fluconazole, requires the development of novel therapeutic strategies. In that context, this study aimed to assess the antifungal potential of fluoxetine, sertraline, and paroxetine against fluconazole-resistant Candida spp. planktonic cells, as well as to assess the mechanism of action and the viability of biofilms treated with fluoxetine. After 24 h, the fluconazole-resistant Candida spp. strains showed minimum inhibitory concentration (MIC) in the ranges of 20-160 μg/mL for fluoxetine, 10-20 μg/mL for sertraline, and 10-100.8 μg/mL for paroxetine by the broth microdilution method (M27-A3). According to our data by flow cytometry, each of the SSRIs cause fungal death after damaging the plasma and mitochondrial membrane, which activates apoptotic signaling pathways and leads to dose-dependant cell viability loss. Regarding biofilm-forming isolates, the fluoxetine reduce mature biofilm of all the species tested. Therefore, it is concluded that SSRIs are capable of inhibit the growth in vitro of Candida spp., both in planktonic form, as biofilm, inducing cellular death by apoptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vivo quantification of brain metabolites by 1H-MRS using water as an internal standard.

PubMed

Christiansen, P; Henriksen, O; Stubgaard, M; Gideon, P; Larsson, H B

1993-01-01

The reliability of absolute quantification of average metabolite concentrations in the human brain in vivo by 1H-MRS using the fully relaxed water signal as an internal standard was tested in a number of in vitro as well as in vivo measurements. The experiments were carried out on a SIEMENS HELICON SP 63/84 wholebody MR-scanner operating at 1.5 T using a STEAM sequence. In vitro studies indicate a very high correlation between metabolite signals (area under peaks) and concentration, R = 0.99 as well as between metabolite signals and the volume of the selected voxel, R = 1.00. The error in quantification of N-acetyl aspartate (NAA) concentration was about 1-2 mM (6-12%). Also in vivo a good linearity between water signal and selected voxel size was seen. The same was true for the studied metabolites, N-acetyl aspartate (NAA), creatine/phosphocreatine (Cr/PCr), and choline (Cho). Calculated average concentrations of NAA, Cr/PCr, and Cho in the occipital lobe of the brain in five healthy volunteers were (mean +/- 1 SD) 11.6 +/- 1.3 mM, 7.6 +/- 1.4 mM, and 1.7 +/- 0.5 mM. The results indicate that the method presented offers reasonable estimation of metabolite concentrations in the brain in vivo and therefore is useful in clinical research.

Design and Optimization of Floating Drug Delivery System of Acyclovir

PubMed Central

Kharia, A. A.; Hiremath, S. N.; Singhai, A. K.; Omray, L. K.; Jain, S. K.

2010-01-01

The purpose of the present work was to design and optimize floating drug delivery systems of acyclovir using psyllium husk and hydroxypropylmethylcellulose K4M as the polymers and sodium bicarbonate as a gas generating agent. The tablets were prepared by wet granulation method. A 32 full factorial design was used for optimization of drug release profile. The amount of psyllium husk (X1) and hydroxypropylmethylcellulose K4M (X2) were selected as independent variables. The times required for 50% (t50%) and 70% (t70%) drug dissolution were selected as dependent variables. All the designed nine batches of formulations were evaluated for hardness, friability, weight variation, drug content uniformity, swelling index, in vitro buoyancy, and in vitro drug release profile. All formulations had floating lag time below 3 min and constantly floated on dissolution medium for more than 24 h. Validity of the developed polynomial equation was verified by designing two check point formulations (C1 and C2). The closeness of predicted and observed values for t50% and t70% indicates validity of derived equations for the dependent variables. These studies indicated that the proper balance between psyllium husk and hydroxypropylmethylcellulose K4M can produce a drug dissolution profile similar to the predicted dissolution profile. The optimized formulations followed Higuchi's kinetics while the drug release mechanism was found to be anomalous type, controlled by diffusion through the swollen matrix. PMID:21694992

Design and optimization of floating drug delivery system of acyclovir.

PubMed

Kharia, A A; Hiremath, S N; Singhai, A K; Omray, L K; Jain, S K

2010-09-01

The purpose of the present work was to design and optimize floating drug delivery systems of acyclovir using psyllium husk and hydroxypropylmethylcellulose K4M as the polymers and sodium bicarbonate as a gas generating agent. The tablets were prepared by wet granulation method. A 3(2) full factorial design was used for optimization of drug release profile. The amount of psyllium husk (X1) and hydroxypropylmethylcellulose K4M (X2) were selected as independent variables. The times required for 50% (t(50%)) and 70% (t(70%)) drug dissolution were selected as dependent variables. All the designed nine batches of formulations were evaluated for hardness, friability, weight variation, drug content uniformity, swelling index, in vitro buoyancy, and in vitro drug release profile. All formulations had floating lag time below 3 min and constantly floated on dissolution medium for more than 24 h. Validity of the developed polynomial equation was verified by designing two check point formulations (C1 and C2). The closeness of predicted and observed values for t(50%) and t(70%) indicates validity of derived equations for the dependent variables. These studies indicated that the proper balance between psyllium husk and hydroxypropylmethylcellulose K4M can produce a drug dissolution profile similar to the predicted dissolution profile. The optimized formulations followed Higuchi's kinetics while the drug release mechanism was found to be anomalous type, controlled by diffusion through the swollen matrix.

Evaluation of retentive strength of four luting cements with stainless steel crowns in primary molars: An in vitro study

PubMed Central

Parisay, Iman; Khazaei, Yegane

2018-01-01

Background: Stainless steel crown (SSC) is the most reliable restoration for primary teeth with extensive caries. Retention is of great importance for a successful restoration and is provided by various factors such as luting cements. The aim of this study was to evaluate the retentive strength of SSC cemented with four different luting cements. Materials and Methods: In this in vitro study, A total of 55 extracted primary first molars were selected. Following crown selection and cementation (one with no cement and four groups cemented with resin, glass ionomer, zinc phosphate, and polycarboxylate), all the specimens were incubated and thermocycled in 5°C–55°C. Retentive properties of SSCs were tested with a mechanical test machine. First dislodgement of each specimen and full crown removal were recorded. One-way ANOVA test followed by least significant difference test and Kruskal–Wallis test was used for retentive strength comparison at the level of significance of P < 0.05. Results: The results of the study showed that the specimens cemented with zinc phosphate exhibited higher retentive strength as compared to glass ionomer and polycarboxylate (P < 0.001 and P = 0.023, respectively). Conclusion: Zinc phosphate cement showed the most promising results; thus, it can be preferably used for cementation of the teeth with no grossly broken down crowns. PMID:29922339

Plasma stable, pH-sensitive non-ionic surfactant vesicles simultaneously enhance antiproliferative effect and selectivity of Sirolimus.

PubMed

Ghanbarzadeh, Saeed; Khorrami, Arash; Pourmoazzen, Zhaleh; Arami, Sanam

2015-05-01

The purpose of the present investigation was to prepare a plasma stable, pH-sensitive niosomal formulation to enhance Sirolimus efficacy and selectivity. pH-sensitive niosomal formulations bearing PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymers and cholesteryl hemisuccinate (CHEMS) were prepared by a modified ethanol injection method and characterized with regard to pH-responsiveness and stability in human serum. The ability of pH-sensitive niosomes to enhance the Sirolimus cytotoxicity was evaluated in vitro using human erythromyeloblastoid leukemia cell line (K562) and compared with cytotoxicity effect on human umbilical vein endothelial cells (HUVEC). This study showed that both formulations can be rendered pH-sensitive property and were found to rapidly release their contents under mildly acidic conditions. However, the CHEMS-based niosomes lost their pH-sensitivity after incubation in plasma, whereas, PEG-PMMI-CholC6 niosomes preserved their ability to respond to pH change. Sirolimus encapsulated in pH-sensitive niosomes exhibited a higher cytotoxicity than the control conventional formulation on K562 cell line. On the other hand, both pH-sensitive niosomes showed lower antiproliferative effect on HUVEC cells. Plasma stable, pH-sensitive PEG-PMMI-CholC6-based niosomes can improve the in vitro efficiency and also reduce the side effects of Sirolimus.

Formulation and optimisation of raft-forming chewable tablets containing H2 antagonist

PubMed Central

Prajapati, Shailesh T; Mehta, Anant P; Modhia, Ishan P; Patel, Chhagan N

2012-01-01

Purpose: The purpose of this research work was to formulate raft-forming chewable tablets of H2 antagonist (Famotidine) using a raft-forming agent along with an antacid- and gas-generating agent. Materials and Methods: Tablets were prepared by wet granulation and evaluated for raft strength, acid neutralisation capacity, weight variation, % drug content, thickness, hardness, friability and in vitro drug release. Various raft-forming agents were used in preliminary screening. A 23 full-factorial design was used in the present study for optimisation. The amount of sodium alginate, amount of calcium carbonate and amount sodium bicarbonate were selected as independent variables. Raft strength, acid neutralisation capacity and drug release at 30 min were selected as responses. Results: Tablets containing sodium alginate were having maximum raft strength as compared with other raft-forming agents. Acid neutralisation capacity and in vitro drug release of all factorial batches were found to be satisfactory. The F5 batch was optimised based on maximum raft strength and good acid neutralisation capacity. Drug–excipient compatibility study showed no interaction between the drug and excipients. Stability study of the optimised formulation showed that the tablets were stable at accelerated environmental conditions. Conclusion: It was concluded that raft-forming chewable tablets prepared using an optimum amount of sodium alginate, calcium carbonate and sodium bicarbonate could be an efficient dosage form in the treatment of gastro oesophageal reflux disease. PMID:23580933

Selective spectroscopic imaging of hyperpolarized pyruvate and its metabolites using a single-echo variable phase advance method in balanced SSFP

PubMed Central

Varma, Gopal; Wang, Xiaoen; Vinogradov, Elena; Bhatt, Rupal S.; Sukhatme, Vikas; Seth, Pankaj; Lenkinski, Robert E.; Alsop, David C.; Grant, Aaron K.

2015-01-01

Purpose In balanced steady state free precession (bSSFP), the signal intensity has a well-known dependence on the off-resonance frequency, or, equivalently, the phase advance between successive radiofrequency (RF) pulses. The signal profile can be used to resolve the contributions from the spectrally separated metabolites. This work describes a method based on use of a variable RF phase advance to acquire spatial and spectral data in a time-efficient manner for hyperpolarized 13C MRI. Theory and Methods The technique relies on the frequency response from a bSSFP acquisition to acquire relatively rapid, high-resolution images that may be reconstructed to separate contributions from different metabolites. The ability to produce images from spectrally separated metabolites was demonstrated in-vitro, as well as in-vivo following administration of hyperpolarized 1-13C pyruvate in mice with xenograft tumors. Results In-vivo images of pyruvate, alanine, pyruvate hydrate and lactate were reconstructed from 4 images acquired in 2 seconds with an in-plane resolution of 1.25 × 1.25mm2 and 5mm slice thickness. Conclusions The phase advance method allowed acquisition of spectroscopically selective images with high spatial and temporal resolution. This method provides an alternative approach to hyperpolarized 13C spectroscopic MRI that can be combined with other techniques such as multi-echo or fluctuating equilibrium bSSFP. PMID:26507361

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 fertilization experiments using sockeye salmon reveal that bigger eggs are more fertilizable under sperm limitation

PubMed Central

Macfarlane, Christopher P.; Hoysak, Drew J.; Liley, N. Robin; Gage, Matthew J.G.

2009-01-01

Although theory and widespread evidence show that the evolution of egg size is driven primarily by offspring and maternal fitness demands, an additional explanation invokes sperm limitation as a selective force that could also influence egg size optima. Levitan proposed that constraints from gamete encounter in external fertilization environments could select for enlargement of ova to increase the physical size of the fertilization target. We test this theory using in vitro fertilization experiments in an externally fertilizing fish. Sockeye salmon (Onchorhyncus nerka) females show considerable between-individual variation in ovum size, and we explored the consequences of this natural variation for the fertilization success of individual eggs under conditions of sperm limitation. By engineering consistent conditions where in vitro fertilization rate was always intermediate, we were able to compare the sizes of fertilized and unfertilized eggs across 20 fertilization replicates. After controlling for any changes in volume through incubation, results showed that successfully fertilized eggs were significantly larger than the eggs that failed to achieve fertilization. Under conditions without sperm limitation, fertility was unaffected by egg size. Our findings therefore support Levitan's theory, demonstrating empirically that some element of egg size variation could be selected by fertilization demands under sperm limitation. However, further research on sperm limitation in natural spawnings is required to assess the selective importance of these results. PMID:19364734

In vitro fertilization experiments using sockeye salmon reveal that bigger eggs are more fertilizable under sperm limitation.

PubMed

Macfarlane, Christopher P; Hoysak, Drew J; Liley, N Robin; Gage, Matthew J G

2009-07-07

Although theory and widespread evidence show that the evolution of egg size is driven primarily by offspring and maternal fitness demands, an additional explanation invokes sperm limitation as a selective force that could also influence egg size optima. Levitan proposed that constraints from gamete encounter in external fertilization environments could select for enlargement of ova to increase the physical size of the fertilization target. We test this theory using in vitro fertilization experiments in an externally fertilizing fish. Sockeye salmon (Onchorhyncus nerka) females show considerable between-individual variation in ovum size, and we explored the consequences of this natural variation for the fertilization success of individual eggs under conditions of sperm limitation. By engineering consistent conditions where in vitro fertilization rate was always intermediate, we were able to compare the sizes of fertilized and unfertilized eggs across 20 fertilization replicates. After controlling for any changes in volume through incubation, results showed that successfully fertilized eggs were significantly larger than the eggs that failed to achieve fertilization. Under conditions without sperm limitation, fertility was unaffected by egg size. Our findings therefore support Levitan's theory, demonstrating empirically that some element of egg size variation could be selected by fertilization demands under sperm limitation. However, further research on sperm limitation in natural spawnings is required to assess the selective importance of these results.

Derivatization of labetalol hydrochloride for its spectrofluorimetric and spectrophotometric determination inhuman plasma: Application to stability study

NASA Astrophysics Data System (ADS)

Omar, Mahmoud A.; Derayea, Sayed M.; Abdel-Lateef, Mohamed A.; El Hamd, Mohamed A.

2018-02-01

Two simple, selective and accurate methods were developed for the determination of Labetalol hydrochloride in pure form and pharmaceutical tablets. Both methods are based on derivatization of the studied drug with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBDsbnd Cl) in alkaline medium (pH 7.5).The reaction product was measured spectrofluorimetrically at 540 nm after excitation at 476 nm (method I) or spectrophotometrically at 480 nm (method II). The calibration graphs were rectilinear over the concentration ranges of 0.10-2.0 and 1.0-11.0 μg mL- 1 for methods I and II, respectively. The proposed methods were successfully applied to the analysis of commercial tablets without interference from common excipients. Furthermore, the spectrofluorimetric method was utilized for the in vitro determination of labetalol in spiked human plasma, with a percent mean recovery (n = 3) of 97.80 ± 1.29%. Moreover, the spectrofluorimetric method was extended to examine the stability study of LBT under different stress conditions such as alkaline, acidic, oxidative, photolytic and a thermal degradation.

Derivatization of labetalol hydrochloride for its spectrofluorimetric and spectrophotometric determination inhuman plasma: Application to stability study.

PubMed

Omar, Mahmoud A; Derayea, Sayed M; Abdel-Lateef, Mohamed A; El Hamd, Mohamed A

2018-02-05

Two simple, selective and accurate methods were developed for the determination of Labetalol hydrochloride in pure form and pharmaceutical tablets. Both methods are based on derivatization of the studied drug with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBDCl) in alkaline medium (pH7.5).The reaction product was measured spectrofluorimetrically at 540nm after excitation at 476nm (method I) or spectrophotometrically at 480nm (method II). The calibration graphs were rectilinear over the concentration ranges of 0.10-2.0 and 1.0-11.0μgmL -1 for methods I and II, respectively. The proposed methods were successfully applied to the analysis of commercial tablets without interference from common excipients. Furthermore, the spectrofluorimetric method was utilized for the in vitro determination of labetalol in spiked human plasma, with a percent mean recovery (n=3) of 97.80±1.29%. Moreover, the spectrofluorimetric method was extended to examine the stability study of LBT under different stress conditions such as alkaline, acidic, oxidative, photolytic and a thermal degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

PubMed

Osborn, Stephanie L; Kurzrock, Eric A

2018-01-01

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

Radiosynthesis and initial characterization of a PDE10A specific PET tracer [18F]AMG 580 in non-human primates.

PubMed

Hwang, Dah-Ren; Hu, Essa; Allen, Jennifer R; Davis, Carl; Treanor, James; Miller, Silke; Chen, Hang; Shi, Bingzhi; Narayanan, Tanjorie K; Barret, Olivier; Alagille, David; Yu, Zhigang; Slifstein, Mark

2015-08-01

Phosphodiesterase 10A (PDE10A) is an intracellular enzyme responsible for the breakdown of cyclic nucleotides which are important second messengers for neurotransmission. Inhibition of PDE10A has been identified as a potential target for treatment of various neuropsychiatric disorders. To assist drug development, we have identified a selective PDE10A positron emission tomography (PET) tracer, AMG 580. We describe here the radiosynthesis of [(18)F]AMG 580 and in vitro and in vivo characterization results. The potency and selectivity were determined by in vitro assay using [(3)H]AMG 580 and baboon brain tissues. [(18)F]AMG 580 was prepared by a 1-step [(18)F]fluorination procedure. Dynamic brain PET scans were performed in non-human primates. Regions-of-interest were defined on individuals' MRIs and transferred to the co-registered PET images. Data were analyzed using two tissue compartment analysis (2TC), Logan graphical (Logan) analysis with metabolite-corrected input function and the simplified reference tissue model (SRTM) method. A PDE10A inhibitor and unlabeled AMG 580 were used to demonstrate the PDE10A specificity. KD was estimated by Scatchard analysis of high and low affinity PET scans. AMG 580 has an in vitro KD of 71.9 pM. Autoradiography showed specific uptake in striatum. Mean activity of 121 ± 18 MBq was used in PET studies. In Rhesus, the baseline BPND for putamen and caudate was 3.38 and 2.34, respectively, via 2TC, and 3.16, 2.34 via Logan, and 2.92, and 2.01 via SRTM. A dose dependent decrease of BPND was observed by the pre-treatment with a PDE10A inhibitor. In baboons, 0.24 mg/kg dose of AMG 580 resulted in about 70% decrease of BPND. The in vivo KD of [(18)F]AMG 580 was estimated to be around 0.44 nM in baboons. [(18)F]AMG 580 is a selective and potent PDE10A PET tracer with excellent specific striatal binding in non-human primates. It warrants further evaluation in humans. Copyright © 2015 Elsevier Inc. All rights reserved.