Adenovirus-specific T-cell Subsets in Human Peripheral Blood and After IFN-γ Immunomagnetic Selection.

PubMed

Qian, Chongsheng; Wang, Yingying; Cai, Huili; Laroye, Caroline; De Carvalho Bittencourt, Marcelo; Clement, Laurence; Stoltz, Jean-François; Decot, Véronique; Reppel, Loïc; Bensoussan, Danièle

2016-01-01

Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

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.

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

Self-assembled pentablock copolymers for selective and sustained gene delivery

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

Zhang, Bingqi

2011-05-15

The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomesmore » of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.« less

Diagnostic efficacy of in vitro methods vs. skin testing in patients with inhalant allergies

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

Corey, J.P.; Liudahl, J.J.; Young, S.A.

1991-03-01

The purpose of our study was to investigate the diagnostic efficacy of two selected methods of in vitro allergy testing. Specifically, the PRIST/modified RAST I125 isotope systems and the Quantizyme/modified EAST alkaline phosphatase method were compared. The time, expense, convenience, and diagnostic efficacy of the two procedures are discussed. Special attention is given to the practicality of each method for the practicing physician.

Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica.

PubMed

Ravanfar, Seyed Ali; Orbovic, Vladimir; Moradpour, Mahdi; Abdul Aziz, Maheran; Karan, Ratna; Wallace, Simon; Parajuli, Saroj

2017-04-01

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

NASA Astrophysics Data System (ADS)

Duan, J.; Lu, X.; He, G.

2017-01-01

In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

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.

In vitro meat production system: why and how?

PubMed

Sharma, Shruti; Thind, Sukhcharanjit Singh; Kaur, Amarjeet

2015-12-01

Due to the nutritional importance and the sustained popularity of meat as a foodstuff, the livestock production sector has been expanding incessantly. This exponential growth of livestock meat sector poses a gigantic challenge to the sustainability of food production system. A new technological breakthrough is being contemplated to develop a substitute for livestock meat. The idea is to grow meat in a culture in the lab and manipulate its composition selectively. This paper aims to discuss the concept of In Vitro Meat production system, articulate the underlying technology and analyse the context of its implications, as proposed by several scientists and stakeholders. The challenges facing this emerging technology have also been discussed.

In vitro-in vivo correlation strategy applied to an immediate-release solid oral dosage form with a biopharmaceutical classification system IV compound case study.

PubMed

Bredael, Gerard M; Bowers, Niya; Boulineau, Fabien; Hahn, David

2014-07-01

The ability to predict in vivo response of an oral dosage form based on an in vitro technique has been a sought after goal of the pharmaceutical scientist. Dissolution testing that demonstrates discrimination to various critical formulations or process attributes provides a sensitive quality check that may be representative or may be overpredictive of potential in vivo changes. Dissolution methodology with an established in vitro-in vivo relationship or correlation may provide the desired in vivo predictability. To establish this in vitro-in vivo link, a clinical study must be performed. In this article, recommendations are given in the selection of batches for the clinical study followed by potential outcome scenarios. The investigation of a Level C in vitro-in vivo correlation (IVIVC), which is the most common correlation for immediate-release oral dosage forms, is presented. Lastly, an IVIVC case study involving a biopharmaceutical classification system class IV compound is presented encompassing this strategy and techniques. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

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.

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.

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

PubMed Central

Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

2015-01-01

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

In vitro digestion-assisted development of a β-cryptoxanthin-rich functional beverage; in vivo validation using systemic response and faecal content.

PubMed

Hernández-Alvarez, E; Blanco-Navarro, I; Pérez-Sacristán, B; Sánchez-Siles, L M; Granado-Lorencio, F

2016-10-01

Bioavailability of carotenoids is low and significant amounts reach the colon where they may be biologically active. We aimed to optimize a previously developed beverage designed to improve cardiovascular and bone remodelling markers in post-menopausal women. By assessing different lipid emulsions (soy lecithin, milkfat globule membrane (MFGM) and olive oil) on the in vitro bioaccessibility of β-Cryptoxanthin and phytosterols, a MFGM containing beverage was selected and resulted stable over time (recovery >95%) under in vitro digestion and simulated anaerobic conditions. This beverage was tested in a randomized human trial (n=38) by evaluating systemic response and the colonic availability of β-Cryptoxanthin. Consumption for six weeks provoked an increment in serum β-Cryptoxanthin of 38.9μg/dl (CI 95%; 31.0, 46.8; p<0.001). In faeces, free β-Cryptoxanthin, tentatively identified β-Cryptoxanthin esters and the ratio cis-/trans-β-carotene approached the profile in the beverage and after in vitro digestion but it was different from serum. In conclusion, in vitro digestion-assisted approach appears adequate to develop functional foods although in vivo validation should consider both systemic response and the availability at the colon. Copyright © 2016 Elsevier Ltd. All rights reserved.

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 ...

Use of in vivo Expression Technology for the Identification of Putative Host Adaptation Factors of the Lyme Disease Spirochete.

PubMed

Casselli, Timothy; Bankhead, Troy

2015-01-01

The causative agent of Lyme disease, Borrelia burgdorferi, is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of B. burgdorferi sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned B. burgdorferi genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with B. burgdorferi. © 2015 S. Karger AG, Basel.

Pharmacokinetics and analgesic effect of ketorolac floating delivery system.

PubMed

Radwan, Mahasen A; Abou El Ela, Amal El Sayeh F; Hassan, Maha A; El-Maraghy, Dalia A

2015-05-01

The efficacy of ketorolac tromethamine (KT) floating alginate beads as a drug delivery system for better control of KT release was investigated. The formulation with the highest drug loading, entrapment efficiency, swelling, buoyancy, and in vitro release would be selected for further in vivo analgesic effect in the mice and pharmacokinetics study in rats compared to the tablet dosage form. KT floating alginate beads were prepared by extrusion congealing technique. KT in plasma samples was analyzed using a UPLC MS/MS assay. The percentage yield, drug loading and encapsulation efficiency were increased proportionally with the hydroxypropylmethyl cellulose (HPMC) polymer amount in the KT floating beads. A reverse relationship was observed between HPMC amount in the beads and the KT in vitro release rate. F3-floating beads were selected, due to its better in vitro results (continued floating for >8 h) than others. A longer analgesic effect was observed for F3 in fed mice as compared to the tablets. After F3 administration to rats, the Cmax (2.2 ± 0.3 µg/ml) was achieved at ∼2 h and the decline in KT concentration was slower. F3 showed a significant increase in the AUC (1.89 fold) in rats as compared to the tablets. KT was successfully formulated as floating beads with prolonged in vitro release extended to a better in vivo characteristic with higher bioavailability in rats. KT in floating beads shows a superior analgesic effect over tablets, especially in fed mice.

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

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.

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

PubMed

Ruiz, Christian; Kustermann, Stefan; Pietilae, Elina; Vlajnic, Tatjana; Baschiera, Betty; Arabi, Leila; Lorber, Thomas; Oeggerli, Martin; Savic, Spasenija; Obermann, Ellen; Singer, Thomas; Rothschild, Sacha I; Zippelius, Alfred; Roth, Adrian B; Bubendorf, Lukas

2016-01-01

The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

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 ...

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.

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.

Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability.

PubMed

Hirabayashi, Miki; Taira, Shu; Kobayashi, Suzuko; Konishi, Kaoru; Katoh, Kaoru; Hiratsuka, Yuichi; Kodaka, Masato; Uyeda, Taro Q P; Yumoto, Noboru; Kubo, Tai

2006-06-20

We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel. 2006 Wiley Periodicals, Inc.

Biodegradable injectable in situ implants and microparticles for sustained release of montelukast: in vitro release, pharmacokinetics, and stability.

PubMed

Ahmed, Tarek A; Ibrahim, Hany M; Samy, Ahmed M; Kaseem, Alaa; Nutan, Mohammad T H; Hussain, Muhammad Delwar

2014-06-01

The objective of this study was to investigate the sustained release of a hydrophilic drug, montelukast (MK), from two biodegradable polymeric drug delivery systems, in situ implant (ISI) and in situ microparticles (ISM). N-Methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), triacetin, and ethyl acetate were selected as solvents. The release of 10% (w/v) MK from both systems containing poly-lactic-co-glycolic acid (PLGA) as the biodegradable polymer was compared. Upon contact with the aqueous medium, the PLGA in ISI and ISM systems solidified resulting in implants and microparticles, respectively. The in vitro drug release from the ISI system showed marked difference from miscible solvents (NMP and DMSO) than the partially miscible ones (triacetin and ethyl acetate), and the drug release decreased with increased PLGA concentration. In the ISM system, the initial in vitro drug release decreased with decreased ratio of polymer phase to external oil phase. In vivo studies in rats showed that ISM had slower drug release than the drug release from ISI. Also, the ISM system when compared to ISI system had significantly reduced initial burst effect. In vitro as well as the in vivo studies for both ISI and ISM systems showed sustained release of MK. The ISM system is suitable for sustained release of MK over 4-week period with a lower initial burst compared to the ISI system. Stability studies of the ISI and ISM formulations showed that MK is stable in the formulations stored at 4°C for more than 2 years.

Antibacterial, anti-inflammatory, and bone-regenerative dual-drug-loaded calcium phosphate nanocarriers-in vitro and in vivo studies.

PubMed

Madhumathi, K; Rubaiya, Y; Doble, Mukesh; Venkateswari, R; Sampath Kumar, T S

2018-05-01

A dual local drug delivery system (DDS) composed of calcium phosphate bioceramic nanocarriers aimed at treating the antibacterial, anti-inflammatory, and bone-regenerative aspects of periodontitis has been developed. Calcium-deficient hydroxyapatite (CDHA, Ca/P = 1.61) and tricalcium phosphate (β-TCP) were prepared by microwave-accelerated wet chemical synthesis method. The phase purity of the nanocarriers was confirmed by x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR), while the transmission electron microscopy (TEM) confirmed their nanosized morphology. CDHA was selected as carrier for the antibiotic (tetracycline) while TCP was chosen as the anti-inflammatory drug (ibuprofen) carrier. Combined drug release profile was studied in vitro from CDHA/TCP (CTP) system and compared with a HA/TCP (BCP) biphasic system. The tetracycline and ibuprofen release rate was 71 and 23% from CTP system as compared to 63 and 20% from BCP system. CTP system also showed a more controlled drug release profile compared to BCP system. Modeling of drug release kinetics from CTP system indicated that the release follows Higuchi model with a non-typical Fickian diffusion profile. In vitro biological studies showed the CTP system to be biocompatible with significant antibacterial and anti-inflammatory activity. In vivo implantation studies on rat cranial defects showed greater bone healing and new bone formation in the drug-loaded CTP system compared to control (no carrier) at the end of 12 weeks. The in vitro and in vivo results suggest that the combined drug delivery platform can provide a comprehensive management for all bone infections requiring multi-drug therapy.

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.

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.

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.

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...

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.

Non-unity molecular heritability demonstrated by continuous evolution in vitro

NASA Technical Reports Server (NTRS)

Schmitt, T.; Lehman, N.

1999-01-01

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

In vitro and in vivo evaluation of N-{2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[(11) C]methoxybenz-amide, a positron emission tomography (PET) radioligand for dopamine D4 receptors, in rodents.

PubMed

Leopoldo, Marcello; Selivanova, Svetlana V; Müller, Adrienne; Lacivita, Enza; Schetz, John A; Ametamey, Simon M

2014-09-01

The D4 dopamine receptor belongs to the D2 -like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D4 receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D4 receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high-affinity D4 receptor-selective ligand N-{2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[(11) C]methoxybenzamide ([(11) C]2) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D4 radioligand should possess in order to have improved in vivo utility in rodents. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

In vitro non-homologous DNA end joining assays—The 20th anniversary

PubMed Central

Pastwa, Elzbieta; Somiari, Richard I.; Malinowski, Mariusz; Somiari, Stella B.; Winters, Thomas A.

2010-01-01

DNA double-strand breaks (DSBs) are the most serious forms of DNA damage in cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to mutations or cell death, and consequently, to cancer predisposition. In human cells non-homologous DNA end joining (NHEJ) is the main repair mechanism of these breaks. Systems for DNA end joining study have been developing during the last 20 years. New assays have some advantages over earlier in vitro DSBs repair assays because they are less time-consuming, allow the use of clinical material and examination of the joining DNA ends produced physiologically in mammalian cells. Proteins involved in NHEJ repair pathway can serve as biomarkers or molecular targets for anticancer drugs. Results of studies on NHEJ in cancer could help to select potent repair inhibitors that may selectively sensitize tumor cells to ionizing radiation (IR) and chemotherapy. Here, we review the principles and practice of in vitro NHEJ assays and provide some insights into the future prospects of this assay in cancer diagnosis and treatment. PMID:19110069

In vitro models.

PubMed

Mather, Jennie Powell

2012-02-01

The current resurgence of interest in the cancer stem cell (CSC) hypothesis as possibly providing a unifying theory of cancer biology is fueled by the growing body of work on normal adult tissue stem cells and the promise that CSC may hold the key to one of the central problems of clinical oncology: tumor recurrence. Many studies suggest that the microenvironment plays a role, perhaps a seminal one, in cancer development and progression. In addition, the possibility that the stem cell-like component of tumors is capable of rapid and reversible changes of phenotype raises questions concerning studies with these populations and the application of what we learn to the clinical situation. These types of questions are extremely difficult to study using in vivo models or freshly isolated cells. Established cell lines grown in defined conditions provide important model systems for these studies. There are three types of in vitro models for CSCs: (a) selected subpopulations of existing tumor lines (derived from serum-containing medium; (b) creation of lines from tumor or normal cells by genetic manipulation; or (c) direct in vitro selection of CSC from tumors or sorted tumor cells using defined serum-free conditions. We review the problems associated with creating and maintaining in vitro cultures of CSCs and the progress to date on the establishment of these important models. Copyright © 2011 AlphaMed Press.

Effects of humic acids in vitro.

PubMed

Vašková, Janka; Veliká, Beáta; Pilátová, Martina; Kron, Ivan; Vaško, Ladislav

2011-06-01

Humic acids are known for their overall positive health and productivity effects in animal feeding trials and, controversially, as an aetiological factor of cancer. We tried to assess the in vitro effect of humic acids from a selected source in Slovakia when used at recommended prophylactic dosage. We investigated antioxidant properties, enzymatic and non-enzymatic antioxidant defence system in liver mitochondria and cultured cancer cell lines in vitro. We observed a significant decrease in superoxide dismutase activity after humic acids treatment irrespective of dissolving in dimethyl sulphoxide or direct addition to mitochondria suspension in a respiration medium. Activities of other antioxidant enzymes measured, such as glutathione peroxidase and glutathione reductase, showed no significant differences from the control as well as the reduced glutathione content. Percentage of inhibition by humic acids of superoxide radical indicated lower efficacy compared with that of hydroxyl radical. Survival of six different cancer cells lines indicated that only the acute T lymphoblastic leukaemia cell line was sensitive to the tested humic acids. Despite relatively low solubility in aqueous solutions, humic acids from the selected source participated in redox regulation. By recapturing the radicals, humic acids reloaded the antioxidant defensive mechanism. Results from in vitro study conducted with humic acids from the natural source showed potential of these substances as promising immunity enhancing agents.

Self-microemulsifying smaller molecular volume oil (Capmul MCM) using non-ionic surfactants: a delivery system for poorly water-soluble drug.

PubMed

Bandivadeka, Mithun Mohanraor; Pancholi, Shyam Sundar; Kaul-Ghanekar, Ruchika; Choudhari, Amit; Koppikar, Soumya

2012-07-01

The main purpose of this work is to formulate self-microemulsifying drug delivery system (SMEDDS) using smaller molecular oil with Atorvastatin calcium as a model drug. Solubility of the selected drug was accessed in oils and surfactants. Percent transmittance (%T) test study was performed to identify the efficient self-microemulsifying formulations. Those formulations which showed higher value for %T were evaluated for droplet size, polydispersity index, ζ potential, refractive index and cloud point measurement. Effect of drug loading on droplet size, increasing dilution in different media, thermodynamic stability and in vitro dissolution was performed to observe the performance of the selected formulation. Further cytotoxicity and permeation enhancement studies were carried out on Caco2 cell lines. Of all the oils accessed for drug solubility, Capmul MCM showed higher solubility capacity for Atorvastatin calcium. Capmul MCM was better microemulsified using combination of Tween 20 and Labrasol surfactant. Droplet size was as low as 86.93 nm with polydispersity index and ζ potential at 0.195 ± 0.011 and -7.27 ± 3.11 mV respectively. The selected undiluted formulation showed refractive index values ranging from 1.40 to 1.47 indicating the isotropicity of the formulation. The selected formulation was robust to dilution in different media and thermodynamically stable. Dissolution profile was enhanced for the selected drug as compared to marketed formulation with t85% and DE values at 10 min and 80.15 respectively. Also cytotoxicity measurement showed minimum effect with good permeation enhancing capacity. Thus our study demonstrates the use of smaller molecular oil (Capmul MCM) for developing self-microemulsifying drug delivery system for better in vitro and in vivo performance.

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors.

PubMed

Šilar, Radoslav; Holátko, Jiří; Rucká, Lenka; Rapoport, Andrey; Dostálová, Hana; Kadeřábková, Pavla; Nešvera, Jan; Pátek, Miroslav

2016-09-01

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

In vitro cell system for studying molecular mechanisms of action associated with low intensity focused ultrasound

NASA Astrophysics Data System (ADS)

Babakhanian, Meghedi; Fan, Richard E.; Mulgaonkar, Amit P.; Singh, Rahul; Culjat, Martin O.; Danesh, Shahab M.; Toro, Ligia; Grundfest, Warren; Melega, William P.

2012-03-01

Low intensity focused ultrasound (LIFU) is now being considered as a noninvasive brain therapy for clinical applications. We maintain that LIFU can efficiently deliver energy from outside the skull to target specific brain regions, effecting localized neuromodulation. However, the underlying molecular mechanisms that drive this LIFU-induced neuromodulation are not well-defined due, in part, to our lack of understanding of how particular sets of LIFU delivery parameters affect the outcome. To efficiently conduct multiple sweeps of different parameters and determine their effects, we have developed an in-vitro system to study the effects of LIFU on different types of cells grown in culture. Presently, we are evaluating how LIFU affects the ionic flux that may underlie neuronal excitation and inhibition observed in-vivo. The results of our in-vitro studies will provide a rationale for selection of optimal LIFU parameter to be used in subsequent in-vivo applications. Thus, a prototype ultrasound cell assay system has been developed to conduct these studies, and is described in this work.

Scaling Pharmacodynamics from In Vitro and Preclinical Animal Studies to Humans

PubMed Central

Mager, Donald E.; Woo, Sukyung; Jusko, William J.

2013-01-01

Summary An important feature of mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) models is the identification of drug- and system-specific factors that determine the intensity and time-course of pharmacological effects. This provides an opportunity to integrate information obtained from in vitro bioassays and preclinical pharmacological studies in animals to anticipate the clinical and adverse responses to drugs in humans. The fact that contemporary PK/PD modeling continues to evolve and seeks to emulate systems level properties should provide enhanced capabilities to scale-up pharmacodynamic data. Critical steps in drug discovery and development, such as lead compound and first in human dose selection, may become more efficient with the implementation and further refinement of translational PK/PD modeling. In this review, we highlight fundamental principles in pharmacodynamics and the basic expectations for in vitro bioassays and traditional allometric scaling in PK/PD modeling. Discussion of PK/PD modeling efforts for recombinant human erythropoietin is also included as a case study showing the potential for advanced systems analysis to facilitate extrapolations and improve understanding of inter-species differences in drug responses. PMID:19252333

Tailoring in vitro evolution for protein affinity or stability

PubMed Central

Jermutus, Lutz; Honegger, Annemarie; Schwesinger, Falk; Hanes, Jozef; Plückthun, Andreas

2001-01-01

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible. PMID:11134506

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

NASA Technical Reports Server (NTRS)

Rothschild, Lynn J.; Greenberg, Daniel T.; Takahashi, Jack R.; Thompson, Kirsten A.; Maheshwari, Akshay J.; Kent, Ryan E.; McCutcheon, Griffin; Shih, Joseph D.; Calvet, Charles; Devlin, Tyler D.;

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

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

PubMed Central

Skamel, Claudia; Aller, Stephen G.; Bopda Waffo, Alain

2014-01-01

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets. PMID:25393763

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

PubMed Central

Gubbens, Jacob; Kim, Soo Jung; Yang, Zhongying; Johnson, Arthur E.; Skach, William R.

2010-01-01

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies. PMID:20581130

Effects of Actinobolin on Growth and Some Metabolic Activities of Cariogenic Streptococci In Vitro and In Vivo

PubMed Central

Keele, Bernard B.; Powell, Hubert L.; Navia, Juan M.; McGhee, Jerry

1971-01-01

Actinobolin, a known inhibitor of protein synthesis, has been shown not to interfere selectively with acid production or dextransucrase activity in a cariogenic streptococcus when the antibiotic is added to a concentration of 500 μg/ml. It has also been shown that actinobolin does not alter the total in vivo flora of the oral cavity of the rat when tested in a rat caries model system. A culture of cariogenic streptococci, adapted to in vitro growth in the presence of 1 mg of actinobolin per ml, has also been isolated. PMID:4944810

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

PubMed

McLaughlin, M; Albertini, D F; Wallace, W H B; Anderson, R A; Telfer, E E

2018-03-01

Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 μm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 μm diameter were selected for IVM in SAGE medium (Step 4) then fixed for analysis. Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 μm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end of Step 3, 32 complexes contained oocytes >100 μm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimization with morphological evaluation and fertilization potential of IVG oocytes is required to determine whether they are normal. The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilization would benefit fertility preservation practice. Funded by MRC Grants (G0901839 and MR/L00299X/1). No competing interests.

Using Human-Derived Neural Cells as an In Vitro Model for Developmental Neurotoxicity Following Exposure to Pesticides

EPA Science Inventory

Agricultural, industrial and commercial use of pesticides continues to increase with an estimated annual usage nearing a billion lbs/year. Many of these compounds target the nervous system of nuisance animals and due to their lack of selectivity, casue adverse effects in non-targ...

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro

NASA Astrophysics Data System (ADS)

Ishijima, A.; Minamihata, K.; Yamaguchi, S.; Yamahira, S.; Ichikawa, R.; Kobayashi, E.; Iijima, M.; Shibasaki, Y.; Azuma, T.; Nagamune, T.; Sakuma, I.

2017-03-01

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid-gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

Development of Y-shaped peptide for constructing nanoparticle systems targeting tumor-associated macrophages in vitro and in vivo

NASA Astrophysics Data System (ADS)

Yan, Lu; Gao, Yunxiang; Pierce, Ryan; Dai, Liming; Kim, Julian; Zhang, Mei

2014-04-01

Tumor-associated macrophage (TAM) is increasingly being viewed as a target of great interest in tumor microenvironment due to its important role in the progression and metastasis of cancers. It has been shown that TAM indeed overexpresses unique surface marker legumain. In this study, we designed and synthesized a Y-shaped legumain-targeting peptide (Y-Leg) with functional groups allowing for further conjugation with imaging and therapeutic moieties (vide infra). The in vitro cell experiments using FITC-conjugated Y-Leg revealed its specific and selective interaction with M2-polarized macrophages (i.e., TAMs) with preference to M1 macrophages, and that the interaction was not interfered with by conjugating FITC to its functional group. Further, we constructed a nanotube system by grafting Y-Leg onto oxidized carbon nanotubes (OCNTs) loaded with paramagnetic Fe3O4 nanoparticles. The intravenous injection of the resultant Y-Leg-OCNT/Fe3O4 nanotubes to 4T1 mammary tumor-bearing mouse led to the magnetic resonance imaging (MRI) of TAM-infiltrated tumor microenvironment, revealing the targeting specificity of Y-Leg-conjugated nanotubes in vivo. The Y shape of peptide and its functional groups containing amines and imidazole can protonate at different pHs, contributing to the in vitro and in vivo targeting specificity. This study represents the first development of novel peptide and peptide-grafted nanotube system targeting M2-polarized TAMs in vivo. The methodology developed in this study is applicable to the construction of various multifunctional nanoparticle systems for selectively targeting, imaging and manipulating of TAMs for the diagnosis and treatment of cancers and inflammatory diseases identified with macrophage-infiltrated disease tissue.

In vitro techniques for the assessment of neurotoxicity.

PubMed Central

Harry, G J; Billingsley, M; Bruinink, A; Campbell, I L; Classen, W; Dorman, D C; Galli, C; Ray, D; Smith, R A; Tilson, H A

1998-01-01

Risk assessment is a process often divided into the following steps: a) hazard identification, b) dose-response assessment, c) exposure assessment, and d) risk characterization. Regulatory toxicity studies usually are aimed at providing data for the first two steps. Human case reports, environmental research, and in vitro studies may also be used to identify or to further characterize a toxic hazard. In this report the strengths and limitations of in vitro techniques are discussed in light of their usefulness to identify neurotoxic hazards, as well as for the subsequent dose-response assessment. Because of the complexity of the nervous system, multiple functions of individual cells, and our limited knowledge of biochemical processes involved in neurotoxicity, it is not known how well any in vitro system would recapitulate the in vivo system. Thus, it would be difficult to design an in vitro test battery to replace in vivo test systems. In vitro systems are well suited to the study of biological processes in a more isolated context and have been most successfully used to elucidate mechanisms of toxicity, identify target cells of neurotoxicity, and delineate the development and intricate cellular changes induced by neurotoxicants. Both biochemical and morphological end points can be used, but many of the end points used can be altered by pharmacological actions as well as toxicity. Therefore, for many of these end points it is difficult or impossible to set a criterion that allows one to differentiate between a pharmacological and a neurotoxic effect. For the process of risk assessment such a discrimination is central. Therefore, end points used to determine potential neurotoxicity of a compound have to be carefully selected and evaluated with respect to their potential to discriminate between an adverse neurotoxic effect and a pharmacologic effect. It is obvious that for in vitro neurotoxicity studies the primary end points that can be used are those affected through specific mechanisms of neurotoxicity. For example, in vitro systems may be useful for certain structurally defined compounds and mechanisms of toxicity, such as organophosphorus compounds and delayed neuropathy, for which target cells and the biochemical processes involved in the neurotoxicity are well known. For other compounds and the different types of neurotoxicity, a mechanism of toxicity needs to be identified first. Once identified, by either in vivo or in vitro methods, a system can be developed to detect and to evaluate predictive ability for the type of in vivo neurotoxicity produced. Therefore, in vitro tests have their greatest potential in providing information on basic mechanistic processes in order to refine specific experimental questions to be addressed in the whole animal. Images Figure 1 PMID:9539010

Use of in-vitro experimental results to model in-situ experiments: bio-denitrification under geological disposal conditions.

PubMed

Masuda, Kaoru; Murakami, Hiroshi; Kurimoto, Yoshitaka; Kato, Osamu; Kato, Ko; Honda, Akira

2013-01-01

Some of the low level radioactive wastes from reprocessing of spent nuclear fuels contain nitrates. Nitrates can be present in the form of soluble salts and can be reduced by various reactions. Among them, reduction by metal compounds and microorganisms seems to be important in the underground repository. Reduction by microorganism is more important in near field area than inside the repository because high pH and extremely high salt concentration would prevent microorganism activities. In the near field, pH is more moderate (pH is around 8) and salt concentration is lower. However, the electron donor may be limited there and it might be the control factor for microorganism's denitrification activities. In this study, in-vitro experiments of the nitrate reduction reaction were conducted using model organic materials purported to exist in underground conditions relevant to geological disposal. Two kinds of organic materials were selected. A super plasticizer was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Nitrates were reduced almost to N2 gas in the existence of super plasticizer. In the case of humic acids, although nitrates were reduced, the rate was much lower and, in this case, dead organism was used as an electron donor instead of humic acids. A reaction model was developed based on the in-vitro experiments and verified by running simulations against data obtained from in-situ experiments using actual groundwaters and microorganisms. The simulation showed a good correlation with the experimental data and contributes to the understanding of microbially mediated denitrification in geological disposal systems.

[Comparative study on transdermal osmosis in vitro of Aconitum brachypodium liniment, gel and patcher].

PubMed

Lin, Ya-ping; Zhao, Ying; Zhang, Yong-ping; Liang, Guang-yi

2007-02-01

To study the transdermal osmosis process of Aconitum brachypodum's liniment, gel and patcher to provide basis for selecting dosage form and controlling the quality. Taking the cumulate rate of transdermal as index, a imitated Fick's diffusion device was used for the investigating the transdermal osmosis course of the three preparations. The best transdermal mathematics models are obtained and the relations between the transdermal course and the release course are analysed. The three preparations have different characteristics of transdermal osmosis course. The liniment meets dynamics 0 order process, the gel and the patcher meet dynamic 0 order process of non-corroded drug system. And the relation is good cubic equation between their transdermal course and release course. The transdermal osmosis experiment in vitro for three preparations can provide basis for selecting dosage form and the quality control in future studies.

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.

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.

Activation of the endogenous nociceptin system by selective nociceptin receptor agonist SCH 221510 produces antitransit and antinociceptive effect: a novel strategy for treatment of diarrhea-predominant IBS.

PubMed

Fichna, J; Sobczak, M; Mokrowiecka, A; Cygankiewicz, A I; Zakrzewski, P K; Cenac, N; Sałaga, M; Timmermans, J-P; Vergnolle, N; Małecka-Panas, E; Krajewska, W M; Storr, M

2014-11-01

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, defined by the presence of loose stools and abdominal pain. In search for a novel anti-IBS-D therapy, here we investigated the nociceptin receptor (NOP)-dependent effects in the GI tract. A novel potent and selective NOP agonist SCH 221510 was used in the study. The effect of NOP activation on mouse intestinal motility was characterized in vitro and in vivo, in physiological conditions and in animal models of hypermotility and diarrhea. Well-established mouse models of visceral pain were used to characterize the antinociceptive effect of the NOP activation. To provide additional evidence that the endogenous nociceptin system is a relevant target for IBS, NOP expression and nociceptin levels were quantified in serum and colonic biopsies from IBS-D patients. SCH 221510 produced a potent NOP-mediated inhibitory effect on mouse intestinal motility in vitro and in vivo in physiological conditions. The NOP agonist displayed an antidiarrheal and analgesic action after oral administration in animal models mimicking the symptoms of IBS-D. Studies on human samples revealed a strong decrease in endogenous nociceptin system expression in IBS-D patients compared with healthy controls. Collectively, mouse and human data suggest that the endogenous nociceptin system is involved in IBS-D and may become a target for anti-IBS-D treatments using potent and selective synthetic NOP agonists. © 2014 John Wiley & Sons Ltd.

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.

Administration of the antitumor drug mitoguazone protects normal thymocytes against spontaneous and etoposide-induced apoptosis.

PubMed

Ferioli, M E; Bottone, M G; Soldani, C; Pellicciari, C

2004-11-01

The suggestion has been made that polyamines may be involved in the control of cell death, since exceedingly high or low levels induce apoptosis in different cell systems. For a deeper insight into the relationship between apoptosis and polyamine metabolism, we investigated in vitro the effect on rat thymocytes of mitoguazone (MGBG, which inhibits S-adenosylmethionine decarboxylase, i.e. a key enzyme in the polyamine biosynthetic pathway). Thymocytes were selected as an especially suitable model system, since they undergo spontaneous apoptosis in vivo and can be easily induced to apoptose in vitro by etoposide, used here as an apoptogenic agent. MGBG protected thymocytes from both spontaneous and drug-induced apoptosis, and this protective effect was associated with a decrease in polyamine oxidase activity and total polyamine levels.

Impact of head and neck radiotherapy on the mechanical behavior of composite resins and adhesive systems: A systematic review.

PubMed

Madrid Troconis, Cristhian Camilo; Santos-Silva, Alan Roger; Brandão, Thaís Bianca; Lopes, Marcio Ajudarte; de Goes, Mario Fernando

2017-11-01

To analyze the evidence regarding the impact of head and neck radiotherapy (HNRT) on the mechanical behavior of composite resins and adhesive systems. Searches were conducted on PubMed, Embase, Scopus and ISI Web of Science databases using "Radiotherapy", "Composite resins" and "Adhesive systems" as keywords. Selected studies were written in English and assessed the mechanical behavior of composite resins and/or adhesive systems when bonding procedure was conducted before and/or after a maximum radiation dose ≥50Gy, applied under in vitro or in vivo conditions. In total, 115 studies were found but only 16 were included, from which five evaluated the effect of in vitro HNRT on microhardness, wear resistance, diametral tensile and flexural strength of composite resins, showing no significant negative effect in most of reports. Regarding bond strength of adhesive systems, 11 studies were included from which five reported no meaningful negative effect when bonding procedure was conducted before simulated HNRT. Conversely, five studies showed that bond strength diminished when adhesive procedure was done after in vitro radiation therapy. Only two studies about dental adhesion were conducted after in vivo radiotherapy but the results were not conclusive. The mechanical behavior of composite resins and adhesive systems seems not to be affected when in vitro HNRT is applied after bonding procedure. However, bond strength of adhesive systems tends to decrease when simulated radiotherapy is used immediately before bonding procedure. Studies assessing dentin bond strength after in-vivo HNRT were limited and controversial. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Mechanistic models enable the rational use of in vitro drug-target binding kinetics for better drug effects in patients.

PubMed

de Witte, Wilhelmus E A; Wong, Yin Cheong; Nederpelt, Indira; Heitman, Laura H; Danhof, Meindert; van der Graaf, Piet H; Gilissen, Ron A H J; de Lange, Elizabeth C M

2016-01-01

Drug-target binding kinetics are major determinants of the time course of drug action for several drugs, as clearly described for the irreversible binders omeprazole and aspirin. This supports the increasing interest to incorporate newly developed high-throughput assays for drug-target binding kinetics in drug discovery. A meaningful application of in vitro drug-target binding kinetics in drug discovery requires insight into the relation between in vivo drug effect and in vitro measured drug-target binding kinetics. In this review, the authors discuss both the relation between in vitro and in vivo measured binding kinetics and the relation between in vivo binding kinetics, target occupancy and effect profiles. More scientific evidence is required for the rational selection and development of drug-candidates on the basis of in vitro estimates of drug-target binding kinetics. To elucidate the value of in vitro binding kinetics measurements, it is necessary to obtain information on system-specific properties which influence the kinetics of target occupancy and drug effect. Mathematical integration of this information enables the identification of drug-specific properties which lead to optimal target occupancy and drug effect in patients.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

PubMed

Urrego, R; Bernal-Ulloa, S M; Chavarría, N A; Herrera-Puerta, E; Lucas-Hahn, A; Herrmann, D; Winkler, S; Pache, D; Niemann, H; Rodriguez-Osorio, N

2017-04-01

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

alpha7 and non-alpha7 nicotinic acetylcholine receptors modulate dopamine release in vitro and in vivo in the rat prefrontal cortex.

PubMed

Livingstone, Phil D; Srinivasan, Jayaraman; Kew, James N C; Dawson, Lee A; Gotti, Cecilia; Moretti, Milena; Shoaib, Mohammed; Wonnacott, Susan

2009-02-01

Nicotine enhances attentional and working memory aspects of executive function in the prefrontal cortex (PFC) where dopamine plays a major role. Here, we have determined the nicotinic acetylcholine receptor (nAChR) subtypes that can modulate dopamine release in rat PFC using subtype-selective drugs. Nicotine and 5-Iodo-A-85380 (beta2* selective) elicited [(3)H]dopamine release from both PFC and striatal prisms in vitro and dopamine overflow from medial PFC in vivo. Blockade by dihydro-beta-erythroidine supports the participation of beta2* nAChRs. However, insensitivity of nicotine-evoked [(3)H]dopamine release to alpha-conotoxin-MII in PFC prisms suggests no involvement of alpha6beta2* nAChRs, in contrast to the striatum, and this distinction is supported by immunoprecipitation of nAChR subunits from these tissues. The alpha7 nAChR-selective agonists choline and Compound A also promoted dopamine release from PFC in vitro and in vivo, and their effects were enhanced by the alpha7 nAChR-selective allosteric potentiator PNU-120596 and blocked by specific antagonists. DNQX and MK801 inhibited [(3)H]dopamine release evoked by choline and PNU-120596, suggesting crosstalk between alpha7 nAChRs, glutamate and dopamine in the PFC. In vivo, systemic (but not local) administration of PNU-120596, in the absence of agonist, facilitated dopamine overflow in the medial PFC, consistent with the activation of extracortical alpha7 nAChRs by endogenous acetylcholine or choline. These data establish that both beta2* and alpha7 nAChRs can modulate dopamine release in the PFC in vitro and in vivo. Through their distinct actions on dopamine release, these nAChR subtypes could contribute to executive function, making them specific therapeutic targets for conditions such as schizophrenia and attention deficit hyperactivity disorder.

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.

Inhibition of decay fungi using cotton cellulose hydrolysis as a model for wood decay

Treesearch

Frederick Green

2000-01-01

Environmental pressures to replace chromium and arsenic in fixed waterborne preservatives have been increasing. Potential inhibitors of brown-, white- and soft-rot fungi need to be evaluated as alternative preservatives by screening and testing in, in vitro model systems. This paper reports the inhibition of cellulose depolymerization and weight loss of selected decay...

Inhibition of Thyroid Hormone Release from Cultured Amphibian Thyroid Glands by Methimazole, 6-Propylthiouracil, and Perchlorate

EPA Science Inventory

The research presented here is the development of an in vitro thyroid gland culture system to test the effect of chemicals directly on the gland without influence of other parts of the HPT axis. . . This information can then be used to select chemicals for further evaluation in v...

Bioaccessibility in vitro of nutraceuticals from bark of selected Salix species.

PubMed

Gawlik-Dziki, Urszula; Sugier, Danuta; Dziki, Dariusz; Sugier, Piotr

2014-01-01

The aim of this study was to investigate and to compare the extractability, bioaccessibility, and bioavailability in vitro of antioxidative compounds from bark of selected Salix species: S. alba (SA), S. daphnoides (SD), S. purpurea (SP), and S. daphnoides x purpurea (SDP) hybrid willow clones originating from their natural habitats and cultivated on the sandy soil. The highest amount of phenolic glycosides was found in the bark of SDP and SD. The best source of phenolics was bark of SDP. The highest content of flavonoids were found in SD bark samples, whereas the highest concentration of bioaccessible and bioavailable phenolic acids was determined in SDP bark. Bark of all tested Salix species showed significant antiradical activity. This properties are strongly dependent on extraction system and genetic factors. Regardless of Salix genotypes, the lowest chelating power was found for chemically-extractable compounds. Bark of all Salix species contained ethanol-extractable compounds with reducing ability. Besides this, high bioaccessibility and bioavailability in vitro of Salix bark phytochemicals were found. Obtained results indicate that extracts from bark tested Salix genotypes can provide health promoting benefits to the consumers; however, this problem requires further study.

Bioaccessibility In Vitro of Nutraceuticals from Bark of Selected Salix Species

PubMed Central

2014-01-01

The aim of this study was to investigate and to compare the extractability, bioaccessibility, and bioavailability in vitro of antioxidative compounds from bark of selected Salix species: S. alba (SA), S. daphnoides (SD), S. purpurea (SP), and S. daphnoides x purpurea (SDP) hybrid willow clones originating from their natural habitats and cultivated on the sandy soil. The highest amount of phenolic glycosides was found in the bark of SDP and SD. The best source of phenolics was bark of SDP. The highest content of flavonoids were found in SD bark samples, whereas the highest concentration of bioaccessible and bioavailable phenolic acids was determined in SDP bark. Bark of all tested Salix species showed significant antiradical activity. This properties are strongly dependent on extraction system and genetic factors. Regardless of Salix genotypes, the lowest chelating power was found for chemically-extractable compounds. Bark of all Salix species contained ethanol-extractable compounds with reducing ability. Besides this, high bioaccessibility and bioavailability in vitro of Salix bark phytochemicals were found. Obtained results indicate that extracts from bark tested Salix genotypes can provide health promoting benefits to the consumers; however, this problem requires further study. PMID:24696660

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

PubMed

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

2014-09-01

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

An investigation into the characteristics and drug release properties of multiple W/O/W emulsion systems containing low concentration of lipophilic polymeric emulsifier.

PubMed

Vasiljevic, Dragana; Parojcic, Jelena; Primorac, Marija; Vuleta, Gordana

2006-02-17

Multiple W/O/W emulsions with high content of inner phase (Phi1=Phi2=0.8) were prepared using relatively low concentrations of lipophilic polymeric primary emulsifier, PEG 30-dipolyhydroxystearate, and diclofenac diethylamine (DDA) as a model drug. The investigated formulations were characterized and their stability over the time was evaluated by dynamic and oscillatory rheological measurements, microscopic analysis and in vitro drug release study. In vitro release profiles of the selected model drug were evaluated in terms of the effective diffusion coefficients and flux of the released drug. The multiple emulsion samples exhibited good stability during the ageing time. Concentration of the lipophilic primary emulsifier markedly affected rheological behaviour as well as the droplet size and in vitro drug release kinetics of the investigated systems. The multiple emulsion systems with highest concentration (2.4%, w/w) of the primary emulsifier had the lowest droplet size and the highest apparent viscosity and highest elastic characteristics. Drug release data indicated predominately diffusional drug release mechanism with sustained and prolonged drug release accomplished with 2.4% (w/w) of lipophilic emulsifier employed.

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.

In vitro and In vivo characterization of quercetin loaded multiphase hydrogel for wound healing application.

PubMed

Jangde, Rajendra; Srivastava, Shikha; Singh, Manju R; Singh, Deependra

2018-05-03

The present work aim to prepare and evaluate multiphase hydrogel system incorporated with quercetin loaded liposomes (QLH), for wound healing. The quercetin loaded liposomal hydrogel were prepared by taking 15% carbopol and varying gelatin ratio. The clear and transparent hydrogel was obtained by taking ratio of gelatin to carbapol (6/4) compared to other ratios. The best prepared hydrogel were characterized for surface morphology, water vapor transmission rate (WVTR), swelling ratio, hemocompatibility, stability, in-vitro release and in-vivo studies. The evaluated results of (QLH) for surface morphology, WVTR, swelling ratio, hemocompatibility and in-vitro release were found to be significant compared to other prepared formulations. Consequently, on basis of optimized hydrogel was selected to study wound healing activity in albino rats. The results demonstrated accelerated wound-healing with significant decrease in wound closure time compared to conventional dosage form. The results of in-vitro and in-vivo promises reliable mode of treatment for connective tissue disorder as wound healing. Copyright © 2018. Published by Elsevier B.V.

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.

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.

Evaluation in vitro and in vivo of dimethicone transdermal therapeutic systems. Influence of propylene glycol on drug release.

PubMed

Ritschel, W A; Nayak, P M

1987-03-01

Coumarin-containing transdermal drug delivery systems were studied in vitro for drug release and in vivo in rats for drug absorption. The matrix of the transdermal delivery system, dimethicone, was a commercially available silicone elastomer. The devices containing 1, 3 and 5% coumarin released in vitro 8.8 (87.4%), 23.4 (74.5%) and 31.6 mg (63.3%) of drug within 24 h. The device containing 5% coumarin was selected for further studies in which 5, 10, 20, 30, 50 and 70% propylene glycol was added. Up to 20% propylene glycol content did not change the amount released. The preparations with 30, 50 and 70% propylene glycol released 69.3, 73.6 and 87.9%, respectively. The 50 and 70% preparations were physically not acceptable. Only the preparations containing 5% coumarin without propylene glycol and 5% coumarin and 30% propylene glycol in the elastomer were evaluated in vivo. The area under the blood level-time curve of the propylene glycol-containing system was twice that of the device without propylene glycol. Blood levels were maintained between about 2 micrograms/ml and 5 micrograms/ml during the time the device was kept on the skin (24 h).

Translating in vitro ligand bias into in vivo efficacy.

PubMed

Luttrell, Louis M; Maudsley, Stuart; Gesty-Palmer, Diane

2018-01-01

It is increasingly apparent that ligand structure influences both the efficiency with which G protein-coupled receptors (GPCRs) engage their downstream effectors and the manner in which they are activated. Thus, 'biased' agonists, synthetic ligands whose intrinsic efficacy differs from the native ligand, afford a strategy for manipulating GPCR signaling in ways that promote beneficial signals while blocking potentially deleterious ones. Still, there are significant challenges in relating in vitro ligand efficacy, which is typically measured in heterologous expression systems, to the biological response in vivo, where the ligand is acting on natively expressed receptors and in the presence of the endogenous ligand. This is particularly true of arrestin pathway-selective 'biased' agonists. The type 1 parathyroid hormone receptor (PTH 1 R) is a case in point. Parathyroid hormone (PTH) is the principal physiological regulator of calcium homeostasis, and PTH 1 R expressed on cells of the osteoblast lineage are an established therapeutic target in osteoporosis. In vitro, PTH 1 R signaling is highly sensitive to ligand structure, and PTH analogs that affect the selectivity/kinetics of G protein coupling or that engage arrestin-dependent signaling mechanisms without activating heterotrimeric G proteins have been identified. In vivo, intermittent administration of conventional PTH analogs accelerates the rate of osteoblastic bone formation, largely through known cAMP-dependent mechanisms. Paradoxically, both intermittent and continuous administration of an arrestin pathway-selective PTH analog, which in vivo would be expected to antagonize endogenous PTH 1 R-cAMP signaling, also increases bone mass. Transcriptomic analysis of tissue from treated animals suggests that conventional and arrestin pathway-selective PTH1R ligands act in largely different ways, with the latter principally affecting pathways involved in the regulation of cell cycle, survival, and migration/cytoskeletal dynamics. Such multi-dimensional in vitro and in vivo analyses of ligand bias may provide insights into the physiological roles of non-canonical arrestin-mediated signaling pathways in vivo, and provide a conceptual framework for translating arrestin pathway-selective ligands into viable therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Preparation and characterization of metoprolol tartrate containing matrix type transdermal drug delivery system.

PubMed

Malipeddi, Venkata Ramana; Awasthi, Rajendra; Ghisleni, Daniela Dal Molim; de Souza Braga, Marina; Kikuchi, Irene Satiko; de Jesus Andreoli Pinto, Terezinha; Dua, Kamal

2017-02-01

The present study aimed to develop matrix-type transdermal drug delivery system (TDDS) of metoprolol tartrate using polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA). The transdermal films were evaluated for physical parameters, Fourier transform infrared spectroscopy analysis (FTIR), differential scanning calorimetry (DSC), in vitro drug release, in vitro skin permeability, skin irritation test and stability studies. The films were found to be tough, non-sticky, easily moldable and possess good tensile strength. As the concentration of PVA was increased, the tensile strength of the films was also increased. Results of FTIR spectroscopy and DSC revealed the absence of any drug-polymer interactions. In vitro release of metoprolol followed zero-order kinetics and the mechanism of release was found to be diffusion rate controlled. In vitro release studies of metoprolol using Keshary-Chein (vertical diffusion cell) indicated 65.5 % drug was released in 24 h. In vitro skin permeation of metoprolol transdermal films showed 58.13 % of the drug was released after 24 h. In vitro skin permeation of metoprolol followed zero-order kinetics in selected formulations. The mechanism of release was found to be diffusion rate controlled. In a 22-day skin irritation test, tested formulation of transdermal films did not exhibit any allergic reactions, inflammation, or contact dermatitis. The transdermal films showed good stability in the 180-day stability study. It can be concluded that the TDDS of MPT can help in bypassing the first-pass effect and will provide patient improved compliance, without sacrificing the therapeutic advantages of the drugs.

Optimizing novel implant formulations for the prolonged release of biopharmaceuticals using in vitro and in vivo imaging techniques.

PubMed

Beyer, Susanne; Xie, Li; Schmidt, Mike; de Bruin, Natasja; Ashtikar, Mukul; Rüschenbaum, Sabrina; Lange, Christian M; Vogel, Vitali; Mäntele, Werner; Parnham, Michael J; Wacker, Matthias G

2016-08-10

As a rapidly growing class of therapeutics, biopharmaceuticals have conquered the global market. Despite the great potential from a therapeutic perspective, such formulations often require frequent injections due to their short half-life. Aiming to establish a parenteral dosage form with prolonged release properties, a biodegradable implant was developed, based on a combination of nanoencapsulation of protein-heparin complexes, creation of a slow release matrix by freeze-drying, and compression using hyaluronan and methylcellulose. In order to investigate this novel delivery system, formulations containing IFN-β-1a and trypsinogen as model proteins were developed. No degradation of the proteins was observed at any stage of the formulation processing. The potential of the delivery system was evaluated in vivo and in vitro after fluorescence-labeling of the biopharmaceuticals. An optimized agarose gel was utilized as in vitro release medium to simulate the subcutaneous environment in a biorelevant manner. In addition, the formulations were administered to female SJL mice and release was innovatively tracked by fluorescence imaging, setting up an in vitro-in vivo correlation. A prolonged time of residence of approximately 12days was observed for the selected formulation design. Copyright © 2016 Elsevier B.V. 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

Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro

NASA Astrophysics Data System (ADS)

Yoshida, Mari; Reyes, Sabrina Galiñanes; Tsuda, Soichiro; Horinouchi, Takaaki; Furusawa, Chikara; Cronin, Leroy

2017-06-01

Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using this data, we develop a simple deterministic model, which allows us to predict the fate of multi-drug evolution in this system. Furthermore, we are able to reverse established drug resistance based on the model prediction by modulating antibiotic selection stresses. Our results support the idea that the development of antibiotic resistance may be potentially controlled via continuous switching of drugs.

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.

Accelerated killing of cancer cells using a multifunctional single-walled carbon nanotube-based system for targeted drug delivery in combination with photothermal therapy.

PubMed

Jeyamohan, Prashanti; Hasumura, Takashi; Nagaoka, Yutaka; Yoshida, Yasuhiko; Maekawa, Toru; Kumar, D Sakthi

2013-01-01

The photothermal effect of single-walled carbon nanotubes (SWCNTs) in combination with the anticancer drug doxorubicin (DOX) for targeting and accelerated destruction of breast cancer cells is demonstrated in this paper. A targeted drug-delivery system was developed for selective killing of breast cancer cells with polyethylene glycol biofunctionalized and DOX-loaded SWCNTs conjugated with folic acid. In our work, in vitro drug-release studies showed that the drug (DOX) binds at physiological pH (pH 7.4) and is released only at a lower pH, ie, lysosomal pH (pH 4.0), which is the characteristic pH of the tumor environment. A sustained release of DOX from the SWCNTs was observed for a period of 3 days. SWCNTs have strong optical absorbance in the near-infrared (NIR) region. In this special spectral window, biological systems are highly transparent. Our study reports that under laser irradiation at 800 nm, SWCNTs exhibited strong light-heat transfer characteristics. These optical properties of SWCNTs open the way for selective photothermal ablation in cancer therapy. It was also observed that internalization and uptake of folate-conjugated NTs into cancer cells was achieved by a receptor-mediated endocytosis mechanism. Results of the in vitro experiments show that laser was effective in destroying the cancer cells, while sparing the normal cells. When the above laser effect was combined with DOX-conjugated SWCNTs, we found enhanced and accelerated killing of breast cancer cells. Thus, this nanodrug-delivery system, consisting of laser, drug, and SWCNTs, looks to be a promising selective modality with high treatment efficacy and low side effects for cancer therapy.

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

PubMed Central

Yoshida, Kimiko; Goto, Naoko; Ohnami, Shumpei; Aoki, Kazunori

2012-01-01

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:23029088

In vitro selection of shape-changing DNA nanostructures capable of binding-induced cargo release.

PubMed

Oh, Seung Soo; Plakos, Kory; Xiao, Yi; Eisenstein, Michael; Soh, H Tom

2013-11-26

Many biological systems employ allosteric regulatory mechanisms, which offer a powerful means of directly linking a specific binding event to a wide spectrum of molecular functionalities. There is considerable interest in generating synthetic allosteric regulators that can perform useful molecular functions for applications in diagnostics, imaging and targeted therapies, but generating such molecules through either rational design or directed evolution has proven exceptionally challenging. To address this need, we present an in vitro selection strategy for generating conformation-switching DNA nanostructures that selectively release a small-molecule payload in response to binding of a specific trigger molecule. As an exemplar, we have generated a DNA nanostructure that hybridizes with a separate 'cargo strand' containing an abasic site. This abasic site stably sequesters a fluorescent cargo molecule in an inactive state until the DNA nanostructure encounters an ATP trigger molecule. This ATP trigger causes the nanostructure to release the cargo strand, thereby liberating the fluorescent payload and generating a detectable fluorescent readout. Our DNA nanostructure is highly sensitive, with an EC50 of 30 μM, and highly specific, releasing its payload in response to ATP but not to other chemically similar nucleotide triphosphates. We believe that this selection approach could be generalized to generate synthetic nanostructures capable of selective and controlled release of other small-molecule cargos in response to a variety of triggers, for both research and clinical applications.

Selection, proliferation and differentiation of bone marrow-derived liver stem cells with a culture system containing cholestatic serum in vitro.

PubMed

Cai, Yun-Feng; Zhen, Zuo-Jun; Min, Jun; Fang, Tian-Ling; Chu, Zhong-Hua; Chen, Ji-Sheng

2004-11-15

To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission.

PubMed

Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

2015-11-11

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell-cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105-106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT-BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

PubMed Central

Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

2015-01-01

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105–106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies. PMID:26569290

The Lambda Select cII Mutation Detection System.

PubMed

Besaratinia, Ahmad; Tommasi, Stella

2018-04-26

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Precision-cut liver slices of Atlantic cod (Gadus morhua): an in vitro system for studying the effects of environmental contaminants.

PubMed

Eide, M; Karlsen, O A; Kryvi, H; Olsvik, P A; Goksøyr, A

2014-08-01

The Atlantic cod (Gadus morhua) is an economically important species commonly consumed by humans. The widespread distribution of cod in the North Atlantic Ocean makes it vulnerable to effluents from human activities, such as coastal industries and offshore petroleum exploration. It has been demonstrated that many effluents have adverse effects on cod reproduction and health, e.g. by disrupting endocrine signaling pathways. The liver, expressing important components of the biotransformation and the endocrine system, is one of the main target organs. Thus, reliable and reproducible in vitro systems of the liver are important for studying effects of environmental contaminants. The aim of this study was to investigate precision-cut liver slices (PCLS) as an alternative in vitro system for toxicological studies of the Atlantic cod liver. Slices of 8 mm in diameter and 250 μm thickness were prepared and cultivated from immature cod. Several analyses to measure the liver slice viability were performed: enzyme assays, histology, and morphometric analysis, all confirming cell viability for up to 72 h in culture. The liver slices were also exposed to two well-known model environmental contaminants, β-naphthoflavone (BNF) and 17α-ethynylestradiol (EE2), representing established agonists for the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER), respectively. The results showed increased transcription of the target genes cytochrome P450 1A (CYP1A) and vitellogenin (VTG), both well-established biomarkers for exposure of fish to the selected compounds. In conclusion, PCLS is a promising in vitro system for toxicological studies of cod liver cells. The liver slices are viable in culture for several days and respond to environmental contaminants in a dose- and time-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.

Radiation and chemical mutagen induced somaclonal variations through in vitro organogenesis of cotton (Gossypium hirsutum L.).

PubMed

Muthusamy, Annamalai; Jayabalan, Narayanasamy

2014-12-01

The purpose of the investigation was to induce somaclonal variations by gamma rays (GR), ethylmethane sulphonate (EMS) and sodium azide (SA) during in vitro organogenesis of cotton. The shoot tip explants were irradiated with 5-50 Gray (Gy) GR (Cobalt 60), 0.5-5.0 mM EMS and SA separately, and inoculated on Murashige and Skoog (MS) medium fortified with plant growth regulator (PGR) for organogenesis. The plantlets with well-developed root systems were acclimatized and transferred into the experimental field to screen the somaclonal variations during growth and development. The number of somaclonal variations was observed in growth of irradiated/treated shoot tips, multiplication, plantlet regeneration and growth in vitro and ex vitro. The lower doses/concentrations of mutagenic treatments showed significant enhancement in selected agronomical characters and they showed decreased trends with increasing doses/concentrations of mutagenic agents. The results of the present study revealed the influence of lower doses/concentrations of mutagenic treatments on in vitro and ex vitro growth of cotton plantlets and their significant improvement in agronomical characters which needs further imperative stability analysis. The present observations showed the platform to use lower doses/concentrations of mutagenic agents to induce variability for enhanced agronomical characters, resistant and tolerant cotton varieties.

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

One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system.

PubMed

Zhang, Xing; Wu, Hui; Huang, Bing; Li, Zhimin; Ye, Qin

2017-01-10

In vitro cascade catalysis using enzyme-based system is becoming a promising biomanufacturing platform for biofuels and biochemicals production. Glutathione is a pivotal non-protein thiol compound and widely applied in food and pharmaceutical industries. In this study, glutathione was synthesized by a bifunctional glutathione synthetase together with a thermophilic ATP regeneration system through a two-enzyme cascade in vitro. Four bifunctional glutathione synthetases from Streptococcus sanguinis, S. gordonii, S. uberis and Bacillus cereus were applied for glutathione synthesis. The bifunctional glutathione synthetase from S. sanguinis was selected and coupled with the polyphosphate kinase from Thermosynechococcus elongatus BP-1 for regenerating ATP to produce glutathione in one pot. In the optimized system, 28.5mM glutathione was produced within 5h due to efficient ATP regeneration from low-cost polyphosphate. The yield based on added l-cysteine reached 81.4% and the productivity of glutathione achieved 5.7mM/h. The one-pot system indicated a potential biotransformation platform for industrial production of glutathione. Copyright © 2016 Elsevier B.V. All rights reserved.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2008-02-12

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2012-01-31

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

PubMed Central

Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

2016-01-01

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

Selection of a discriminant and biorelevant in vitro dissolution test for the development of fenofibrate self-emulsifying lipid-based formulations.

PubMed

Pestieau, Aude; Krier, Fabrice; Brouwers, Adeline; Streel, Bruno; Evrard, Brigitte

2016-09-20

Fenofibrate, a BCS class II compound, has a low bioavailability especially when taken orally on an empty stomach. The challenge to find a new formulation for providing bioavailability, independent of food, is still ongoing. If the development of a suitable oral delivery formulation of BCS class II compounds is a frequent and great challenge to formulation scientists, the in vitro evaluation of these new formulations is also a great challenge. The purpose of this study was therefore to select an in vitro dissolution test that would be useful and as biorelevant as possible for the development of fenofibrate self-emulsifying lipid-based formulations. In this context, three different fenofibrate formulations, for which in vivo data are available in the literature, were tested using different dissolution tests until we found the one that was the most suitable. As part of this approach, we started with the simplest in vitro dissolution tests and progressed to tests that were increasingly more complex. The first tests were different single phase dissolution tests: a test under sink conditions based on the USP monograph, and different tests under non-sink conditions in non-biorelevant and biorelevant media. Given the inconclusive results obtained with these tests, biphasic dissolution systems were then tested: one with USP apparatus type II alone and another which combined USP apparatus types II and IV. This last combined test seemed the most suitable in vitro dissolution test for the development of the future fenofibrate lipid-based formulations we intend to develop in our own laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Boronate-Based Fluorescent Probes: Imaging Hydrogen Peroxide in Living Systems

PubMed Central

Lin, Vivian S.; Dickinson, Bryan C.; Chang, Christopher J.

2014-01-01

Hydrogen peroxide, a reactive oxygen species with unique chemical properties, is produced endogenously in living systems as a destructive oxidant to ward off pathogens or as a finely tuned second messenger in dynamic cellular signaling pathways. In order to understand the complex roles that hydrogen peroxide can play in biological systems, new tools to monitor hydrogen peroxide in its native settings, with high selectivity and sensitivity, are needed. Knowledge of organic synthetic reactivity provides the foundation for the molecular design of selective, functional hydrogen peroxide probes. A palette of fluorescent and luminescent probes that react chemoselectively with hydrogen peroxide has been developed, utilizing a boronate oxidation trigger. These indicators offer a variety of colors and in cellulo characteristics and have been used to examine hydrogen peroxide in a number of experimental setups, including in vitro fluorometry, confocal fluorescence microscopy, and flow cytometry. In this chapter, we provide an overview of the chemical features of these probes and information on their behavior to help researchers select the optimal probe and application. PMID:23791092

Flow cytometric investigations of diploid and tetraploid plants and in vitro cultures of Datura stramonium and Hyoscyamus niger.

PubMed

Weber, Jost; Georgiev, Vasil; Pavlov, Atanas; Bley, Thomas

2008-10-01

Plant in vitro systems are valuable sources for the production of biological active substances. However, changed profiles of secondary metabolites, and low, variable yields possibly caused by genetic instabilities complicate their industrial implementation. DNA profiling of plant in vitro cultures may provide data for the selection of highly producing in vitro cultures. Diploid and tetraploid Datura stramonium and Hyoscyamus niger plant as well as calli, and hairy root lines derived from them were analyzed by flow cytometry. Plant in vitro cultures undergo several cycles of endoreduplication more than the explants from which they were obtained. The highest cycle values were observed in calli (e.g. 1.19 for diploid H. niger) possibly induced by the growth factors. However, hairy roots cultivated without growth factor exhibited significant degrees of endoreduplication (cycle value 0.88 for diploid H. niger). Sets of five hairy root lines from each plant and ploidy level showed consistent within-set ploidy patterns. The ploidy profiles of investigated plant in vitro and in vivo differ. For the first time we report that hairy roots of two Solanaceae species undergo endoreduplication. Theploidy profiles of in vitro cultures (hairy roots and calli) seem to be influenced by the genome size, the growth factors applied, and the type of in vitro culture. The transformation of several hairy root lines showed no differences in the ploidy patterns. Copyright 2008 International Society for Advancement of Cytometry.

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.

PubMed

Jakobsson, Lars; Franco, Claudio A; Bentley, Katie; Collins, Russell T; Ponsioen, Bas; Aspalter, Irene M; Rosewell, Ian; Busse, Marta; Thurston, Gavin; Medvinsky, Alexander; Schulte-Merker, Stefan; Gerhardt, Holger

2010-10-01

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

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.

Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations.

PubMed

Hullahalli, Karthik; Rodrigues, Marinelle; Palmer, Kelli L

2017-06-23

CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis , which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.

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.

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

Modified Dietary Fiber from Cassava Pulp and Assessment of Mercury Bioaccessibility and Intestinal Uptake Using an In Vitro Digestion/Caco-2 Model System.

PubMed

Kachenpukdee, Natta; Santerre, Charles R; Ferruzzi, Mario G; Oonsivilai, Ratchadaporn

2016-07-01

The ability of modified dietary fiber (MDF) generated from cassava pulp to modulate the bioaccessibility and intestinal absorption of heavy metals may be helpful to mitigate health risk associated with select foods including select fish high in methyl mercury. Using a coupled in vitro digestion/Caco-2 human intestinal cell model, the reduction of fish mercury bioaccessibility and intestinal uptake by MDF was investiaged. MDF was prepared from cassava pulp, a byproduct of tapioca production. The highest yield (79.68%) of MDF was obtained by enzymatic digestion with 0.1% α-amylase (w/v), 0.1% amyloglucosidase (v/v) and 1% neutrase (v/v). MDF and fish tissue were subjected to in vitro digestion and results suggest that MDF may reduce mercury bioaccessibility from fish to 34% to 85% compared to control in a dose-dependent manner. Additionally, accumulation of mercury from digesta containing fish and MDF was only modestly impacted by the presence of MDF. In conclusion, MDF prepared from cassava pulp may be useful as an ingredient to reduce mercury bioavailability from food such as fish specifically by inhibiting mercury transfer to the bioaccessibile fraction during digestion. © 2016 Institute of Food Technologists®

In vitro and in vivo antiviral activity of 2'-fluorinated arabinosides of 5-(2-haloalkyl)uracil.

PubMed

Rosenwirth, B; Streicher, W; De Clercq, E; Wanek, E; Schwarz, W; Griengl, H

1987-06-01

5-(2-Fluoroethyl)-2'-deoxyuridine (FEDU), its 2'-fluoroarabinofuranosyl analog (FEFAU) and the 2'-fluoroarabinofuranosyl analog (CEFAU) of the potent anti-herpesvirus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU) were evaluated for activity against herpes simplex virus type 1 (HSV-1) and HSV-2 in vitro and in vivo. FEDU, FEFAU and CEFAU proved to be potent and selective anti-herpesvirus agents in vitro. Their potency is evident from their low minimum inhibitory concentrations for HSV-1 and HSV-2, and their selectivity is attested by the marginal inhibition of cell proliferation at relatively high concentrations, and by the high concentrations at which DNA-, RNA- or protein synthesis in normal uninfected host cells is inhibited. Their activity spectrum is broader than that of CEDU: in addition to being highly effective against HSV-1 replication, these derivatives, in particular FEFAU, inhibit HSV-2 replication at concentrations comparable to acyclovir (ACV). In the systemic and cutaneous HSV-1 infection models in mice, FEDU, FEFAU and CEFAU were markedly less potent than CEDU in suppressing the development of lesions and in reducing the mortality rate. In HSV-2 infections in mice and in guinea pigs FEDU, FEFAU and CEFAU were virtually ineffective. CEDU, however, exerted a protective effect in these animal models, albeit at relatively high concentrations.

Formulation of microspheres containing Crataegus monogyna Jacq. extract with free radical scavenging activity.

PubMed

Lucconi, Giulia; Chlapanidas, Theodora; Martino, Emanuela; Gaggeri, Raffaella; Perteghella, Sara; Rossi, Daniela; Faragò, Silvio; Vigo, Daniele; Faustini, Massimo; Collina, Simona; Torre, Maria Luisa

2014-02-01

Extracts of Crataegus monogyna Jacq. (hawthorn) show an interesting free radical scavenging (FRS) effect, related to their flavonoids content. Unfortunately, their oral administration is affected by their low bioavailability. The aim of this work is to obtain a multiparticulate drug delivery system for hawthorn extracts for oral administration. The extracts from flowering tops (FL) or fruits (FR) of hawthorn were obtained with maceration, using ethanol as an extraction solvent, and their antioxidant activity was evaluated. FL extract showed the highest FRS activity (EC50 3.72 ± 1.21 µg/ml), so it was selected to prepare microparticulate systems by a spray-drying technique, which were characterized by granulometric analysis, scanning electron microscopy-energy dispersive X-ray spectroscopy, confocal fluorescence microscopy and hyperoside content. Antioxidant activity was evaluated before and after gastrointestinal transit in vitro simulation. Results indicate that the microparticulate systems maintained the antioxidant activity of hawthorn also after gastrointestinal transit in vitro simulation, exhibiting properties suitable for oral administration.

Phage and Yeast Display.

PubMed

Sheehan, Jared; Marasco, Wayne A

2015-02-01

Despite the availability of antimicrobial drugs, the continued development of microbial resistance--established through escape mutations and the emergence of resistant strains--limits their clinical utility. The discovery of novel, therapeutic, monoclonal antibodies (mAbs) offers viable clinical alternatives in the treatment and prophylaxis of infectious diseases. Human mAb-based therapies are typically nontoxic in patients and demonstrate high specificity for the intended microbial target. This specificity prevents negative impacts on the patient microbiome and avoids driving the resistance of nontarget species. The in vitro selection of human antibody fragment libraries displayed on phage or yeast surfaces represents a group of well-established technologies capable of generating human mAbs. The advantage of these forms of microbial display is the large repertoire of human antibody fragments present during a single selection campaign. Furthermore, the in vitro selection environments of microbial surface display allow for the rapid isolation of antibodies--and their encoding genes--against infectious pathogens and their toxins that are impractical within in vivo systems, such as murine hybridomas. This article focuses on the technologies of phage display and yeast display, as these strategies relate to the discovery of human mAbs for the treatment and vaccine development of infectious diseases.

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.

Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

PubMed

Petrie, Katherine L; Joyce, Gerald F

2014-10-01

The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

Design of composite microparticle systems based on pectin and waste material of propolis for modified l-alanyl-l-glutamine release and with immunostimulant activity.

PubMed

Villa Nova, Mônica; Ratti, Bianca A; Herculano, Leandro S; Bittencourt, Paulo R S; Novello, Cláudio R; Bazotte, Roberto Barbosa; Lautenschlager, Sueli de Oliveira Silva; Bruschi, Marcos Luciano

2017-12-12

Catabolic conditions like acquired immunodeficiency syndrome, cancer, and burn can cause immunosuppression. Amino acids such as alanine and glutamine are essential for the activity of the immune system. Propolis is immunostimulant and the waste of propolis extraction has been reused with technological and therapeutic purposes. Therefore, this study describes the association of propolis byproduct extract (BPE) with pectin to prepare spray-dried microparticles containing the dipeptide l-alanyl-l-glutamine as stimulant systems of neutrophils. The use of a factorial design allowed selecting the best formulation, which was characterized by morphology, size, and entrapment efficiency analyses. In addition, the systems were characterized by thermal and X-ray diffraction analysis, Fourier-transform infrared spectroscopy, in vitro drug release, and in vitro cytotoxicity and stimulation test of neutrophils. Small well-structured microparticles with good entrapment efficiency values were achieved. Thermal stability of formulation was observed, and it was proved that pectin, BPE and l-alanyl-l-glutamine were dispersed throughout the matrix. The drug was released from the microparticles during 24 h governed by swelling and diffusion. The drug-loaded formulations showed a significant stimulating effect on neutrophils. These structures could increase the activity of immune cells, and other in vitro and in vivo studies should be performed in the future.

Guidance on Nanomaterial Hazards and Risks

DTIC Science & Technology

2015-05-21

and at room temperature and 37 C°– solid separation by centrifugation, filtration , or chemical techniques (more experimental techniques combining...members in this potency sequence using bolus in vivo testing, verify the bolus results with selective inhalation testing. The potency of members of...measures in in vitro and limited in vivo experimental systems would facilitate the characterization of dose-response relationships across a set of ENMs

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

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...

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.

A genetic replacement system for selection-based engineering of essential proteins

PubMed Central

2012-01-01

Background Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches. Results We established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation. The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains. Conclusions We propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies. PMID:22898007

Biotransformation of prednisone and dexamethasone by cytochrome P450 based systems - Identification of new potential drug candidates.

PubMed

Putkaradze, Natalia; Kiss, Flora Marta; Schmitz, Daniela; Zapp, Josef; Hutter, Michael C; Bernhardt, Rita

2017-01-20

Prednisone and dexamethasone are synthetic glucocorticoids widely used as anti-inflammatory and immunosuppressive drugs. Since their hydroxylated derivatives could serve as novel potential drug candidates, our aim was to investigate their biotransformation by the steroid hydroxylase CYP106A2 from Bacillus megaterium ATCC13368. In vitro we were able to demonstrate highly selective 15β-hydroxylation of the steroids with a reconstituted CYP106A2 system. The reactions were thoroughly characterized, determining the kinetic parameters and the equilibrium dissociation constant. The observed lower conversion rate in the case of dexamethasone hydroxylation was clarified by quantum chemical calculations, which suggest a rearrangement of the intermediately formed radical species. To identify the obtained conversion products with NMR, CYP106A2-based Bacillus megaterium whole-cell systems were applied resulting in an altered product pattern for prednisone, yet no significant change for dexamethasone conversion compared to in vitro. Even the MS941 control strain performed a highly selective biotransformation of prednisone producing the known metabolite 20β-dihydrocortisone. The identified novel prednisone derivatives 15β, 17, 20β, 21-tetrahydroxy-preg-4-en-3,11-dione and 15β, 17, 20β, 21-tetrahydroxy-preg-1,4-dien-3,11-dione as well as the 15β-hydroxylated variants of both drugs are promising candidates for drug-design and development approaches. Copyright © 2016 Elsevier B.V. All rights reserved.

A framework for in vitro systems toxicology assessment of e-liquids

PubMed Central

Iskandar, Anita R.; Gonzalez-Suarez, Ignacio; Majeed, Shoaib; Marescotti, Diego; Sewer, Alain; Xiang, Yang; Leroy, Patrice; Guedj, Emmanuel; Mathis, Carole; Schaller, Jean-Pierre; Vanscheeuwijck, Patrick; Frentzel, Stefan; Martin, Florian; Ivanov, Nikolai V.; Peitsch, Manuel C.; Hoeng, Julia

2016-01-01

Abstract Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air–liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols. PMID:27117495

A framework for in vitro systems toxicology assessment of e-liquids.

PubMed

Iskandar, Anita R; Gonzalez-Suarez, Ignacio; Majeed, Shoaib; Marescotti, Diego; Sewer, Alain; Xiang, Yang; Leroy, Patrice; Guedj, Emmanuel; Mathis, Carole; Schaller, Jean-Pierre; Vanscheeuwijck, Patrick; Frentzel, Stefan; Martin, Florian; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia

2016-07-01

Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air-liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

PubMed

Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

2015-10-16

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

Transdermal delivery of alprazolam from a monolithic patch: formulation based on in vitro characterization.

PubMed

Soler, L I; Boix, A; Lauroba, J; Colom, H; Domenech, J

2012-10-01

Alprazolam, a benzodiazepine widely used for the treatment of psychiatric disorders, has been aimed to be formulated in a transdermal delivery system (TDS) prototype. A series of TDS prototypes dosed in all cases at 0.35 mg·cm(-2) of alprazolam were prepared as a monolithic drug in adhesive matrix using acrylic pressure-sensitive adhesives (PSA) of acrylate vinyl acetate (Duro-tack(®)). The effects of several permeation enhancers as azone, transcutol, propylene glycol, dodecyl alcohol, decyl alcohol, diethanolamine, N-methyl pyrrolidone and lauric acid were studied. Prototypes have been characterized based on adhesion parameters (peel adhesion and shear adhesion), in vitro human skin permeation and in vitro drug release according to European Pharmacopoeia for the selected prototype. Best results show that a combination of permeation enhancers from different chemical groups is able to provide almost a 33 fold increase in the transdermal alprazolam flux of an aqueous saturated dispersion (from 0.054 ± 0.019 to 1.76 ± 0.21 μg h.cm(-2)). Based on these in vitro flux data, a predictive simulation of the achievable plasmatic levels was performed assuming a constant systemic infusion of drug. In summary, it is possible to obtain a prototype of a TDS of alprazolam with adequate adhesive properties (peel adhesion and shear adhesion) and able to predict sustained therapeutic plasmatic levels.

Selection of genes of Mycobacterium tuberculosis upregulated during residence in lungs of infected mice.

PubMed

Srivastava, Vikas; Jain, Anamika; Srivastava, Brahm S; Srivastava, Ranjana

2008-05-01

In sequel to previous report [Srivastava V, Rouanet C, Srivastava R, Ramalingam B, Locht C, Srivastava BS. Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection. Microbiology 2007;153:659-66], the genes of Mycobacterium tuberculosis upregulated during residence in lungs of infected mice were identified in an in vivo expression system based on kanamycin resistance. A promoter library of M. tuberculosis was constructed in a promoter trap shuttle vector pLL192 containing an artificial bicistronic operon composed of promoterless green fluorescent protein gene followed by kanamycin resistance gene. The library was introduced in M. bovis BCG and then infected in mice by intravenous route. Mice were treated twice daily with 40 mg/kg dose of kanamycin by intramuscular route for 21 days. Recombinant BCG recovered from the lungs were reinfected in mice to enrich clones surviving kanamycin treatment in the lung but sensitive to killing by kanamycin in vitro. After nucleotide sequencing of inserts from these clones, 20 genes belonging to fatty acids metabolism, membrane transport, nitric oxide defence and PE_PGRS/PPE family were identified. Real-time PCR analysis using RNA isolated from M. tuberculosis grown in vitro and from the lungs, confirmed upregulation of genes from 2 to 20-fold in vivo compared to growth in vitro. Several of these select 20 genes were also found upregulated ex vivo in macrophage-like cell line J774A.1, thus, suggesting a correlation in mycobacterial gene expression between ex vivo and in vivo conditions.

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

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...

Research in drug development against viral diseases of military importance (biological testing). Volume 1. Final report, 15 November 1985-31 January 1991

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

Shannon, W.M.; Arnett, G.; Brazier, A.D.

1991-03-01

The purpose of this program is to evaluate the efficacy of candidate antiviral compounds against a spectrum of viruses of military importance. This program involves (a) primary testing of chemical compounds and natural products for antiviral efficacy in vitro using standard CPE-inhibition assays, (b) primary testing of compounds for antiviral efficacy in vivo in animal model systems, and (c) secondary evaluation of the active candidate antiviral compounds. The target viruses for in vitro testing are Vaccinia Virus (VV), Adenovirus (AD2), Vesicular Stomatitis Virus (VSV), Punta Toro Virus (PT), Sandfly Fever Virus (SF), Yellow Fever Virus (YF), Venezuelan Equine Encephalomyelitis Virusmore » (VE), Japanese Encephalitis Virus and Vaccinia Virus infections of mice. Approximately 10,000 compounds have been received for in vitro evaluation and over 66,000 assays have been performed on this contract. Compounds have been identified in nearly all virus systems that have confirmed antiviral activity equal or exceeding that of the various positive control compounds (Ribavirin, Selenazofurin, Carbocyclic-3-deaza-adenosine, Adenosine dialdehyde, Ara-A, ddC and AZT). Many of these compounds represent potent and selective new antiviral agents.« less

Systems Toxicology: Real World Applications and Opportunities.

PubMed

Hartung, Thomas; FitzGerald, Rex E; Jennings, Paul; Mirams, Gary R; Peitsch, Manuel C; Rostami-Hodjegan, Amin; Shah, Imran; Wilks, Martin F; Sturla, Shana J

2017-04-17

Systems Toxicology aims to change the basis of how adverse biological effects of xenobiotics are characterized from empirical end points to describing modes of action as adverse outcome pathways and perturbed networks. Toward this aim, Systems Toxicology entails the integration of in vitro and in vivo toxicity data with computational modeling. This evolving approach depends critically on data reliability and relevance, which in turn depends on the quality of experimental models and bioanalysis techniques used to generate toxicological data. Systems Toxicology involves the use of large-scale data streams ("big data"), such as those derived from omics measurements that require computational means for obtaining informative results. Thus, integrative analysis of multiple molecular measurements, particularly acquired by omics strategies, is a key approach in Systems Toxicology. In recent years, there have been significant advances centered on in vitro test systems and bioanalytical strategies, yet a frontier challenge concerns linking observed network perturbations to phenotypes, which will require understanding pathways and networks that give rise to adverse responses. This summary perspective from a 2016 Systems Toxicology meeting, an international conference held in the Alps of Switzerland, describes the limitations and opportunities of selected emerging applications in this rapidly advancing field. Systems Toxicology aims to change the basis of how adverse biological effects of xenobiotics are characterized, from empirical end points to pathways of toxicity. This requires the integration of in vitro and in vivo data with computational modeling. Test systems and bioanalytical technologies have made significant advances, but ensuring data reliability and relevance is an ongoing concern. The major challenge facing the new pathway approach is determining how to link observed network perturbations to phenotypic toxicity.

Systems Toxicology: Real World Applications and Opportunities

PubMed Central

2017-01-01

Systems Toxicology aims to change the basis of how adverse biological effects of xenobiotics are characterized from empirical end points to describing modes of action as adverse outcome pathways and perturbed networks. Toward this aim, Systems Toxicology entails the integration of in vitro and in vivo toxicity data with computational modeling. This evolving approach depends critically on data reliability and relevance, which in turn depends on the quality of experimental models and bioanalysis techniques used to generate toxicological data. Systems Toxicology involves the use of large-scale data streams (“big data”), such as those derived from omics measurements that require computational means for obtaining informative results. Thus, integrative analysis of multiple molecular measurements, particularly acquired by omics strategies, is a key approach in Systems Toxicology. In recent years, there have been significant advances centered on in vitro test systems and bioanalytical strategies, yet a frontier challenge concerns linking observed network perturbations to phenotypes, which will require understanding pathways and networks that give rise to adverse responses. This summary perspective from a 2016 Systems Toxicology meeting, an international conference held in the Alps of Switzerland, describes the limitations and opportunities of selected emerging applications in this rapidly advancing field. Systems Toxicology aims to change the basis of how adverse biological effects of xenobiotics are characterized, from empirical end points to pathways of toxicity. This requires the integration of in vitro and in vivo data with computational modeling. Test systems and bioanalytical technologies have made significant advances, but ensuring data reliability and relevance is an ongoing concern. The major challenge facing the new pathway approach is determining how to link observed network perturbations to phenotypic toxicity. PMID:28362102

In vitro-in vivo correlation in skin permeation.

PubMed

Mohammed, D; Matts, P J; Hadgraft, J; Lane, M E

2014-02-01

In vitro skin permeation studies have been used extensively in the development and optimisation of delivery of actives in vivo. However, there are few reported correlations of such in vitro studies with in vivo data. The aim of this study was to investigate the skin permeation of a model active, niacinamide, both in vitro and in vivo. Conventional diffusion cell studies were conducted in human skin to determine niacinamide permeation from a range of vehicles which included dimethyl isosorbide (DMI), propylene glycol (PG), propylene glycol monolaurate (PGML), N-methyl 2-pyrrolidone (NMP), Miglyol 812N® (MG), and mineral oil (MO). Single, binary or ternary systems were examined. The same vehicles were subsequently examined to investigate niacinamide delivery in vivo. For this proof-of-concept study one donor was used for the in vitro studies and one volunteer for the in vivo investigations to minimise biovariability. Analysis of in vitro samples was conducted using HPLC and in vivo uptake of niacinamide was evaluated using Confocal Raman spectroscopy (CRS). The amount of niacinamide permeated through skin in vitro was linearly proportional to the intensity of the niacinamide signal determined in the stratum corneum in vivo. A good correlation was observed between the signal intensities of selected vehicles and niacinamide signal intensity. The findings provide further support for the use of CRS to monitor drug delivery into and across the skin. In addition, the results highlight the critical role of the vehicle and its disposition in skin for effective dermal delivery.

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

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

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations. To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing.

Rabies virus quasispecies: Implications for pathogenesis

PubMed Central

Morimoto, Kinjiro; Hooper, D. Craig; Carbaugh, Heather; Fu, Zhen Fang; Koprowski, Hilary; Dietzschold, Bernhard

1998-01-01

Passage of the mouse-adapted rabies virus strain CVS-24 (where CVS is challenge virus standard) in BHK cells results in the rapid selection of a dominant variant designated CVS-B2c that differs genotypically and phenotypically from the dominant variant CVS-N2c present in mouse-brain- or neuroblastoma-cell-passaged CVS-24. The glycoprotein of CVS-B2c has 10 amino acid substitutions compared with that of CVS-N2c. Because CVS-B2c can be reproducibly selected in BHK cells, it is likely to be a conserved minor subpopulation of CVS-24. CVS-N2c is more neurotropic in vitro and in vivo than CVS-B2c, which replicates more readily in nonneuronal cells in vitro and in vivo. These characteristics appear to be relevant to the pathogenicity of the two variants. CVS-N2c is more pathogenic for adult mice than CVS-B2c. In contrast, CVS-B2c is more pathogenic for neonatal mice. These differences in pathogenicity are reflected in the selection pattern when mixtures of CVS-N2c and CVS-B2c were used to infect neonatal and adult mice. Although CVS-N2c was highly selected in adult mice, no selection for either variant was seen in neonates, suggesting that certain aspects of development, such as maturation of the nervous and immune systems, may contribute to the selection process. We speculate that the existence of different variants within a rabies virus strain may facilitate the virus in overcoming barriers to its spread, both within the host and between species. PMID:9501231

Rabies virus quasispecies: implications for pathogenesis.

PubMed

Morimoto, K; Hooper, D C; Carbaugh, H; Fu, Z F; Koprowski, H; Dietzschold, B

1998-03-17

Passage of the mouse-adapted rabies virus strain CVS-24 (where CVS is challenge virus standard) in BHK cells results in the rapid selection of a dominant variant designated CVS-B2c that differs genotypically and phenotypically from the dominant variant CVS-N2c present in mouse-brain- or neuroblastoma-cell-passaged CVS-24. The glycoprotein of CVS-B2c has 10 amino acid substitutions compared with that of CVS-N2c. Because CVS-B2c can be reproducibly selected in BHK cells, it is likely to be a conserved minor subpopulation of CVS-24. CVS-N2c is more neurotropic in vitro and in vivo than CVS-B2c, which replicates more readily in nonneuronal cells in vitro and in vivo. These characteristics appear to be relevant to the pathogenicity of the two variants. CVS-N2c is more pathogenic for adult mice than CVS-B2c. In contrast, CVS-B2c is more pathogenic for neonatal mice. These differences in pathogenicity are reflected in the selection pattern when mixtures of CVS-N2c and CVS-B2c were used to infect neonatal and adult mice. Although CVS-N2c was highly selected in adult mice, no selection for either variant was seen in neonates, suggesting that certain aspects of development, such as maturation of the nervous and immune systems, may contribute to the selection process. We speculate that the existence of different variants within a rabies virus strain may facilitate the virus in overcoming barriers to its spread, both within the host and between species.

Selection of new lactic acid bacteria strains bearing probiotic features from mucosal microbiota of healthy calves: Looking for immunobiotics through in vitro and in vivo approaches for immunoprophylaxis applications.

PubMed

Sandes, Sávio; Alvim, Luige; Silva, Bruno; Acurcio, Leonardo; Santos, Cinara; Campos, Márcia; Santos, Camila; Nicoli, Jacques; Neumann, Elisabeth; Nunes, Álvaro

2017-07-01

From the birth, since their mucosal microbiota and immune system are not fully developed, newborn calves are susceptible to several mucosal pathogenic microorganisms. Operating through humoral and non-humoral mechanisms in the host, several lactic acid bacteria strains bearing probiotic features are often employed in livestock as food supplement, improving animal production performance, promoting health and reducing the severity of mucosal infections. Accordingly, we isolated, species-level identified and screened for their probiotic potentials seventy lactic acid bacteria strains from upper airway, vaginal and intestinal mucosa of healthy calves. Based on in vitro approaches, we selected three strains: Lactobacillus fermentum V3B-08 isolated from upper airway mucosa, Weissella hellenica V1V-30 isolated from vaginal mucosa and Lactobacillus farciminis B4F-06 isolated from intestinal mucosa were used to mono-colonize germ-free mice in the same site in which these strains were isolated, aiming to characterize their immunomodulatory features. These strains were able to colonize germ-free mice mucosa and trigger sIgA synthesis at a local level, in addition to stimulating, in different ways, adaptive immune responses at a systemic level. Copyright © 2017 Elsevier GmbH. All rights reserved.

Pharmacophore modeling for identification of anti-IGF-1R drugs and in-vitro validation of fulvestrant as a potential inhibitor

PubMed Central

Hanif, Rumeza; Jabeen, Ishrat; Mansoor, Qaisar; Ismail, Muhammad

2018-01-01

Insulin-like growth factor 1 receptor (IGF-1R) is an important therapeutic target for breast cancer treatment. The alteration in the IGF-1R associated signaling network due to various genetic and environmental factors leads the system towards metastasis. The pharmacophore modeling and logical approaches have been applied to analyze the behaviour of complex regulatory network involved in breast cancer. A total of 23 inhibitors were selected to generate ligand based pharmacophore using the tool, Molecular Operating Environment (MOE). The best model consisted of three pharmacophore features: aromatic hydrophobic (HyD/Aro), hydrophobic (HyD) and hydrogen bond acceptor (HBA). This model was validated against World drug bank (WDB) database screening to identify 189 hits with the required pharmacophore features and was further screened by using Lipinski positive compounds. Finally, the most effective drug, fulvestrant, was selected. Fulvestrant is a selective estrogen receptor down regulator (SERD). This inhibitor was further studied by using both in-silico and in-vitro approaches that showed the targeted effect of fulvestrant in ER+ MCF-7 cells. Results suggested that fulvestrant has selective cytotoxic effect and a dose dependent response on IRS-1, IGF-1R, PDZK1 and ER-α in MCF-7 cells. PDZK1 can be an important inhibitory target using fulvestrant because it directly regulates IGF-1R. PMID:29787591

Pharmacophore modeling for identification of anti-IGF-1R drugs and in-vitro validation of fulvestrant as a potential inhibitor.

PubMed

Khalid, Samra; Hanif, Rumeza; Jabeen, Ishrat; Mansoor, Qaisar; Ismail, Muhammad

2018-01-01

Insulin-like growth factor 1 receptor (IGF-1R) is an important therapeutic target for breast cancer treatment. The alteration in the IGF-1R associated signaling network due to various genetic and environmental factors leads the system towards metastasis. The pharmacophore modeling and logical approaches have been applied to analyze the behaviour of complex regulatory network involved in breast cancer. A total of 23 inhibitors were selected to generate ligand based pharmacophore using the tool, Molecular Operating Environment (MOE). The best model consisted of three pharmacophore features: aromatic hydrophobic (HyD/Aro), hydrophobic (HyD) and hydrogen bond acceptor (HBA). This model was validated against World drug bank (WDB) database screening to identify 189 hits with the required pharmacophore features and was further screened by using Lipinski positive compounds. Finally, the most effective drug, fulvestrant, was selected. Fulvestrant is a selective estrogen receptor down regulator (SERD). This inhibitor was further studied by using both in-silico and in-vitro approaches that showed the targeted effect of fulvestrant in ER+ MCF-7 cells. Results suggested that fulvestrant has selective cytotoxic effect and a dose dependent response on IRS-1, IGF-1R, PDZK1 and ER-α in MCF-7 cells. PDZK1 can be an important inhibitory target using fulvestrant because it directly regulates IGF-1R.

Drawbacks of the ancient RNA-based life-like system under primitive earth conditions.

PubMed

Kawamura, Kunio

2012-07-01

Following the discovery of ribozymes, the "RNA world" hypothesis has become the most accepted hypothesis concerning the origin of life and genetic information. However, this hypothesis has several drawbacks. Verification of the hypothesis from different viewpoints led us to proposals from the viewpoint of the hydrothermal origin of life, solubility of RNA and related biopolymers, and the possibility of creating an evolutionary system comparable to the in vitro selection technique for functional RNA molecules based on molecular biology. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Nanostructured lipid system as a strategy to improve the anti-Candida albicans activity of Astronium sp.

PubMed Central

Bonifácio, Bruna Vidal; Ramos, Matheus Aparecido dos Santos; da Silva, Patrícia Bento; Negri, Kamila Maria Silveira; de Oliveira Lopes, Érica; de Souza, Leonardo Perez; Vilegas, Wagner; Pavan, Fernando Rogério; Chorilli, Marlus; Bauab, Taís Maria

2015-01-01

The genus Astronium (Anacardiaceae) includes species, such as Astronium fraxinifolium, Astronium graveolens, and Astronium urundeuva, which possess anti-inflammatory, anti-ulcerogenic, healing, and antimicrobial properties. Nanostructured lipid systems are able to potentiate the action of plant extracts, reducing the required dose and side effects and improving antimicrobial activity. This work aims to evaluate a nanostructured lipid system that was developed as a strategy to improve the anti-Candida albicans activity of hydroethanolic extracts of stems and leaves from Astronium sp. The antifungal activity against C. albicans (ATCC 18804) was evaluated in vitro by a microdilution technique. In addition to the in vitro assays, the Astronium sp. that showed the best antifungal activity and selectivity index was submitted to an in vivo assay using a model of vulvovaginal candidiasis infection. In these assays, the extracts were either used alone or were incorporated into the nanostructured lipid system (comprising 10% oil phase, 10% surfactant, and 80% aqueous phase). The results indicated a minimal inhibitory concentration of 125.00 µg/mL before incorporation into the nanostructured system; this activity was even more enhanced when this extract presented a minimal inhibitory concentration of 15.62 µg/mL after its incorporation. In vivo assay dates showed that the nanostructure-incorporated extract of A. urundeuva leaves was more effective than both the unincorporated extract and the antifungal positive control (amphotericin B). These results suggest that this nanostructured lipid system can be used in a strategy to improve the in vitro and in vivo anti-C. albicans activity of hydroethanolic extracts of Astronium sp. PMID:26300640

Screening and selection of artificial riboswitches.

PubMed

Harbaugh, Svetlana V; Martin, Jennifer; Weinstein, Jenna; Ingram, Grant; Kelley-Loughnane, Nancy

2018-05-17

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system. Copyright © 2018. Published by Elsevier Inc.

Sex in a test tube: testing the benefits of in vitro recombination.

PubMed

Pesce, Diego; Lehman, Niles; de Visser, J Arjan G M

2016-10-19

The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'. © 2016 The Author(s).

Toward global standards for comparator pharmaceutical products: case studies of amoxicillin, metronidazole, and zidovudine in the Americas.

PubMed

Löbenberg, Raimar; Chacra, Nadia B; Stippler, Erika S; Shah, Vinod P; DeStefano, Anthony J; Hauck, Walter W; Williams, Roger L

2012-09-01

This study compared in vitro dissolution characteristics and other quality measures of different amoxicillin, metronidazole, and zidovudine products purchased in the Americas to a comparator pharmaceutical product (CPP). These three drugs are classified as Biopharmaceutics Classification System Class I drugs with the possibility that dissolution findings might be used to document bioequivalence. All investigated zidovudine products were found to be in vitro equivalent to the CPP. Only 3 of 12 tested amoxicillin products were found to be in vitro equivalent to the CPP. None of the tested metronidazole products were in vitro equivalent to the CPP. These findings suggest but do not confirm bioinequivalence where in vitro comparisons failed, given that an in vivo blood level study might have confirmed bioequivalence. At times, identifying a CPP in one of the selected markets proved difficult. The study demonstrates that products sold across national markets may not be bioequivalent. When coupled with the challenge of identifying a CPP in different countries, the results of this study suggest the value of an international CPP as well as increased use of BCS approaches as means of either documenting bioequivalence or signaling the need for further in vivo studies. Because of increased movement of medicines across national borders, practitioners and patients would benefit from these approaches.

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.

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.

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.

Effects of fenoterol on the skeletal system depend on the androgen level.

PubMed

Śliwiński, Leszek; Cegieła, Urszula; Pytlik, Maria; Folwarczna, Joanna; Janas, Aleksandra; Zbrojkiewicz, Małgorzata

2017-04-01

The role of sympathetic nervous system in the osseous tissue remodeling is not clear enough. The effects of fenoterol, a selective β 2 -adrenomimetic drug, on the skeletal system of normal and androgen deficient (orchidectomized) rats were studied in vivo. Osteoclastogenesis and mRNA expression in osteoblasts were investigated in vitro in mouse cell cultures. Fenoterol administered to animals with physiological androgen level unfavorably affected the skeletal system, damaging the bone microarchitecture. Androgen deficiency induced osteoporotic changes, and fenoterol protected the osseous tissue from consequences of androgen deficiency. The results of in vitro studies correlated with the in vivo observations. A significantly increased number of osteoclasts in bone marrow cell cultures to which testosterone and fenoterol were added simultaneously was demonstrated. In cultures without the addition of testosterone, fenoterol significantly inhibited osteoclastogenesis in comparison with control cultures. The results indicate the favorable action of fenoterol in conditions of testosterone deficiency, and its destructive influence upon the skeleton in the presence of androgens. The results confirm the key role of sympathetic nervous system in the regulation of bone remodeling. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Formulation and characterization of lipid-based drug delivery system of raloxifene-microemulsion and self-microemulsifying drug delivery system

PubMed Central

Thakkar, Hetal; Nangesh, Jitesh; Parmar, Mayur; Patel, Divyakant

2011-01-01

Background: Raloxifene, a second-generation selective estrogen receptor modulator (SERM) used to prevent osteoporosis in postmenopausal women is administered orally in the form of a tablet. The absolute bioavailability of the drug is only 2% because of extensive hepatic first-pass metabolism. Lipid-based formulations are reported to reduce the first-pass metabolism by promoting its lymphatic uptake. Materials and Methods: In the present investigation, microemulsion and Self-Microemulsifying Drug Delivery System (SMEDDS) formulations of Raloxifene were prepared. The prepared formulations were characterized for drug loading, size, transparency, zeta potential, Transmission Electron Microscopy (TEM) and in vitro intestinal permeability. Results: The results indicated that high drug loading, optimum size and desired zeta potential and transparency could be achieved with both SMEDDS and microemulsion. The TEM studies indicated the absence of aggregation with both the systems. The in vitro intestinal permeability results showed that the permeation of the drug from the microemulsion and SMEDDs was significantly higher than that obtained from the drug dispersion and marketed formulation. Conclusion: Lipid based formulations such as microemulsion and Self Microemulsifying drug delivery systems are expected to increase the oral bioavailability as evidenced by the increased intestinal permeation. PMID:21966167

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.

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.

Towards active capsular endoscopy: preliminary results on a legged platform.

PubMed

Menciassi, Arianna; Stefanini, Cesare; Orlandi, Giovanni; Quirini, Marco; Dario, Paolo

2006-01-01

This paper illustrates the problem of active locomotion in the gastrointestinal tract for endoscopic capsules. Authors analyze the problem of locomotion in unstructured, flexible and tubular environments and explain the reasons leading to the selection of a legged system. They present a theoretical simulation of legged capsule locomotion, which is used to define the optimal parameters for capsule design and gait selection. Finally, a legged capsule--about 3 cm3 in volume--is presented; it consists of 4 back legs whose actuation is achieved thanks to a miniaturized DC brushless motor. In vitro tests demonstrate good performance in terms of achievable speed (92 mm/min).

Lecithin/TPGS-based spray-dried self-microemulsifying drug delivery systems: In vitro pulmonary deposition and cytotoxicity.

PubMed

Ishak, Rania A H; Osman, Rihab

2015-05-15

The aim of the present work was to develop a new solid self-microemulsifying drug delivery system (SMEDDS) for the pulmonary delivery of the poorly water-soluble anti-cancer drug atorvastatin (AVT). Microemulsion (ME) was first developed using isopropyl myristate (IPM), a combination of 2 biocompatible surfactants: lecithin/d-α-tocopheryl polyethylene glycol succinate (TPGS) and ethanol as co-surfactant. Two types of lecithin with different phosphatidylcholine (PC) contents were compared. Phase diagram, physico-chemical characterization and stability studies were used to investigate ME region. Solid SMEDDS were then prepared by spray-drying the selected ME using a combination of carriers composed of sugars, leucine as dispersibility enhancer with or without polyethylene glycol (PEG) 6000. Yield, flow properties, particle size and in vitro pulmonary deposition were used to characterize the spray-dried powders. Reconstituted MEs were characterized in terms of morphology, particle size and size distribution. In vitro cytotoxicity study was undertaken on lung cancer cell line for the selected MEs and SD-SMEDDS formulae. Results showed that the most satisfactory MEs properties were obtained with 1:3 lecithin/TPGS, 1:1 lecithin/oil and 1:1 surfactant/co-surfactant ratios. A larger ME area was obtained with lecithin containing 100% PC compared to the less expensive lecithin containing 20% PC. By manipulating spray drying parameters, carrier composition and ratio of ME lipids to carrier, microparticles with more than 70% of respirable fraction could be prepared. The ME was efficiently recovered in simulated lung fluid even after removal of alcohol. The concurrent delivery of AVT with TPGS in solid SMEDDS greatly enhanced the cytotoxic activity on lung cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Micromatricial metronidazole benzoate film as a local mucoadhesive delivery system for treatment of periodontal diseases.

PubMed

El-Kamel, Amal Hassan; Ashri, Lubna Y; Alsarra, Ibrahim A

2007-09-14

The main objective of this study was to develop a local, oral mucoadhesive metronidazole benzoate (MET) delivery system that can be applied and removed by the patient for the treatment of periodontal diseases. Mucoadhesive micromatricial chitosan/poly(epsilon-caprolactone) (CH/PCL) films and chitosan films were prepared. Thermal behavior, morphology, and particle size measurements were used to evaluate the prepared films. The effect of different molar masses of CH and different ratios of medium Mwt molar mass chitosan (MCH):PCL on water absorption, in vitro bioadhesion, mechanical properties, and in vitro drug release was examined. In vivo performance of the selected formulation was also evaluated. Differential scanning calorimetry examination revealed that MET existed mainly in amorphous form. Under microscopic examination, PCL microparticles were homogeneously dispersed in the films. The use of different molar masses of CH and different ratios of (MCH):PCL affected the size of the entrapped particles. Addition of PCL significantly decreased percentage water uptake and bioadhesion force compared with pure CH film. With regard to mechanical properties, the 2-layered film containing 1:0.625 MCH:PCL had the best tensile properties. At fixed CH:PCL ratio (1:1.25), the slowest drug release was obtained from films containing high molar mass CH. On the other hand, the 2-layered film that consisted of 1:0.625 MCH:PCL had the slowest MET release. In vivo evaluation of the selected film revealed that metronidazole concentration in saliva over 6 hours ranged from 5 to 15 microg/mL, which was within and higher than the reported range of minimum inhibitory concentration for metronidazole. A significant in vitro/in vivo correlation under the adopted experimental conditions was obtained.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 4: proposing a new lipid formulation performance classification system.

PubMed

Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Calderone, Marilyn; Igonin, Annabel; Jule, Eduardo; Vertommen, Jan; Blundell, Ross; Benameur, Hassan; Müllertz, Anette; Porter, Christopher J H; Pouton, Colin W

2014-08-01

The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF performance, including the degree of supersaturation generated on dispersion/digestion. Stresses evaluated included decreasing LBF concentration (↓LBF), increasing bile salt, and decreasing pH. Their capacity to stress LBFs was dependent on LBF composition and drug type: ↓LBF was a stressor to medium-chain glyceride-rich LBFs, but not more hydrophilic surfactant-rich LBFs, whereas decreasing pH stressed tolfenamic acid LBFs, but not fenofibrate LBFs. Lastly, a new Performance Classification System, that is, LBF composition independent, is proposed to promote standardized LBF comparisons, encourage robust LBF development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance grading. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

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.

Self-Assembled Tb3+ Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo.

PubMed

Jung, Sung Ho; Kim, Ka Young; Lee, Ji Ha; Moon, Cheol Joo; Han, Noh Soo; Park, Su-Jin; Kang, Dongmin; Song, Jae Kyu; Lee, Shim Sung; Choi, Myong Yong; Jaworski, Justyn; Jung, Jong Hwa

2017-01-11

To more accurately assess the pathways of biological systems, a probe is needed that may respond selectively to adenosine triphosphate (ATP) for both in vitro and in vivo detection modes. We have developed a luminescence probe that can provide real-time information on the extent of ATP, ADP, and AMP by virtue of the luminescence and luminescence lifetime observed from a supramolecular polymer based on a C 3 symmetrical terpyridine complex with Tb 3+ (S1-Tb). The probe shows remarkable selective luminescence enhancement in the presence of ATP compared to other phosphate-displaying nucleotides including adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), H 2 PO 4 - (Pi), and pyrophosphate (PPi). In addition, the time-resolved luminescence lifetime and luminescence spectrum of S1-Tb could facilitate the quantitative measurement of the exact amount of ATP and similarly ADP and AMP within living cells. The time-resolved luminescence lifetime of S1-Tb could also be used to quantitatively monitor the amount of ATP, ADP, and AMP in vitro following the enzymatic hydrolysis of ATP. The long luminescence lifetime, which was observed into the millisecond range, makes this S1-Tb-based probe particularly attractive for monitoring biological ATP levels in vivo, because any short lifetime background fluorescence arising from the complex molecular environment may be easily eliminated.

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...

Research in drug development against viral diseases of military importance (biological testing). Volume 2. Final report, 15 November 1985-31 January 1991

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

Shannon, W.M.; Arnett, G.; Brazier, A.D.

1991-03-01

The purpose of this program is to evaluate the efficacy of candidate antiviral compounds against a spectrum of viruses of military importance. This program involves (a) primary testing of chemical compounds and natural products for antiviral efficacy in vitro using standard CPE-inhibition assays, (b) primary testing of compounds for antiviral efficacy in vivo in animal model systems, and (c) secondary evaluation of the active candidate antiviral compounds. The target viruses for in vitro testing are Vaccinia Virus (VV), Adenovirus (AD2), Vesicular Stomatitis Virus (VSV), Punta Toro Virus (PT), Sandfly fever Virus (SF), Yellow Fever Virus (YF), Venezuelan Equine Encephalomyelitis Virusmore » (VE), Japanese Encephalitis Virus, Pichinde Virus (PIC), Hantaan Virus (HTN), and Human Immunodeficiency Virus (HIV). The in vivo systems are Pichinde Virus infection of hamsters, Venezuelan Equine Encephalomyelitis Virus, Japanese Encephalitis Virus and Vaccinia virus infections of mice. Approximately 10,000 compounds have been received for in vitro evaluation and over 66,000 assays have been performed on this contract. Compounds have been identified in nearly all virus systems that have confirmed antiviral activity equal or exceeding that of the various positive control compounds (ribavirin, selenazofurin, carbocyclic-3-aza-adenosine, adenosine dialdehyde, Ara-A, ddC and AZT). Many of these compounds represent potent and selective new antiviral agents.« less

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.

Formulation and evaluation of an in situ gel forming system for controlled delivery of triptorelin acetate.

PubMed

Abashzadeh, Sh; Dinarvand, R; Sharifzadeh, M; Hassanzadeh, G; Amini, M; Atyabi, F

2011-11-20

The novel physical hydrogels composed of chitosan or its water soluble derivatives such as carboxymethyl chitosan (CMCh) and sodium carboxymethyl chitosan (NaCMCh) and opened ring polyvinyl pyrrolidone (OP-PVP) were used as a controlled delivery system for triptorelin acetate, a luteinizing-releasing hormone agonist. The in situ gel forming system designed according to physical interactions such as chains entanglements and hydrophilic attractions especially h-bonds of chitosan and/or NaCMCh and OR-PVP. In order to increase in situ gel forming rate the chitosan microspheres prepared through spray drying technique. The chitosan or NaCMCh/OR-PVP blends prepared at different ratios (0.05, 0.10, 0.12, 0.16, 0.20 and 0.24) and suspended in sesame oil as non-aqueous vehicle at different solid content (10-30%). The suitable ratio of polymers with faster in situ gel forming rate was selected for in vivo studies. The gel formation and drug release from the system was evaluated both in vitro and in vivo. In vitro and in vivo results were compared with Diphereline SR 3.75mg, a commercially available controlled delivery system of triptorelin. In vitro release studies showed a sustained release profile for about 192h with first order kinetics. In vivo studies on male rats by determination of serum testosterone were confirmed the acceptable performance of in situ gel forming system compared with Diphereline SR in decreasing the serum testosterone level for 35days, demonstrating the potential of the novel in situ gel forming system for controlled delivery of peptides. Copyright © 2011 Elsevier B.V. All rights reserved.

Selective nitrergic neurodegeneration in diabetes mellitus–a nitric oxide-dependent phenomenon

PubMed Central

Cellek, Selim; Rodrigo, José; Lobos, Edgar; Fernández, Patricia; Serrano, Julia; Moncada, Salvador

1999-01-01

In vitro and in vivo studies have demonstrated a dysfunctional nitrergic system in diabetes mellitus, thus explaining the origin of diabetic impotence. However, the mechanism of this nitrergic defect is not understood.In the penises of streptozotocin (STZ)-induced diabetic rats, here, we show by immunohistochemistry that nitrergic nerves undergo selective degeneration since the noradrenergic nerves which have an anti-erectile function in the penis remained intact.Nitrergic relaxation responses in vitro and erectile responses to cavernous nerve stimulation in vivo were attenuated in these animals, whereas noradrenergic responses were enhanced.Activity and protein amount of neuronal nitric oxide synthase (nNOS) were also reduced in the penile tissue of diabetic rats.We, thus, hypothesized that NO in the nitrergic nerves may be involved in the nitrergic nerve damage, since only the nerves which contain neuronal NO synthase underwent degeneration.We administered an inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), in the drinking water of rats for up to 12 weeks following the establishment of diabetes with STZ.Here we demonstrate that this compound protected the nitrergic nerves from morphological and functional impairment. Our results show that selective nitrergic degeneration in diabetes is NO-dependent and suggest that inhibition of NO synthase is neuroprotective in this condition. PMID:10588937

Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

PubMed Central

Takayama, Naoya; Nishimura, Satoshi; Nakamura, Sou; Shimizu, Takafumi; Ohnishi, Ryoko; Endo, Hiroshi; Yamaguchi, Tomoyuki; Otsu, Makoto; Nishimura, Ken; Nakanishi, Mahito; Sawaguchi, Akira; Nagai, Ryozo; Takahashi, Kazutoshi; Yamanaka, Shinya; Nakauchi, Hiromitsu

2010-01-01

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095

In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae▿

PubMed Central

Bialek-Davenet, Suzanne; Marcon, Estelle; Leflon-Guibout, Véronique; Lavigne, Jean-Philippe; Bert, Frédéric; Moreau, Richard; Nicolas-Chanoine, Marie-Hélène

2011-01-01

The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression. PMID:21464248

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.

Possible mechanisms of dose-dependent cough suppressive effect of Althaea officinalis rhamnogalacturonan in guinea pigs test system.

PubMed

Sutovská, M; Nosálová, G; Sutovský, J; Franová, S; Prisenznáková, L; Capek, P

2009-07-01

The rhamnogalacturonan, isolated from the roots of medicinal plant Althaea officinalis L., showed various biological effects on the citric acid-induced cough reflex and reactivity of airways smooth muscle in vitro and in vivo conditions. It possessed dose-dependent cough suppression effect comparable with opioid agonist codeine. However, reactivity of the airways smooth muscle, measured in vitro as well as in vivo conditions was not significantly affected by rhamnogalacturonan and thus bronchodilatory activity did not participate in the cough suppression effect of polysaccharide tested. Moreover, the cough suppression effect of the polymer was not significantly modified by pretreatment of K(+)(ATP) ion channels with selective antagonist and therefore activation of this type of ion channels is not involved in the mechanism of rhamnogalacturonan cough suppressive ability. On the contrary, pretreatment of animals with selective 5-HT(2) receptors antagonist significantly decreased rhamnogalacturonan antitussive efficacy. From this point of view it seems that the cough suppression effect of the polymer is associated with the serotonergic 5-HT(2) receptor's function.

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

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.

Time lapse imaging: is it time to incorporate this technology into routine clinical practice?

PubMed

Bhide, Priya; Maheshwari, Abha; Cutting, Rachel; Seenan, Susan; Patel, Anita; Khan, Khalid; Homburg, Roy

2017-06-01

Time-lapse imaging (TLI) systems for embryo incubation, assessment and selection are a novel technology available to in vitro fertilization (IVF) clinics. However, there is uncertainty about their clinical and cost-effectiveness and insufficient good quality evidence to warrant their routine use. Despite this, enthusiastic commercial marketing and slipping clinical equipoise have led to the widespread hasty introduction of this technology into practice, often at a considerable expense to the patient. We have reviewed the published literature and aim to summarize the strengths, weaknesses, opportunities and threats of these systems. These specialized incubators provide undisturbed embryo culture conditions and, by almost continuous monitoring of embryo development, generate morphokinetic parameters to aid embryo selection. They are thus hypothesized to improve outcomes following IVF. Although literature reports improved reproductive outcomes, these outcomes are largely surrogate and there is a paucity of studies reporting live births. The use of time lapse systems may reduce early pregnancy loss, increase elective single embryo transfers and limit multiple pregnancies through better embryo selection. However, the quality of the studies and hence the evidence so far, is low to moderate quality. We recommend further research producing robust high-quality evidence for and against the use of these systems.

Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

PubMed Central

Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

2018-01-01

The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool.

PubMed

Moore, Simon J; Lai, Hung-En; Needham, Hannah; Polizzi, Karen M; Freemont, Paul S

2017-04-01

Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Fold or hold: experimental evolution in vitro

PubMed Central

Collins, S; Rambaut, A; Bridgett, S J

2013-01-01

We introduce a system for experimental evolution consisting of populations of short oligonucleotides (Oli populations) evolving in a modified quantitative polymerase chain reaction (qPCR). It is tractable at the genetic, genomic, phenotypic and fitness levels. The Oli system uses DNA hairpins designed to form structures that self-prime under defined conditions. Selection acts on the phenotype of self-priming, after which differences in fitness are amplified and quantified using qPCR. We outline the methodological and bioinformatics tools for the Oli system here and demonstrate that it can be used as a conventional experimental evolution model system by test-driving it in an experiment investigating adaptive evolution under different rates of environmental change. PMID:24003997

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease.

PubMed

Frikha-Gargouri, Olfa; Ben Abdallah, Dorra; Bhar, Ilhem; Tounsi, Slim

2017-01-01

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes ( sfp, ituC and bmyB ) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight ( p = 0.000) and the antibacterial activity in vitro ( p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol ( p = 0.004) and the reduction in gall weight ( p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo . Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens . These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains.

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease

PubMed Central

Frikha-Gargouri, Olfa; Ben Abdallah, Dorra; Bhar, Ilhem; Tounsi, Slim

2017-01-01

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes (sfp, ituC and bmyB) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight (p = 0.000) and the antibacterial activity in vitro (p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol (p = 0.004) and the reduction in gall weight (p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo. Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens. These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains. PMID:28855909

The effect of antimicrobial agents on bond strength of orthodontic adhesives: a meta-analysis of in vitro studies.

PubMed

Altmann, A S P; Collares, F M; Leitune, V C B; Samuel, S M W

2016-02-01

Antimicrobial orthodontic adhesives aim to reduce white spot lesions' incidence in orthodontic patients, but they should not jeopardizing its properties. Systematic review and meta-analysis were performed to answer the question whether the association of antimicrobial agents with orthodontic adhesives compromises its mechanical properties and whether there is a superior antimicrobial agent. PubMed and Scopus databases. In vitro studies comparing shear bond strength of conventional photo-activated orthodontic adhesives to antimicrobial photo-activated orthodontic adhesives were considered eligible. Search terms included the following: orthodontics, orthodontic, antimicrobial, antibacterial, bactericidal, adhesive, resin, resin composite, bonding agent, bonding system, and bond strength. The searches yielded 494 citations, which turned into 467 after duplicates were discarded. Titles and abstracts were read and 13 publications were selected for full-text reading. Twelve studies were included in the meta-analysis. The global analysis showed no statistically significant difference between control and experimental groups. In the subgroup analysis, only the chlorhexidine subgroup showed a statistically significant difference, where the control groups had higher bond strength than the experimental groups. Many studies on in vitro orthodontic bond strength fail to report test conditions that could affect their outcomes. The pooled in vitro data suggest that adding an antimicrobial agent to an orthodontic adhesive system does not influence bond strength to enamel. It is not possible to state which antimicrobial agent is better to be associated. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

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.

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

PubMed

Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

2013-12-01

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

Pharmacokinetics of the Novel, Selective, Non-steroidal Mineralocorticoid Receptor Antagonist Finerenone in Healthy Volunteers: Results from an Absolute Bioavailability Study and Drug-Drug Interaction Studies In Vitro and In Vivo.

PubMed

Heinig, Roland; Gerisch, Michael; Engelen, Anna; Nagelschmitz, Johannes; Loewen, Stephanie

2018-05-19

Finerenone is a selective, non-steroidal mineralocorticoid receptor antagonist. In vivo and in vitro studies were performed to assess absolute bioavailability of finerenone, the effect of metabolic enzyme inhibitors on the pharmacokinetics of finerenone and its metabolites, the quantitative contribution of the involved enzymes cytochrome P450 (CYP) 3A4 and CYP2C8 and the relevance of gut wall versus liver metabolism. The pharmacokinetics, safety and tolerability of finerenone (1.25-10 mg orally or 0.25-1.0 mg intravenously) were evaluated in healthy male volunteers in four crossover studies. Absolute bioavailability was assessed in volunteers receiving finerenone orally and by intravenous infusion (n = 15) and the effects of erythromycin (n = 15), verapamil (n = 13) and gemfibrozil (n = 16) on finerenone pharmacokinetics were investigated. Finerenone was also incubated with cryopreserved human hepatocytes in vitro in the presence of erythromycin, verapamil or gemfibrozil. Finerenone absolute bioavailability was 43.5% due to first-pass metabolism in the gut wall and liver. The geometric mean AUC 0-∞ ratios of finerenone (drug + inhibitor/drug alone) were 3.48, 2.70 and 1.10 with erythromycin, verapamil and gemfibrozil, respectively. The contribution ratio of CYP3A4 to the metabolic clearance of finerenone derived from these values was 0.88-0.89 and was consistent with estimations based on in vitro data, with the remaining metabolic clearance due to CYP2C8 involvement. Finerenone is predominantly metabolized by CYP3A4 in the gut wall and liver. Increases in systemic exposure upon concomitant administration of inhibitors of this isoenzyme are predictable and consistent with in vitro data. Inhibition of CYP2C8, the second involved metabolic enzyme, has no relevant effect on finerenone in vivo.

Evaluation of an in vitro system to simulate equine foregut digestion and the influence of acidity on protein and fructan degradation in the horse's stomach.

PubMed

Strauch, S; Wichert, B; Greef, J M; Hillegeist, D; Zeyner, A; Liesegang, A

2017-06-01

The aim of this study was to improve an in vitro system in order to gather optimized information on the digestion of different forages in the horse's upper gastrointestinal tract. Therefore, foregut digestion of several forages was simulated in vitro (Part 1). The effect of different pH values on in vitro fructan degradation of two selected grasses (Part 2) was tested subsequently. Part 1: We hypothesized that our system produces representative results simulating digestive processes in the upper alimentary tract, but neglects microbial fermentation. In vitro digestion of six forages (grass mixture for horses, grass mixture for cows (GMC), tall fescue, English perennial ryegrass (ER), white clover, lucerne) was performed in two phases with pepsin and pancreatin. The results are consistent with current data from in vivo studies, including a degradation of crude protein and monosaccharides as well as a relative increase in fibres. Interestingly, a loss of fructan was measured in two feedstuffs (ER/GMC: 4.1/4.4% DM fructan before and 0.59/0.00% DM after simulated foregut digestion). Part 2: As fructans are thought not to be fragmented by digestive enzymes, another hypothesis was developed: acidic hydrolysis leads to a degradation of fructans. To evaluate the influence of gastric pH on the digestion of fructan and protein, different pH values (2, 3 and 4) were adjusted in a second series of in vitro foregut digestion trials with ER and GMC. As expected, the highest degradation of protein was seen at the lowest pH (protein in ER/GMC at pH 2: 6.11/8.28% DM and at pH 4: 7.73/10.64% DM), whereas fructan degradation was highest at pH 4 (fructan in ER/GMC at pH 2: 1.63/1.95% DM and at pH 4: 1.31/0.91% DM). We presume that not only acidic hydrolysis but also plant enzymes cause the loss of fructans in an acidic environment. Journal of Animal Physiology and Animal Nutrition © 2017 Blackwell Verlag GmbH.

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.

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.

DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

PubMed

Rathore, Mangal S; Jha, Bhavanath

2016-03-01

The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

PubMed

Jiang, Ying; Hardie, Joseph; Liu, Yuanchang; Ray, Moumita; Luo, Xiang; Das, Riddha; Landis, Ryan F; Farkas, Michelle E; Rotello, Vincent M

2018-06-05

The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Copyright © 2018 Elsevier B.V. All rights reserved.

In vitro immunomodulatory potential of Artemisia indica Willd. in chicken lymphocytes.

PubMed

Ruwali, Pushpa; Ambwani, Tanuj Kumar; Gautam, Pankaj

2018-01-01

Evaluation of the in vitro immunomodulatory potential of Artemisia indica Willd. methanolic extract in chicken lymphocyte culture system through lymphocyte (B and T cells) proliferation assay, after standardizing the maximum non-cytotoxic dose (MNCD) in chicken lymphocytes. Fresh aerial parts of A. indica Willd. (family: Asteraceae) specimens were collected (altitude 1560 m), gotten authenticated, processed, dried, and Soxhlet extracted to yield methanolic extract (AME). Chicken splenocytes were isolated from spleens collected from healthy birds; lymphocytes were separated by density gradient centrifugation, percentage cell viability determined and final cell count adjusted to 10 7 cells/ml in RPMI-1640 medium. MNCD of AME in chicken lymphocytes was determined through 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide dye reduction assay. Immunomodulatory potential of AME was evaluated through lymphocytes proliferation or B and T cells blastogenesis assay in the presence of appropriate mitogens, namely, lipopolysaccharide (LPS) and concanavalin A (Con A), respectively. Maximum concentration of AME exhibiting 100% cell viability (MNCD) was 200 μg/ml and was selected for further in vitro analysis. The in vitro exposure of chicken lymphocytes to 200 µg/ml dose of AME, resulted in significant (p<0.05) upregulation of 11.76% in B cell proliferation in the presence of B cell mitogen (LPS) and a significant (p<0.05) increase of 12.018% T cells proliferation in the presence of the mitogen (Con A), as compared to the control. The significant upregulation in the proliferation of two major cell types modulating the immune system is an indication of the immunostimulatory potential of the plant. It would be worthwhile to further evaluate A. indica on relevant immunomodulatory aspects, especially the in vivo studies in a poultry system.

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

PubMed Central

He, M; Taussig, M J

1997-01-01

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries. PMID:9396828

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

PubMed

He, M; Taussig, M J

1997-12-15

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.

Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

PubMed

Yamashita, Naoya; Jitsuki-Takahashi, Aoi; Ogawara, Miyuki; Ohkubo, Wataru; Araki, Tomomi; Hotta, Chie; Tamura, Tomohiko; Hashimoto, Shu-ichi; Yabuki, Takashi; Tsuji, Toru; Sasakura, Yukie; Okumura, Hiromi; Takaiwa, Aki; Koyama, Chika; Murakami, Koji; Goshima, Yoshio

2015-09-01

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Dose selection based on physiologically based pharmacokinetic (PBPK) approaches.

PubMed

Jones, Hannah M; Mayawala, Kapil; Poulin, Patrick

2013-04-01

Physiologically based pharmacokinetic (PBPK) models are built using differential equations to describe the physiology/anatomy of different biological systems. Readily available in vitro and in vivo preclinical data can be incorporated into these models to not only estimate pharmacokinetic (PK) parameters and plasma concentration-time profiles, but also to gain mechanistic insight into compound properties. They provide a mechanistic framework to understand and extrapolate PK and dose across in vitro and in vivo systems and across different species, populations and disease states. Using small molecule and large molecule examples from the literature and our own company, we have shown how PBPK techniques can be utilised for human PK and dose prediction. Such approaches have the potential to increase efficiency, reduce the need for animal studies, replace clinical trials and increase PK understanding. Given the mechanistic nature of these models, the future use of PBPK modelling in drug discovery and development is promising, however some limitations need to be addressed to realise its application and utility more broadly.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Inhibition of isoleucyl-tRNA synthetase as a potential treatment for human African Trypanosomiasis.

PubMed

Cestari, Igor; Stuart, Kenneth

2013-05-17

Trypanosoma brucei sp. causes human African trypanosomiasis (HAT; African sleeping sickness). The parasites initially proliferate in the hemolymphatic system and then invade the central nervous system, which is lethal if not treated. New drugs are needed for HAT because the approved drugs are few, toxic, and difficult to administer, and drug resistance is spreading. We showed by RNAi knockdown that T. brucei isoleucyl-tRNA synthetase is essential for the parasites in vitro and in vivo in a mouse model of infection. By structure prediction and experimental analysis, we also identified small molecules that inhibit recombinant isoleucyl-tRNA synthetase and that are lethal to the parasites in vitro and highly selective compared with mammalian cells. One of these molecules acts as a competitive inhibitor of the enzyme and cures mice of the infection. Because members of this class of molecules are known to cross the blood-brain barrier in humans and to be tolerated, they may be attractive as leading candidates for drug development for HAT.

Inhibition of Isoleucyl-tRNA Synthetase as a Potential Treatment for Human African Trypanosomiasis*

PubMed Central

Cestari, Igor; Stuart, Kenneth

2013-01-01

Trypanosoma brucei sp. causes human African trypanosomiasis (HAT; African sleeping sickness). The parasites initially proliferate in the hemolymphatic system and then invade the central nervous system, which is lethal if not treated. New drugs are needed for HAT because the approved drugs are few, toxic, and difficult to administer, and drug resistance is spreading. We showed by RNAi knockdown that T. brucei isoleucyl-tRNA synthetase is essential for the parasites in vitro and in vivo in a mouse model of infection. By structure prediction and experimental analysis, we also identified small molecules that inhibit recombinant isoleucyl-tRNA synthetase and that are lethal to the parasites in vitro and highly selective compared with mammalian cells. One of these molecules acts as a competitive inhibitor of the enzyme and cures mice of the infection. Because members of this class of molecules are known to cross the blood-brain barrier in humans and to be tolerated, they may be attractive as leading candidates for drug development for HAT. PMID:23548908

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

Protamine-based nanoparticles as new antigen delivery systems.

PubMed

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

21 CFR 864.5425 - Multipurpose system for in vitro coagulation studies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Multipurpose system for in vitro coagulation... Hematology Devices § 864.5425 Multipurpose system for in vitro coagulation studies. (a) Identification. A multipurpose system for in vitro coagulation studies is a device consisting of one automated or semiautomated...

21 CFR 864.5425 - Multipurpose system for in vitro coagulation studies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Multipurpose system for in vitro coagulation... Hematology Devices § 864.5425 Multipurpose system for in vitro coagulation studies. (a) Identification. A multipurpose system for in vitro coagulation studies is a device consisting of one automated or semiautomated...

Novel method for in vitro depletion of T cells by monoclonal antibody-targeted photosensitization.

PubMed

Berki, T; Németh, P

1998-02-01

An immunotargeting method (called photo-immunotargeting) has been developed for selective in vitro cell destruction. The procedure combines the photosensitizing (toxic) effect of light-induced dye-molecules, e.g., hematoporphyrin (HP) and the selective binding ability of monoclonal antibodies (mAb) to cell surface molecules. The photosensitizer HP molecules were covalently attached to monoclonal antibodies (a-Thy-1) recognizing an antigen on the surface of T lymphocytes, and used for T cell destruction. To increase the selectivity of the conventional targeting methods, a physical activation step (local light irradiation) as a second degree of specificity was employed. The HP in conjugated form was sufficient to induce T cell (thymocytes, EL-4 cell line) death after irradiation at 400 nm, at tenfold lower concentration compared to the photosensitizing effect of unbound HP. The selective killing of T lymphocytes (bearing the Thy-1 antigen) in a mixed cell population was demonstrated after a treatment with the phototoxic conjugate and light irradiation. This method can be useful for selective destruction of one population (target cell) in an in vitro heterogeneous cell mixture, e.g., in bone marrow transplants for T cell depletion to avoid graft vs. host reaction.

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

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

PubMed

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

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.

Pharmacokinetics and anti-hypertensive effect of metoprolol tartrate rectal delivery system.

PubMed

Abou el Ela, Amal El Sayeh F; Allam, Ayat A; Ibrahim, Ehsan H

2016-01-01

The main aim of this work was to develop rectal suppositories for better delivery of metoprolol tartrate (MT). The various bases used were fatty, water soluble and emulsion bases. The physical properties of the prepared suppositories were characterized such as weight variation, hardness, disintegration time, melting range and the drug content uniformity. The in vitro release of MT from the prepared suppositories was carried out. The evaluation of the pharmacological effects of MT on the blood pressure and heart rate of the healthy rabbits after the rectal administration compared to the oral tablets was studied. Moreover, the formulation with the highest in vitro release and the highest pharmacological effects would be selected for a further pharmacokinetics study compared to the oral tablets. The results revealed that the emulsion bases gave the highest rate of the drug release than the other bases used. The reduction effect of the emulsion MT suppository base on the blood pressure and heart rate was found to be faster and greater than that administered orally. The selected emulsion suppository base (F11) showed a significant increase in the AUC (1.88-fold) in rabbits as compared to the oral tablets. From the above results we can conclude that rectal route can serve as an efficient alternative route to the oral one for systemic delivery of MT which may be due to the avoidance of first-pass effect in the liver.

β-CD-dextran polymer for efficient sequestration of cholesterol from phospholipid bilayers: Mechanistic and safe-toxicity investigations.

PubMed

Stelzl, Dominik; Nielsen, Thorbjørn Terndrup; Hansen, Terkel; di Cagno, Massimiliano

2015-12-30

The aim of this work was to investigate the suitability of β-cyclodextrin-dextran (BCD-dextran) polymer as cholesterol sequestering agent in vitro. For this purpose, BCD-dextran-cholesterol complexation was studied by phase solubility studies as well as with a specifically designed in vitro model based on giant unilamellar vesicles (GUVs) to evaluate the ability of this polymer to sequestrate cholesterol from phospholipid bilayers. Cholesterol-sequestering ability of BCD-dextran was also investigated on different cell lines relevant for the hematopoietic system and results were correlated to cells toxicity. BCD-dextran polymer was capable of extracting significant amount of cholesterol from phospholipid bilayers and to a higher extent in comparison to available β-cyclodextrins (BCDs). The ability of BCD-dextran in sequestering cholesterol resulted also very high on cell lines relevant for the hematopoietic system. Moreover, BCD-dextran resulted less toxic on cell cultures due to higher selectivity in sequestering cholesterol in comparison to MBCD (that sequestrated also significant amounts of cholesteryl esters). In conclusion, BCD-dextran resulted an extremely efficient cholesterol-sequestering agent and BCD-dextran resulted more selective to cholesterol extraction in comparison to other BCDs (therefore of lower cytotoxicity). This phenomenon might play a key role to develop an efficient treatment for hypercholesterolemia based on cholesterol segregation. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc and vitamins B6, B12, D, and E

USDA-ARS?s Scientific Manuscript database

A review of in vitro bioaccessibility and bioavailability methods for polyphenols and selected nutrients is presented. The review focuses on in vitro solubility, dialyzability, the dynamic gastrointestinal model (TIM), and Caco-2 cell models, the latter primarily for uptake and transport, and a disc...

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.

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

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

Implementing oxygen control in chip-based cell and tissue culture systems.

PubMed

Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth

2016-09-21

Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

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.

Evidence-based selection of training compounds for use in the mechanism-based integrated prediction of drug-induced liver injury in man.

PubMed

Dragovic, Sanja; Vermeulen, Nico P E; Gerets, Helga H; Hewitt, Philip G; Ingelman-Sundberg, Magnus; Park, B Kevin; Juhila, Satu; Snoeys, Jan; Weaver, Richard J

2016-12-01

The current test systems employed by pharmaceutical industry are poorly predictive for drug-induced liver injury (DILI). The 'MIP-DILI' project addresses this situation by the development of innovative preclinical test systems which are both mechanism-based and of physiological, pharmacological and pathological relevance to DILI in humans. An iterative, tiered approach with respect to test compounds, test systems, bioanalysis and systems analysis is adopted to evaluate existing models and develop new models that can provide validated test systems with respect to the prediction of specific forms of DILI and further elucidation of mechanisms. An essential component of this effort is the choice of compound training set that will be used to inform refinement and/or development of new model systems that allow prediction based on knowledge of mechanisms, in a tiered fashion. In this review, we focus on the selection of MIP-DILI training compounds for mechanism-based evaluation of non-clinical prediction of DILI. The selected compounds address both hepatocellular and cholestatic DILI patterns in man, covering a broad range of pharmacologies and chemistries, and taking into account available data on potential DILI mechanisms (e.g. mitochondrial injury, reactive metabolites, biliary transport inhibition, and immune responses). Known mechanisms by which these compounds are believed to cause liver injury have been described, where many if not all drugs in this review appear to exhibit multiple toxicological mechanisms. Thus, the training compounds selection offered a valuable tool to profile DILI mechanisms and to interrogate existing and novel in vitro systems for the prediction of human DILI.

Sperm selection in natural conception: what can we learn from Mother Nature to improve assisted reproduction outcomes?

PubMed Central

Sakkas, Denny; Ramalingam, Mythili; Garrido, Nicolas; Barratt, Christopher L.R.

2015-01-01

BACKGROUND In natural conception only a few sperm cells reach the ampulla or the site of fertilization. This population is a selected group of cells since only motile cells can pass through cervical mucus and gain initial entry into the female reproductive tract. In animals, some studies indicate that the sperm selected by the reproductive tract and recovered from the uterus and the oviducts have higher fertilization rates but this is not a universal finding. Some species show less discrimination in sperm selection and abnormal sperm do arrive at the oviduct. In contrast, assisted reproductive technologies (ART) utilize a more random sperm population. In this review we contrast the journey of the spermatozoon in vivo and in vitro and discuss this in the context of developing new sperm preparation and selection techniques for ART. METHODS A review of the literature examining characteristics of the spermatozoa selected in vivo is compared with recent developments in in vitro selection and preparation methods. Contrasts and similarities are presented. RESULTS AND CONCLUSIONS New technologies are being developed to aid in the diagnosis, preparation and selection of spermatozoa in ART. To date progress has been frustrating and these methods have provided variable benefits in improving outcomes after ART. It is more likely that examining the mechanisms enforced by nature will provide valuable information in regard to sperm selection and preparation techniques in vitro. Identifying the properties of those spermatozoa which do reach the oviduct will also be important for the development of more effective tests of semen quality. In this review we examine the value of sperm selection to see how much guidance for ART can be gleaned from the natural selection processes in vivo. PMID:26386468

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.

SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

PubMed

Kaltenbach, Miriam; Stein, Viktor; Hollfelder, Florian

2011-09-19

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo.

PubMed

Donato, M Teresa; Hallifax, David; Picazo, Laura; Castell, José V; Houston, J Brian; Gomez-Lechón, M José; Lahoz, Agustin

2010-09-01

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.

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.

Sunflower (Helianthus annuus L.).

PubMed

Radonic, Laura M; Lewi, Dalia M; López, Nilda E; Hopp, H Esteban; Escandón, Alejandro S; Bilbao, Marisa López

2015-01-01

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

PubMed Central

Grünwald, Geoffrey K; Vetter, Alexandra; Klutz, Kathrin; Willhauck, Michael J; Schwenk, Nathalie; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Zach, Christian; Wagner, Ernst; Göke, Burkhard; Holm, Per S; Ogris, Manfred; Spitzweg, Christine

2013-01-01

We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy. PMID:24193032

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

PubMed Central

Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M.; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico

2015-01-01

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493

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.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Is Selectively Toxic to Primary Dopaminergic Neurons In Vitro

PubMed Central

Griggs, Amy M.; Agim, Zeynep S.; Mishra, Vartika R.; Tambe, Mitali A.; Director-Myska, Alison E.; Turteltaub, Kenneth W.; McCabe, George P.; Rochet, Jean-Christophe; Cannon, Jason R.

2014-01-01

Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4′-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4′-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress. PMID:24718704

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

PubMed

van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

2017-11-25

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

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.

Effect of methylmercury on the rat mast cell degranulation

NASA Astrophysics Data System (ADS)

Graevskaya, E. E.; Yasutake, A.; Aramai, R.; Rubin, A. B.

2003-05-01

Methylmercury is the well-known neurotoxicant as weil as a modulator of the immune system. We investigated the effects of MeHg on the rat mast cell degranulation induced by nonimmunological stimuli (the selective liberator of histamine, compound 48/80, and calcium ionophore A23187) both in vivo and in vitro. In 8, 12 and 15 days afterthe final administration of MeHg we observed the suppression of calcium ionophore A23187-and 48/80-induced histamine release, which enhanced with time. In experiments in vitro incubation of peritoneal mast cells with MeHg alone in the dose range 10^{-8} to 10^{-6} did not induce mast cell degranulation, however modified the activation of mast cells by compound 48/80, and calcium ionophore A23187. We observed activation of stimulated secretion by preliminary incubation with low dose of MeHg 10^{-8} M and inhibition by dose of MeHg 10^{-6} M. These results show that MeHg treatment can modify mast cell function in vivo and in vitro and provide insight into the understanding what role this cell has in the pathogenesis of Minamata disease-comlected disorders.

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.

Limonene enhances the in vitro and in vivo permeation of trimetazidine across a membrane-controlled transdermal therapeutic system.

PubMed

Krishnaiah, Yellela S; Al-Saidan, Saleh M

2008-01-01

The objective of the study was to design membrane-controlled transdermal therapeutic system (TTS) for trimetazidine. The optimization of (i) concentration of ethanol-water solvent system, (ii) HPMC concentration of drug reservoir and (iii) limonene concentration in 2% w/v HPMC gel was done based on the in vitro permeation of trimetazidine across excised rat epidermis. A limonene-based membrane-controlled TTS of trimetazidine was fabricated and evaluated for its in vivo drug release in rabbit model. The in vitro permeation of trimetazidine from water, ethanol and selected concentrations (25, 50 and 75% v/v) of ethanol-water co-solvent systems showed that 50% v/v of ethanol-water solvent system provided an optimal transdermal flux of 233.1+/-3.8 microg/cm(2.)h. The flux of the drug decreased to 194.1+/-7.4 microg/cm(2.)h on adding 2% w/v of HPMC to ethanolic (50% v/v ethanol-water) solution of trimetazidine. However, on adding selected concentrations of limonene (0, 2, 4, 6 and 8% w/v) to 2% w/v HPMC gel drug reservoir, the flux of the drug increased to 365.5+/-7.1 microg/cm(2.)h. Based on these results, 2% w/v HPMC gel drug reservoir containing 6% w/v of limonene was chosen as an optimal formulation for studying the influence of rate-controlling EVA2825 membrane and adhesive-coated EVA2825 membrane. The flux of the drug across EVA2825 membrane (mean thickness 31.2 microm) decreased to 285.8+/-2.2 microg/cm(2.)h indicating that the chosen membrane was effective as rate-controlling membrane. On applying an adhesive coat (mean thickness 10.2 microm) to EVA2825 membrane, the drug flux further decreased to 212.4+/-2.6 microg/cm(2.)h. However, the flux of the drug across adhesive-coated EVA2825 membrane-rat epidermis composite was 185.9+/-2.9 microg/cm(2.)h, which is about 2-times higher than the desired flux. The fabricated limonene-based TTS patch of trimetazidine showed a mean steady state plasma concentration of 71.5 ng/mL for about 14 h with minimal fluctuation when tested in rabbits. It was concluded from the investigation that the limonene-based TTS patch of trimetazidine provided constant drug delivery across the skin in rabbit model.

DHMI: dynamic holographic microscopy interface

NASA Astrophysics Data System (ADS)

He, Xuefei; Zheng, Yujie; Lee, Woei Ming

2016-12-01

Digital holographic microscopy (DHM) is a powerful in-vitro biological imaging tool. In this paper, we report a fully automated off-axis digital holographic microscopy system completed with a graphical user interface in the Matlab environment. The interface primarily includes Fourier domain processing, phase reconstruction, aberration compensation and autofocusing. A variety of imaging operations such as region of interest selection, de-noising mode (filtering and averaging), low frame rate imaging for immediate reconstruction and high frame rate imaging routine ( 27 fps) are implemented to facilitate ease of use.

Microorganisms and biomolecules in space hard environment

NASA Technical Reports Server (NTRS)

Horneck, G.

1981-01-01

Microorganisms and biomolecules exposed to space vacuum and to different intensities of selected wavelengths of solar ultraviolet radiation is studied. The influence of these factors, applied singly or simultaneously, on the integrity of microbial systems and biomolecules is measured. Specifically, this experiment will study in Bacillus subtilis spores (1) disturbances in subsequent germination, outgrowth, and colony formation; (2) photochemical reactions of the DNA and protein in vivo and in vitro and their role in biological injury; and (3) the efficiency of repair processes in these events.

Possibility of Predicting Serotonin Transporter Occupancy From the In Vitro Inhibition Constant for Serotonin Transporter, the Clinically Relevant Plasma Concentration of Unbound Drugs, and Their Profiles for Substrates of Transporters.

PubMed

Yahata, Masahiro; Chiba, Koji; Watanabe, Takao; Sugiyama, Yuichi

2017-09-01

Accurate prediction of target occupancy facilitates central nervous system drug development. In this review, we discuss the predictability of serotonin transporter (SERT) occupancy in human brain estimated from in vitro K i values for human SERT and plasma concentrations of unbound drug (C u,plasma ), as well as the impact of drug transporters in the blood-brain barrier. First, the geometric means of in vitro K i values were compared with the means of in vivo K i values (K i,u,plasma ) which were calculated as C u,plasma values at 50% occupancy of SERT obtained from previous clinical positron emission tomography/single photon emission computed tomography imaging studies for 6 selective serotonin transporter reuptake inhibitors and 3 serotonin norepinephrine reuptake inhibitors. The in vitro K i values for 7 drugs were comparable to their in vivo K i,u,plasma values within 3-fold difference. SERT occupancy was overestimated for 5 drugs (P-glycoprotein substrates) and underestimated for 2 drugs (presumably uptake transporter substrates, although no evidence exists as yet). In conclusion, prediction of human SERT occupancy from in vitro K i values and C u,plasma was successful for drugs that are not transporter substrates and will become possible in future even for transporter substrates, once the transporter activities will be accurately estimated from in vitro experiments. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Patient-specific in vitro models for hemodynamic analysis of congenital heart disease - Additive manufacturing approach.

PubMed

Medero, Rafael; García-Rodríguez, Sylvana; François, Christopher J; Roldán-Alzate, Alejandro

2017-03-21

Non-invasive hemodynamic assessment of total cavopulmonary connection (TCPC) is challenging due to the complex anatomy. Additive manufacturing (AM) is a suitable alternative for creating patient-specific in vitro models for flow measurements using four-dimensional (4D) Flow MRI. These in vitro systems have the potential to serve as validation for computational fluid dynamics (CFD), simulating different physiological conditions. This study investigated three different AM technologies, stereolithography (SLA), selective laser sintering (SLS) and fused deposition modeling (FDM), to determine differences in hemodynamics when measuring flow using 4D Flow MRI. The models were created using patient-specific MRI data from an extracardiac TCPC. These models were connected to a perfusion pump circulating water at three different flow rates. Data was processed for visualization and quantification of velocity, flow distribution, vorticity and kinetic energy. These results were compared between each model. In addition, the flow distribution obtained in vitro was compared to in vivo. The results showed significant difference in velocities measured at the outlets of the models that required internal support material when printing. Furthermore, an ultrasound flow sensor was used to validate flow measurements at the inlets and outlets of the in vitro models. These results were highly correlated to those measured with 4D Flow MRI. This study showed that commercially available AM technologies can be used to create patient-specific vascular models for in vitro hemodynamic studies at reasonable costs. However, technologies that do not require internal supports during manufacturing allow smoother internal surfaces, which makes them better suited for flow analyses. Copyright © 2017 Elsevier Ltd. All rights reserved.

N-acetyl-L-cysteine pre-treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos.

PubMed

Pérez, L; Arias, M E; Sánchez, R; Felmer, R

2015-12-01

Excess of reactive oxygen species (ROS) on in vitro embryo production systems negatively affects the quality and developmental potential of embryos, as result of a decreased sperm quality and increased DNA fragmentation. This issue is of major importance in assisted fertilisation procedures such as intracytoplasmic sperm injection (ICSI), because this technique does not allow the natural selection of competent spermatozoa, and therefore, DNA-damaged spermatozoa might be used to fertilise an egg. The aim of this study was to investigate a new strategy to prevent the potential deleterious effect of ROS on cryopreserved bovine spermatozoa. We evaluated the effect of a sperm pre-treatment with different concentrations of N-acetyl-L-cysteine (NAC) on ROS production, viability and DNA fragmentation and assessed the effect of this treatment on the in vitro developmental potential and quality of embryos generated by ICSI. The results show a strong scavenging effect of 1 and 10 mm NAC after exposure of spermatozoa to a ROS inducer, without compromising the viability and DNA integrity. Importantly, in vitro developmental potential and quality of embryos generated by ICSI with spermatozoa treated with NAC were not affected, confirming the feasibility of using this treatment before an ICSI cycle. © 2015 Blackwell Verlag GmbH.

Development of optimal medium content for bioelements accumulation in Bacopa monnieri (L.) in vitro culture.

PubMed

Łojewski, Maciej; Muszyńska, Bożena; Smalec, Agata; Reczyński, Witold; Opoka, Włodzimierz; Sułkowska-Ziaja, Katarzyna

2014-10-01

Bacopa monnieri is one of the most interesting plants from the Ayurveda system. The aims of present research were, basing on in vitro shoot culture of B. monnieri, to determine content and to evaluate the influence of physiologically important metabolites on the selected bioelements accumulation in biomass. The most significant increase in biomass production was observed in the culture medium enriched with 0.5 mg/L of anthranilic acid. In this medium also, the highest accumulation of Mg was noted. The highest concentration of iron was determined in B. monnieri in vitro culture enriched with 0.25 g/L of serine. The addition of L-tryptophan, magnesium sulfate, and zinc hydroaspartate caused only a small increase in the accumulation of copper in B. monnieri. Increase in Zn accumulation was obtained in biomass from in vitro culture of B. monnieri with the addition of magnesium sulfate and zinc hydroaspartate. In the case of Na, the maximum level of this element was in biomass from medium enriched with zinc hydroaspartate. Twofold increase in K concentration was obtained in biomass from cultures on medium with addition of serine and magnesium sulfate. The concentrations of Ca in biomass of all studied media were at the similar level.

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro.

PubMed

Tommasi, Stella; Bates, Steven E; Behar, Rachel Z; Talbot, Prue; Besaratinia, Ahmad

2017-10-01

Electronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown. We have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene. We observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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.

In vitro screening of selected feed additives, plant essential oils and plant extracts for rumen methane mitigation.

PubMed

Durmic, Zoey; Moate, Peter J; Eckard, Richard; Revell, Dean K; Williams, Richard; Vercoe, Philip E

2014-04-01

Ruminants produce large quantities of methane in their rumen as a by-product of microbial digestion of feed. Antibiotics are added to ruminant feed to reduce wasteful production of methane; however, this practice has some downsides. A search for safer and natural feed additives with anti-methanogenic properties is under way. The objective of this research was to examine selected feed additives, plant essential oils and plant extracts for their anti-methanogenic potential in the rumen using an in vitro batch fermentation system. A significant reduction (P < 0.05) in methane production was observed with nine feed additives (up to 40% reduction), all eight essential oils (up to 75% reduction) and two plant extracts (14% reduction) when compared to their respective controls. Amongst these, only an algal meal high in docosahexaenoic acid, preparations of Nannochloropsis oculata, calcareous marine algae, yeast metabolites and two tannins did not inhibit microbial gas and volatile acid production. The current study identified some potent dietary ingredients or plant compounds that can assist in developing novel feed additives for methane mitigation from the rumen. © 2013 Society of Chemical Industry.

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.

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

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

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

Recapitulation of complex transport and action of drugs at tumor microenvironment using tumor-microenvironment-on-chip

PubMed Central

Han, Bumsoo; Qu, Chunjing; Park, Kinam; Konieczny, Stephen F.; Korc, Murray

2016-01-01

Targeted delivery aims to selectively distribute drugs to targeted tumor tissue but not to healthy tissue. This can address many of clinical challenges by maximizing the efficacy but minimizing the toxicity of anti-cancer drugs. However, complex tumor microenvironment poses various barriers hindering the transport of drugs and drug delivery systems. New tumor models that allow for the systematic study of these complex environments are highly desired to provide reliable test beds to develop drug delivery systems for targeted delivery. Recently, research efforts have yielded new in vitro tumor models, the so called tumor-microenvironment-on-chip, that recapitulate certain characteristics of the tumor microenvironment. These new models show benefits over other conventional tumor models, and have the potential to accelerate drug discovery and enable precision medicines. However, further research is warranted to overcome their limitations and to properly interpret the data obtained from these models. In this article, key features of the in vivo tumor microenvironment that are relevant to drug transport processes for targeted delivery was discussed, and the current status and challenges for developing in vitro transport model systems was reviewed. PMID:26688098

Interaction between drug delivery vehicles and cells under the effect of shear stress.

PubMed

Godoy-Gallardo, M; Ek, P K; Jansman, M M T; Wohl, B M; Hosta-Rigau, L

2015-09-01

Over the last decades, researchers have developed an ever greater and more ingenious variety of drug delivery vehicles (DDVs). This has made it possible to encapsulate a wide selection of therapeutic agents, ranging from proteins, enzymes, and peptides to hydrophilic and hydrophobic small drugs while, at the same time, allowing for drug release to be triggered through a diverse range of physical and chemical cues. While these advances are impressive, the field has been lacking behind in translating these systems into the clinic, mainly due to low predictability of in vitro and rodent in vivo models. An important factor within the complex and dynamic human in vivo environment is the shear flow observed within our circulatory system and many other tissues. Within this review, recent advances to leverage microfluidic devices to better mimic these conditions through novel in vitro assays are summarized. By grouping the discussion in three prominent classes of DDVs (lipidic and polymeric particles as well as inorganic nanoparticles), we hope to guide researchers within drug delivery into this exciting field and advance a further implementation of these assay systems within the development of DDVs.

A new minimally invasive heart surgery instrument for atrial fibrillation treatment: first in vitro and animal tests.

PubMed

Abadie, J; Faure, A; Chaillet, N; Rougeot, P; Beaufort, D; Goldstein, J P; Finlay, P A; Bogaerts, G

2006-06-01

The paper presents a new robotic system for beating heart surgery. The final goal of this project is to develop a tele-operated system for the thoracoscopic treatment of patients with atrial fibrillation. The system consists of a robot that moves an innovative end-effector used to perform lines as in the Cox-Maze technique. The device is an electrode mesh that is introduced in the thorax through a trocar and is deployed inside the left atrium, where it can create selective ablation lines at any atrial region, using radio frequency. The current version of the umbrella has 22 electrodes. Using visual feedback from an ultrasound based navigation system, the surgeon can choose which electrodes on the mesh to activate. Once the umbrella is in contact with the endocardium of the left atrium, at the expected position, the surgeon activates the chosen electrodes sequentially. The umbrella can then be moved to another position. In vitro and in vivo animal tests have been carried out in order to test and improve the instrument, the robotic system and the operative procedure. The performed trials proved the ability of the system to treat atrial fibrillation. More in vivo tests are currently being performed to make the robot and its device ready for clinical use. Copyright 2006 John Wiley & Sons, Ltd.

Bcl-2 antagonists kill plasmacytoid dendritic cells from lupus-prone mice and dampen interferon-α production.

PubMed

Zhan, Yifan; Carrington, Emma M; Ko, Hyun-Ja; Vikstrom, Ingela B; Oon, Shereen; Zhang, Jian-Guo; Vremec, David; Brady, Jamie L; Bouillet, Philippe; Wu, Li; Huang, David C S; Wicks, Ian P; Morand, Eric F; Strasser, Andreas; Lew, Andrew M

2015-03-01

Interferon-α (IFNα)-producing plasmacytoid dendritic cells (PDCs) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). IFNα-related genes are highlighted among SLE susceptibility alleles and are characteristically expressed in the blood of patients with SLE, while in mouse models of lupus, PDC numbers and IFNα production are increased. This study was undertaken to investigate the effects of inhibitors that selectively target different antiapoptotic molecules on the survival of PDCs. PDC numbers, in vitro survival, and expression of antiapoptotic molecules were evaluated in lupus-prone (NZB × NZW)F1 (NZB/NZW) mice. The impact of Bcl-2 antagonists and glucocorticoids on PDCs was evaluated in vitro and in vivo. IFNα production by NZB/NZW mice was evaluated before and after treatment with Bcl-2 antagonists. PDCs, but not lymphoid tissue-resident conventional DCs, largely relied on the antiapoptotic protein Bcl-2 for survival. The enlarged PDC compartment in NZB/NZW mice was associated with selectively prolonged survival and increased Bcl-2 transcription. Functionally, this resulted in enhanced production of IFNα. Bcl-2 inhibitors selectively killed mouse and human PDCs, including PDCs from SLE patients, but not conventional DCs, dampened IFNα production by PDCs, and synergized with glucocorticoids to kill activated PDCs. Enhanced PDC survival is a likely contributing factor to enhanced IFNα production by lupus PDCs. Bcl-2 antagonists potently and selectively kill PDCs and reduce IFNα production. Thus, we believe that they are attractive candidates for treating PDC-associated diseases. Copyright © 2015 by the American College of Rheumatology.

Antimicrobial photodynamic therapy in chronic osteomyelitis induced by Staphylococcus aureus: An in vitro and in vivo study

NASA Astrophysics Data System (ADS)

dos Reis Júnior, João Alves; de Assis, Patrícia Nascimento; Paraguassú, Gardênia Matos; de Vieira de Castro, Isabele Cardoso; Trindade, Renan Ferreira; Marques, Aparecida Maria Cordeiro; Almeida, Paulo Fernando; Pinheiro, Antônio Luiz Barbosa

2012-09-01

Osteomyelitis it is an acute or chronic inflammation in the marrow spaces in the superficial or cortical bone, and associated to bacterial infection. Chronic osteomyelitis represents a major health problem due to its difficult treatment and increased morbidity. Antimicrobial photodynamic therapy (APT) by laser is a treatment based on a cytotoxic photochemical reaction in which, a bright light produced by a laser system and an active photosensitizer absorbed by cells leads an activation that induces a series of metabolic reactions that culminates a bacterial killing. The aim of this study was to assess, both in vitro and in vivo, the effect of lethal laser photosensitization on osteomyelitis. On the in vitro study a diode laser (λ660nm; 40mW; o/ = 0.4 cm2; 5 or 10 J/cm2) and 5, 10 and 15μg/mL toluidine blue (TB) were tested and the best parameter chosen for the in vivo study. The concentration of 5μg/mL was selected to perform the decontamination of infected by Staphylococcus aureus tibial bone defects in rats. The results were performed by ANOVA test. On the in vitro studies all PDTs groups in the different concentrations reduced significantly (p<0,001) the amount of bacteria. On the in vivo study PDT group presented a bacterial reduction of 97,4% (P<0,001). The photodynamic therapy using toluidine blue was effective in reducing the staphiloccocus aureus in both in vitro and in vivo studies.

Extracellular proteome analysis of Leptospira interrogans serovar Lai.

PubMed

Zeng, Lingbing; Zhang, Yunyi; Zhu, Yongzhang; Yin, Haidi; Zhuang, Xuran; Zhu, Weinan; Guo, Xiaokui; Qin, Jinhong

2013-10-01

Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

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

Foot and mouth disease vaccine strain selection: Current approaches and future perspectives.

PubMed

Mahapatra, Mana; Parida, Satya

2018-06-27

Lack of cross protection between foot and mouth disease (FMD) virus (FMDV) serotypes as well as incomplete protection between some subtypes of FMDV affect the application of vaccine in the field. Further, the emergence of new variant FMD viruses periodically makes the existing vaccine inefficient. Consequently, periodical vaccine strain selection either by in vivo methods or in vitro methods become an essential requirement to enable utilisation of appropriate and efficient vaccines. Areas covered: Here we describe the cross reactivity of the existing vaccines with the global pool of circulating viruses and the putative selected vaccine strains for targeting protection against the two major circulating serotype O and A FMD viruses for East Africa, the Middle East, South Asia and South East Asia. Expert Commentary: Although in vivo cross protection studies are more appropriate methods for vaccine matching and selection than in vitro neutralisation test or ELISA, in the face of an outbreak both in vivo and in vitro methods of vaccine matching are not easy, and time consuming. The FMDV capsid contains all the immunogenic epitopes, and therefore vaccine strain prediction models using both capsid sequence and serology data will likely replace existing tools in the future.

Formulation and in vitro and in vivo characterization of a phenytoin self-emulsifying drug delivery system (SEDDS).

PubMed

Atef, Eman; Belmonte, Albert A

2008-11-15

The aim of this study is to develop and characterize a self-emulsifying drug delivery system (SEDDS) of phenytoin, and to compare its relative bioavailability to a commercially available suspension. Four phenytoin SEDDS were prepared and evaluated. Following emulsification, the optimized formula was selected to have the smallest mean particle size and the highest absolute zeta potential, which should yield the formation of a stable emulsion. Its dissolution characteristics were superior to the other SEDDS formulas. In vivo and in vitro tests were run to compare the optimized formula, SEDDS II, to a commercially available Dilantin suspension. The in vitro dissolution indicated a significant improvement in phenytoin release characteristics. The in vivo study using male rats showed a clear enhancement in phenytoin oral absorption from SEDDS compared to Dilantin suspension. The area under the curve AUC((-10min-->10h)) of phenytoin after SEDDS administration increased by 2.3 times compared to Dilantin (p<0.05), and the rate of absorption of phenytoin was significantly faster from the SEDDS. The concentration after 30min (C(30min)) of SEDDS administration was 4.9 times higher than C(30min) after Dilantin administration (p<0.05). A sustained effect of phenytoin in plasma was also observed. After 12 weeks storage, SEDDS II was found to be chemically and physically stable under stressed conditions.

In Vitro Methods To Measure Toxicity Of Chemicals

DTIC Science & Technology

2004-12-01

industrial compounds for toxicity will require high-throughput in vitro assays with which to select candidate compounds for more intensive animal...for estimating the starting dose for the rat oral acute toxicity test, thus reducing and refining the use of animals in the toxicological

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.

A Smart Molecule for Selective Sensing of Nitric Oxide: Conversion of NO to HSNO; Relevance of Biological HSNO Formation.

PubMed

Islam, Abu Saleh Musha; Bhowmick, Rahul; Pal, Kaberi; Katarkar, Atul; Chaudhuri, Keya; Ali, Mahammad

2017-04-17

A smart molecule, QT 490 , containing thiosemicarbazide moiety acts as a highly selective turn-on in vitro NO sensor through the unprecedented NO-induced transformation of thiosemicarbazide moiety to 1,3,4-oxadiazole heterocycle with the concomitant release of HSNO, thereby eliminating any interference from various endogenous biomolecules including dehydroascorbic acid, ascorbic acid, etc. The kinetic studies of the reactions between QT 490 and NO provide a mechanistic insight into formation of HSNO/RSNO from the reaction between H 2 S/RSH and NO in the biological system. This novel probe is non-cytotoxic, cell permeable, water-soluble, and appropriate for intracellular cytoplasmic NO sensing with the possibilities of in vivo applications.

Microleakage of teeth restored with different adhesive dowel systems: an in vitro study.

PubMed

Sarı, Tuğrul; Özyesil, Atilla G

2014-01-01

Commercial fiber-reinforced dowel systems are marketed as having better adhesion and sealing ability than conventional metallic dowel systems. The aim of this in vitro study was to evaluate the microleakage of teeth restored with nine dowel systems. Ninety mandibular second premolar teeth were decoronated, and nine homogenous groups were composed of ten teeth each. Root canal and dowel space preparations were made, and eight fiber-reinforced composite dowel systems and one stainless steel dowel system were used to fabricate dowel restorations. Microleakage measurements of the restored teeth were made with a modified fluid filtration method, and data were collected. One sample Kolmogorov-Smirnov, one-way ANOVA, and Tukey-HSD tests were performed on the relative microleakage data of the groups. Microleakage values were reported relative to those for teeth with unfilled canals. The highest and lowest relative microleakage values were recorded for the metallic Parapost (7.06 × 10(-4) %) and fiber-reinforced Everstick (3.55 × 10(-4) %) groups, respectively. Significant differences in relative microleakage between the fiber-reinforced dowels and stainless steel dowels were observed. Significant differences among the fiber-reinforced dowel groups were observed as well. The sealing ability of all fiber-reinforced composite dowels is not better than that of stainless steel dowels, and there are significant differences among different fiber-reinforced dowel systems as well. Differences among commercial dowel systems must be taken into consideration when making a selection. © 2013 by the American College of Prosthodontists.

Microemulsions as vehicles for topical administration of voriconazole: formulation and in vitro evaluation.

PubMed

El-Hadidy, Gladious Naguib; Ibrahim, Howida Kamal; Mohamed, Magdi Ibrahim; El-Milligi, Mohamed Farid

2012-01-01

This work was undertaken to investigate microemulsion (ME) as a topical delivery system for the poorly water-soluble voriconazole. Different ME components were selected for the preparation of plain ME systems with suitable rheological properties for topical use. Two permeation enhancers were incorporated, namely sodium deoxycholate or oleic acid. Drug-loaded MEs were evaluated for their physical appearance, pH, rheological properties and in vitro permeation studies using guinea pig skin. MEs based on polyoxyethylene(10)oleyl ether (Brij 97) as the surfactant showed pseudoplastic flow with thixotropic behavior and were loaded with voriconazole. Jojoba oil-based MEs successfully prolonged voriconazole release up to 4 h. No significant changes in physical or rheological properties were recorded on storage for 12 months at ambient conditions. The presence of permeation enhancers favored transdermal rather than dermal delivery. Sodium deoxycholate was more effective than oleic acid for enhancing the voriconazole permeation. Voriconazole-loaded MEs, with and without enhancers, showed significantly better antifungal activity against Candida albicans than voriconazole supersaturated solution. In conclusion, the studied ME formulae could be promising vehicles for topical delivery of voriconazole.

A novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da

2009-08-01

The pursuit of efficient and highly targeting-selective transporters is an active topic in cancer-targeting therapy. In this study, a novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes (SWCNTs) was developed to investigate cancer cell targeting in vitro. SWCNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). PL-PEG functionalized SWCNTs were then conjugated with fluorescein isothiocyanate (FITC) labeled integrin αvβ3 monoclonal antibody to construct SWCNT-integrin αvβ3 monoclonal antibody system (denoted as SWCNT-PEG-mAb). In vitro study revealed that the system had a high efficiency in cancer cell targeting in integrin αvβ3 positive U87MG cells. Moreover, the SWCNT-PEG-mAb is stable in physiological media, and can be readily transported into U87MG cells via integrin αvβ3-mediated endocytosis in cell. In summary, the integrin αvβ3 monoclonal antibody labeled SWCNT is a potential carrier-candidate for cancer-imaging and drug-delivering in cancer-targeting therapy.

Alkaloids from psychotria target sirtuins: in silico and in vitro interaction studies.

PubMed

Sacconnay, Lionel; Ryckewaert, Lucie; Dos Santos Passos, Carolina; Guerra, Maria Cristina; Kato, Lucilia; Alves de Oliveira, Cecilia Maria; Henriques, Amélia; Carrupt, Pierre-Alain; Simões-Pires, Claudia; Nurisso, Alessandra

2015-04-01

Epigenetic enzymes such as histone deacetylases play a crucial role in the development of ageing-related diseases. Among the 18 histone deacetylase isoforms found in humans, class III histone deacetylases, also known as sirtuins, seem to be promising targets for treating neurodegenerative conditions. Recently, Psychotria alkaloids, mainly monoterpene indoles, have been reported for their inhibitory properties against central nervous system cholinesterase and monoamine oxidase proteins. Given the multifunctional profile of these alkaloids in the central nervous system, and the fact that the indole scaffold has been previously associated with sirtuin inhibition, we hypothesized that these indole derivatives could also interact with sirtuins. In the present study, alkaloids previously isolated from Psychotria spp. were evaluated for their potential interaction with human sirtuin 1 and sirtuin 2 by molecular docking and molecular dynamics simulation approaches. The in silico results allowed for the selection of five potentially active compounds, namely, prunifoleine, 14-oxoprunifoleine, E-vallesiachotamine, Z-vallesiachotamine, and vallesiachotamine lactone. The sirtuin inhibition of these compounds was confirmed in vitro in a dose-response manner, with preliminary information on their pharmacokinetics properties. Georg Thieme Verlag KG Stuttgart · New York.

Physicochemical characterization of Capstone depleted uranium aerosols IV: in vitro solubility analysis.

PubMed

Guilmette, Raymond A; Cheng, Yung Sung

2009-03-01

As part of the Capstone Depleted Uranium (DU) Aerosol Study, the solubility of selected aerosol samples was measured using an accepted in vitro dissolution test system. This static system was employed along with a SUF (synthetic ultrafiltrate) solvent, which is designed to mimic the physiological chemistry of extracellular fluid. Using sequentially obtained solvent samples, the dissolution behavior over a 46-d test period was evaluated by fitting the measurement data to two- or three-component negative exponential functions. These functions were then compared with Type M and S absorption taken from the International Commission on Radiological Protection Publication 66 Human Respiratory Tract Model. The results indicated that there was a substantial variability in solubility of the aerosols, which in part depended on the type of armor being impacted by the DU penetrator and the particle size fraction being tested. Although some trends were suggested, the variability noted leads to uncertainties in predicting the solubility of other DU-based aerosols. Nevertheless, these data provide a useful experimental basis for modeling the intake-dose relationships for inhaled DU aerosols arising from penetrator impact on armored vehicles.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

PubMed

Flores-Herrera, Patricio; Arredondo-Zelada, Oscar; Marshall, Sergio H; Gómez, Fernando A

2018-06-01

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis. Copyright © 2018. Published by Elsevier B.V.

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

Investigations on the 4-Quinolone-3-Carboxylic Acid Motif. 5. Modulation of the Physicochemical Profile of a Set of Potent and Selective Cannabinoid-2 Receptor Ligands through a Bioisosteric Approach

PubMed Central

Nocerino, Stefania; Pedani, Valentina; Pasquini, Serena; Tafi, Andrea; De Chiaro, Maria; Bellucci, Luca; Valoti, Massimo; Guida, Francesca; Luongo, Livio; Dragoni, Stefania; Ligresti, Alessia; Rosenberg, Avraham; Bolognini, Daniele; Cascio, Maria Grazia; Pertwee, Roger G.; Moaddel, Ruin; Maione, Sabatino; Di Marzo, Vincenzo

2012-01-01

Three heterocyclic systems were selected as potential surrogates of the amide linker for a series of 1,6-disubstituted-4-quinolone-3-carboxamides, potent and selective CB2 ligands exhibiting scarce water solubility, with the aim of improving their physicochemical profile and also of clarifying properties of importance for amide bond mimicry. Among the newly synthesized compounds, the 1,2,3-triazole derivative 11 emerged as the most promising in terms of both physicochemical and pharmacodynamic properties. When assayed in vitro, 11 exhibited inverse agonist activity, whereas, in vivo, in the formalin test in mice, it produced analgesic effects antagonized by a well established inverse agonist. Metabolic studies allowed the identification of the side chain hydroxylated derivative 32 as its only metabolite which, in its racemic form, showed still appreciable CB2 selectivity, but was 150-fold less potent than the parent compound. PMID:22383251

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

Dilts, R.P.; Kalivas, P.W.

The enkephalin analog (2-D-penicillamine, 5-D-penicillamine)enkephalin was radioiodinated (125I-DPDPE) and shown to retain a pharmacological selectivity characteristic of the delta opioid receptor in in vitro binding studies. The distributions of 125I-DPDPE binding, using in vitro autoradiographic techniques, were similar to those previously reported for the delta opioid receptor. The nucleus accumbens, striatum, and medial prefrontal cortex contain dense gradients of 125I-DPDPE binding in regions known to receive dopaminergic afferents emanating from the mesencephalic tegmentum. Selective chemical lesions of the ventral tegmental area and substantia nigra were employed to deduce the location of the 125I-DPDPE binding within particular regions of the mesocorticolimbicmore » dopamine system. Unilateral lesions of dopamine perikarya (A9 and A10) within the ventral tegmental area and substantia nigra produced by mesencephalic injection of 6-hydroxydopamine resulted in significant (20-30%) increases in 125I-DPDPE binding contralateral to the lesion within the striatum and nucleus accumbens. Lesions of the perikarya (dopaminergic and nondopaminergic) of the ventral tegmental area, induced by quinolinic acid injections, caused increases of less magnitude within these same nuclei. No significant alterations in 125I-DPDPE binding were observed within the mesencephalon as a result of either treatment. The specificity of the lesions was confirmed by immunocytochemistry for tyrosine hydroxylase. These results suggest that the enkephalins and opioid agonists acting through delta opioid receptors do not directly modulate dopaminergic afferents but do regulate postsynaptic targets of the mesocorticolimbic dopamine system.« less

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1990-01-01

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

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 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

Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

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

Kim, Jae Ho; Kim, Sang Hie; Kolozsvary, A.

1995-11-01

The purpose of this investigation was to demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses or irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growingmore » in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 {mu}g/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 {plus_minus} 0.1 and 1.3 {plus_minus} 0.1, respectively. Exposure of cells to 10 {mu}g/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 {plus_minus} 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors. 25 refs., 6 figs.« less

New Class of Antitrypanosomal Agents Based on Imidazopyridines.

PubMed

Silva, Daniel G; Gillespie, J Robert; Ranade, Ranae M; Herbst, Zackary M; Nguyen, Uyen T T; Buckner, Frederick S; Montanari, Carlos A; Gelb, Michael H

2017-07-13

The present work describes the synthesis of 22 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and 12 compounds exhibited an in vitro EC 50 ≤ 1 μM against Trypanosoma cruzi ( T. cruzi ) and Trypanosoma brucei ( T. brucei ) parasites, respectively. Based on promising results of in vitro activity (EC 50 < 100 nM), cytotoxicity, metabolic stability, protein binding, and pharmacokinetics (PK) properties, compound 20 was selected as a candidate for in vivo efficacy studies. This compound was screened in an acute mouse model against T.cruzi ( Tulahuen strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an in vivo imaging system (IVIS). Compound 20 demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound 20 represents a potential lead for the development of drugs to treat trypanosomiasis.

Reconstructing Ancient Forms of Life

NASA Technical Reports Server (NTRS)

Benner, Steven A.

1998-01-01

Progress in the past three months has occurred in two areas, reconstruction of ancestral proteins and improved understanding of chemical features that are likely to be universal in generic matter regardless of its genesis. Ancestral ribonucleases have been reconstructed, and an example has been developed that shows how physiological function can be assigned to in vitro behaviors observed in biological systems. Sequence data have been collected to permit the reconstruction of src homology 2 domains that underwent radiative divergence at the time of the radiative divergence of chordates. New studies have been completed that show how genetic matter (or its remnants) might be detected on Mars (or other non-terrean locations.) Last, the first in vitro selection experiments have been completed using a nucleoside library carrying positively charged functionality, illustrating the importance of non-standard nucleotides to those attempting to obtain evidence for an "RNA world" as an early episode of life on earth.

Efficiency of Different Endodontic Irrigation and Activation Systems in Removal of the Smear Layer: A Scanning Electron Microscopy Study.

PubMed

Karade, Priyatam; Chopade, Rutuja; Patil, Suvarna; Hoshing, Upendra; Rao, Madhukar; Rane, Neha; Chopade, Aditi; Kulkarni, Anish

2017-01-01

This in vitro study was designed to evaluate and compare different endodontic irrigation and activation systems for removal of the intracanal smear layer. Forty recently extracted, non-carious human intact single rooted premolars were selected and divided into five groups ( n =10) according to the root canal irrigation systems; syringe and needle irrigation (CTR), sonic irrigation, passive ultrasonic irrigation (PUI) and EndoVac irrigation system. All groups were prepared to #40 apical size with K-files. Each sample was subjected to final irrigation by using four different irrigation/activation systems. After splitting the samples, one half of each root was selected for examination under scanning electron microscope (SEM). The irrigation systems were compared using the Fisher's exact test with the level of significance set at 0.05. The four groups did not differ from each other in the coronal and mid-root parts of the canal. In the apical part of the canal none of the methods could completely remove all the smear layer but EndoVac system showed significantly better removal of smear layer and debris than the other methods. Within the limitations of the present study, the EndoVac system cleaned the apical part of the canal more efficiently than sonic, ultrasonic and syringe and needle irrigation.

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll.

PubMed

Rodríguez Villamil, P; Wei, H; Moreira, G; Caccia, M; Fernandez Taranco, M; Bó, G A

2012-07-01

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production. Copyright © 2012 Elsevier Inc. All rights reserved.

An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

PubMed Central

Janebodin, Kajohnkiart; Buranaphatthana, Worakanya; Ieronimakis, Nicholas; Hays, Aislinn L.; Reyes, Morayma

2013-01-01

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors. PMID:23841093

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.

Unnatural selection in chemical systems

NASA Technical Reports Server (NTRS)

Orgel, Leslie E.

1995-01-01

The theory of evolution through natural selection was proposed by Darwin and Wallace to explain how the characteristics of populations of animals change with time. An examination of their assumptions shows that the theory has much broader application than they originally envisaged. We now know that in appropriate environments RNA molecules or computer viruses, for example, can evolve. The adventure with which we are concerned is the quest for chemical systems that undergo processes analogous to Darwinian selection in the test tube. The search is not restricted to systems that are closely related to nucleic acids, although most of the available experimental evidence concerns such systems. A population of molecules satisfies all the requirements of the theory is there are different kinds of molecules in the population and if each individual molecule can direct the formation of copies of itself, then a population of molecules will adapt to a varying environment by changing its composition so as to maintain as high as possible a rate of replication. Sol Spiegelman is the inventor of 'unnatural selection'. He showed clearly that populations of RNA molecules evolve when replicated repeatedly by Q beta RNA polymerase under a chosen set of adverse reaction conditions. In the systems that he studied, the initial population was fairly homogeneous and much of the variation was created during the course of the experiment by mutation, that is, error of replication. The term 'unnatural selection' will be used loosely to describe evolution of nucleic acids or other replicatable polymers in vitro. The term 'Natural Selection' will be reserved for the evolution of living organisms and their viruses. Natural Section usually involves the coevolution of nucleic acids and proteins, while 'unnatural selection', as practiced so far, allows replicating nucleic acids to evolve but hold constant the enzymes that catalyze replication. It is widely believed that biology based on DNA, RNA, and proteins was preceded by the biology of an 'RNA world' in which enzymes were composed of RNA alone. The origin of RNA replication is thus the central puzzle of the origins of life. Consequently, RNA-catalyzed RNA replication is presently one of the main goals of experimental work on unnatural selection. However, there is also a more distant goal, namely, to achieve replication and selection in systems unrelated to RNA. These different systems are discussed in this article.

Protein and genome evolution in Mammalian cells for biotechnology applications.

PubMed

Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

2009-06-01

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

Application of Rhizobacteria for Plant Growth Promotion Effect and Biocontrol of Anthracnose Caused by Colletotrichum acutatum on Pepper

PubMed Central

Lamsal, Kabir; Kim, Sang Woo; Kim, Yun Seok

2012-01-01

In vitro and greenhouse screening of seven rhizobacterial isolates, AB05, AB10, AB11, AB12, AB14, AB15 and AB17, was conducted to investigate the plant growth promoting activities and inhibition against anthracnose caused by Colletotrichum acutatum in pepper. According to identification based on 16S rDNA sequencing, the majority of the isolates are members of Bacillus and a single isolate belongs to the genus Paenibacillus. All seven bacterial isolates were capable of inhibiting C. acutatum to various degrees. The results primarily showed that antibiotic substances produced by the selected bacteria were effective and resulted in strong antifungal activity against the fungi. However, isolate AB15 was the most effective bacterial strain, with the potential to suppress more than 50% mycelial growth of C. acutatum in vitro. Moreover, antibiotics from Paenibacillus polymyxa (AB15) and volatile compounds from Bacillus subtilis (AB14) exerted efficient antagonistic activity against the pathogens in a dual culture assay. In vivo suppression activity of selected bacteria was also analyzed in a greenhouse with the reference to their prominent in vitro antagonism efficacy. Induced systemic resistance in pepper against C. acutatum was also observed under greenhouse conditions. Where, isolate AB15 was found to be the most effective bacterial strain at suppressing pepper anthracnose under greenhouse conditions. Moreover, four isolates, AB10, AB12, AB15, and AB17, were identified as the most effective growth promoting bacteria under greenhouse conditions, with AB17 inducing the greatest enhancement of pepper growth. PMID:23323049

A scalable engineering approach to improve performance of a miniaturized optical detection system for in vitro point-of-care testing

NASA Astrophysics Data System (ADS)

Robbins, Hannah; Hu, Sijung; Liu, Changqing

2015-03-01

The demand for rapid screening technologies, to be used outside of a traditional healthcare setting, has been vastly expanding. This is requiring a new engineering platform for faster and cost effective techniques to be easily adopted through forward-thinking manufacturing procedures, i.e., advanced miniaturisation and heterogeneous integration of high performance microfluidics based point-of-care testing (POCT) systems. Although there has been a considerable amount of research into POCT systems, there exist tremendous challenges and bottlenecks in the design and manufacturing in order to reach a clinical acceptability of sensitivity and selectivity, as well as smart microsystems for healthcare. The project aims to research how to enable scalable production of such complex systems through 1) advanced miniaturisation of a physical layout and opto-electronic component allocation through an optimal design; and 2) heterogeneous integration of multiplexed fluorescence detection (MFD) for in vitro POCT. Verification is being arranged through experimental testing with a series of dilutions of commonly used fluorescence dye, i.e. Cy5. Iterative procedures will be engaged until satisfaction of the detection limit, of Cy5 dye, 1.209x10-10 M. The research creates a new avenue of rapid screening POCT manufacturing solutions with a particular view on high performance and multifunctional detection systems not only in POCT, but also life sciences and environmental applications.

On prilled Nanotubes-in-Microgel Oral Systems for protein delivery.

PubMed

de Kruif, Jan Kendall; Ledergerber, Gisela; Garofalo, Carla; Fasler-Kan, Elizaveta; Kuentz, Martin

2016-04-01

Newly discovered active macromolecules are highly promising for therapy, but poor bioavailability hinders their oral use. Microencapsulation approaches, such as protein prilling into microspheres, may enable protection from gastrointestinal (GI) enzymatic degradation. This would increase bioavailability mainly for local delivery to GI lumen or mucosa. This work's purpose was to design a novel architecture, namely a Nanotubes-in-Microgel Oral System, by prilling for protein delivery. Halloysite nanotubes (HNT) were selected as orally acceptable clay particles and their lumen was enlarged by alkaline etching. This chemical modification increased the luminal volume to a mean of 216.3 μL g(-1) (+40.8%). After loading albumin as model drug, the HNT were entrapped in microgels by prilling. The formation of Nanoparticles-in-Microsphere Oral System (NiMOS) yielded entrapment efficiencies up to 63.2%. NiMOS shape was spherical to toroidal, with a diameter smaller than 320 μm. Release profiles depended largely on the employed system and HNT type. Protein stability was determined throughout prilling and after in vitro enzymatic degradation. Prilling did not harm protein structure, and NiMOS demonstrated higher enzymatic protection than pure nanotubes or microgels, since up to 82% of BSA remained unscathed after in vitro digestion. Therefore, prilled NiMOS was shown to be a promising and flexible multi-compartment system for oral (local) macromolecular delivery. Copyright © 2016 Elsevier B.V. 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

Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

PubMed Central

Wang, Yingying; Ji, Teng; Sun, Shujuan; Mo, Qingqing; Chen, Pingbo; Fang, Yong; Liu, Jia; Wang, Beibei; Zhou, Jianfeng; Ma, Ding; Wu, Peng

2013-01-01

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells. PMID:24367661

Peptide-functionalized quantum dots for potential applications in the imaging and treatment of obesity.

PubMed

Thovhogi, Ntevheleni; Sibuyi, Nicole Remaliah Samantha; Onani, Martin Opiyo; Meyer, Mervin; Madiehe, Abram Madimabe

2018-01-01

Obesity is a worldwide epidemic affecting millions of people. The current pharmacological treatment of obesity remains limited and ineffective due to drugs' undesirable side effects. Hence, there is a need for novel or improved strategies for long-term therapies that will help prevent the disease progression into other chronic diseases. Nanotechnology holds the future for the treatment of obesity because of its versatility, as shown by improved drug efficiency and safety in cancer clinical trials. Nano-based drug delivery systems could potentially do the same for obesity through targeted drug delivery. This study investigated the use of peptide-functionalized quantum dots (QDs) for the imaging of prohibitin (PHB)-expressing cells in vitro and in diet-induced obese rats, which could potentially be used as nanocarriers of antiobesity drugs. Cadmium (Cd)-based QDs were functionalized with an adipose homing peptide (AHP) and injected intravenously into lean and obese Wistar rats. Biodistribution of the QDs was analyzed by an IVIS ® Lumina XR imaging system and inductively coupled plasma optical emission spectroscopy (ICP-OES). For in vitro studies, PHB-expressing (Caco-2 and MCF-7) and non-PHB-expressing (KMST-6 and CHO) cells were exposed to either unfunctionalized QDs (QD625) or AHP-functionalized QDs (AHP-QD625) and analyzed by fluorescence microscopy. AHP-QD625 accumulated significantly in PHB-expressing cells in vitro when compared with non-PHB-expressing cells. In vivo data indicated that QD625 accumulated mainly in the reticuloendothelial system (RES) organs, while the AHP-QD625 accumulated mostly in the white adipose tissues (WATs). AHP-functionalized QDs were successfully and selectively delivered to the PHB-expressing cells in vitro (Caco-2 and MCF-7 cells) and in the WAT vasculature in vivo. This nanotechnology-based approach could potentially be used for dual targeted drug delivery and molecular imaging of adipose tissues in obese patients in real time.

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

PubMed

Wieben, E D; Nenninger, J M; Pederson, T

1985-05-05

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

Experimental evidence for killing the resistant cells and raising the efficacy and decreasing the toxicity of cytostatics and irradiation by mixtures of the agents of the passive antitumor defense system in the case of various tumor and normal cell lines in vitro.

PubMed

Kulcsár, Gyula

2009-02-01

Despite the substantial decline of the immune system in AIDS, only a few kinds of tumors increase in incidence. This shows that the immune system has no absolute role in the prevention of tumors. Therefore, the fact that tumors do not develop in the majority of the population during their lifetime indicates the existence of other defense system(s). According to our hypothesis, the defense is made by certain substances of the circulatory system. Earlier, on the basis of this hypothesis, we experimentally selected 16 substances of the circulatory system and demonstrated that the mixture of them (called active mixture) had a cytotoxic effect (inducing apoptosis) in vitro and in vivo on different tumor cell lines, but not on normal cells and animals. In this paper, we provide evidence that different cytostatic drugs or irradiation in combination with the active mixture killed significantly more cancer cells, compared with either treatments alone. The active mixture decreased, to a certain extent, the toxicity of cytostatics and irradiation on normal cells, but the most important result was that the active mixture destroyed the multidrug-resistant cells. Our results provide the possibility to improve the efficacy and reduce the side-effects of chemotherapy and radiation therapy and to prevent the relapse by killing the resistant cells.

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...

Effect of solvent on in vitro dissolution: Summary of results for uranium, americium, and cobalt aerosols

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

Guilmette, R.A.; Hoover, M.D.

1995-12-01

The revised 10 CFR Part 20 has adopted the ICRP Publication 30 method for calculating the committed effective dose equivalent from intakes of radionuclides. This dosimetry scheme requires knowledge or assumptions about the chemical form of the radionuclide, its particle size, and its known or assumed solubility. The solubility is classified as being either D (relatively soluble), W, or Y (relatively insoluble), depending on whether the material dissolves over periods of days, weeks, or years. Although Nuclear Regulatory Commission licensees may wish to take advantage of material-specific knowledge in order to adjust annual limits on intake and derived air concentrations,more » relatively few radioactive materials to which workers and the general population may be exposed have been adequately characterized either in terms of physicochemical form or solubility. Experimental measurement of solubility using some type of in vitro dissolution measurement system is therefore needed. However, there is currently no clear consensus regarding the appropriate design of in vitro dissolution systems, particularly when considering the range of different radionuclides to be studied, and the complexity of the biological mechanisms involved in the retention and clearance of inhaled deposited radioactive particles. The purpose of this study was to evaluate the effect of the several solvents on the dissolution of four test aerosols ({sup 57}Co{sub 3}O{sub 4}, {sup 241}AmO{sub 2}, ammonium diuranate [ADU], and U{sub 3}O{sub 8}) selected to encompass a variety of chemical and biochemical properties in vivo. The results of this study provide some guidance on the usefulness of in vitro dissolution tests for estimating the solubility of unknown radionuclide particles within the context of a simple model such as the class D, W, and Y formulation of ICRP 30.« less

A DNA enzyme that cleaves RNA

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

1994-01-01

BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

Apta-nanosensor preparation and in vitro assay for rapid Diazinon detection using a computational molecular approach.

PubMed

Jokar, Mahmoud; Safaralizadeh, Mohammad Hassan; Hadizadeh, Farzin; Rahmani, Fatemeh; Kalani, Mohamad Reza

2017-02-01

Aptamers (ss-DNA or ss-RNA), also known as artificial antibodies, have been selected in vitro median to bind target molecules with high affinity and selectivity. Diazinon is one of the most widely used organophosphorus insecticides in developing and underdeveloped countries as insecticide and acaricide. Diazinon is readily absorbed from the gastrointestinal system and rapidly distributed throughout the body. Thus, the design of clinical and laboratory diagnostics using nanobiosensors is necessary. A computational approach allows us to screen or rank receptor structure and predict interaction outcomes with a deeper understanding, and it is much more cost effective than laboratory attempts. In this research, the best sequence (high affinity bind Diazinon-ssDNA) was ranked among 12 aptamers isolated from SELEX experimentation. Docking results, as the first virtual screening stage and static technique, selected frequent conformation of each aptamer. Then, the quantity and quality of aptamer-Diazinon interaction were simulated using molecular dynamics as a mobility technique. RMSD, RMSF, radius of gyration, and the number of hydrogen bonds formed between Diazinon-aptamer were monitored to assess the quantity and quality of interactions. G-quadruplex DNA aptamer (DF20) showed to be a reliable candidate for Diazinon biosensing. The apta-nanosensor designed using simulation results allowed with linearity detection in the range of .141-.65 nM and a LOD of 17.903 nM, and it was validated using a computational molecular approach.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

PubMed Central

Bao, Xingfeng; Zheng, Wanjun; Sugi, Naoko Hata; Agarwala, Kishan L; Xu, Qunli; Wang, Zichun; Tendyke, Karen; Lee, Winnie; Parent, Lana; Li, Wei; Cheng, Hongsheng; Shen, Yongchun; Taylor, Noel; Dezso, Zoltan; Du, Hong; Kotake, Yoshihiko; Zhao, Nanding; Wang, John; Postema, Maarten; Woodall-Jappe, Mary; Takase, Yasutaka; Uenaka, Toshimitsu; Kingston, David G I; Nomoto, Kenichi

2015-01-01

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment. PMID:25729885

Diagnosis of stinging insect allergy: utility of cellular in-vitro tests.

PubMed

Scherer, Kathrin; Bircher, Andreas J; Heijnen, Ingmar Afm

2009-08-01

Diagnosis of stinging insect allergy is based on a detailed history, venom skin tests, and detection of venom-specific IgE. As an additional diagnostic tool, basophil responsiveness to venom allergens has been shown to be helpful in selected patients. This review summarizes the current diagnostic procedures for stinging insect allergy and discusses the latest developments in cellular in-vitro tests. Cellular assays have been evaluated in patients with Hymenoptera venom allergy. The diagnostic performance of the cellular mediator release test is similar to that of the flow cytometric basophil activation test (BAT), but the BAT has been the most intensively studied. BAT offers the possibility to assess basophil reactivity to allergens in their natural environment and to simultaneously analyze surface marker expression and intracellular signaling. It has been demonstrated that BAT represents a valuable additional diagnostic tool in selected patients when used in combination with other well established tests. A major limitation is the current lack of unified, standardized protocols. Flow cytometry offers huge possibilities to enhance knowledge of basophil functions. The BAT may be used as an additional test to confirm the diagnosis of stinging insect allergy in selected patients, provided that it is performed by an experienced laboratory using a validated assay. Test results have to be interpreted by clinicians familiar with the methodological aspects. The utility of the BAT to confirm allergy diagnosis and to predict the risk of subsequent systemic reactions may be improved by combined analysis of multiple surface markers and intracellular signaling pathways.

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

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.

Quenching-induced deactivation of photosensitizer by nanoencapsulation to improve phototherapy of cancer.

PubMed

Zeisser-Labouèbe, Magali; Mattiuzzo, Marc; Lange, Norbert; Gurny, Robert; Delie, Florence

2009-09-01

Photodynamic therapy has emerged as a promising alternative to current cancer treatment. However, conventional photosensitizers have several limitations due to their unsuitable pharmaceutical formulations and lack of selectivity. Our strategy was to exploit the advantages of nanoparticles and the quenching-induced deactivation of the model photosensitizer hypericin to produce "activatable" drug delivery systems. Efficient fluorescence and activity quenching were achieved by increasing the drug-loading rate of nanoparticles. In vitro assays confirmed the reversibility of hypericin deactivation, as the hypericin fluorescence and photodynamic activity were recovered upon cell internalization.

Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

PubMed

Luke, Garry A; Ryan, Martin D

2018-01-01

To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

Selection of Functional Quorum Sensing Systems by Lysogenic Bacteriophages in Pseudomonas aeruginosa

PubMed Central

Saucedo-Mora, Miguel A.; Castañeda-Tamez, Paulina; Cazares, Adrián; Pérez-Velázquez, Judith; Hense, Burkhard A.; Cazares, Daniel; Figueroa, Wendy; Carballo, Marco; Guarneros, Gabriel; Pérez-Eretza, Berenice; Cruz, Nelby; Nishiyama, Yoshito; Maeda, Toshinari; Belmont-Díaz, Javier A.; Wood, Thomas K.; García-Contreras, Rodolfo

2017-01-01

Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, some of which are used as public goods. Since their production is a cooperative behavior, it is susceptible to social cheating in which non-cooperative QS deficient mutants use the resources without investing in their production. Nevertheless, functional QS systems are abundant; hence, mechanisms regulating the amount of cheating should exist. Evidence that demonstrates a tight relationship between QS and the susceptibility of bacteria against the attack of lytic phages is increasing; nevertheless, the relationship between temperate phages and QS has been much less explored. Therefore, in this work, we studied the effects of having a functional QS system on the susceptibility to temperate bacteriophages and how this affects the bacterial and phage dynamics. We find that both experimentally and using mathematical models, that the lysogenic bacteriophages D3112 and JBD30 select QS-proficient P. aeruginosa phenotypes as compared to the QS-deficient mutants during competition experiments with mixed strain populations in vitro and in vivo in Galleria mellonella, in spite of the fact that both phages replicate better in the wild-type background. We show that this phenomenon restricts social cheating, and we propose that temperate phages may constitute an important selective pressure toward the conservation of bacterial QS. PMID:28912771

Synthesis and in vitro testing of a pyropheophorbide-a-fullerene hexakis adduct immunoconjugate for photodynamic therapy.

PubMed

Rancan, Fiorenza; Helmreich, Matthias; Mölich, Andreas; Ermilov, Eugeny A; Jux, Norbert; Röder, Beate; Hirsch, Andreas; Böhm, Fritz

2007-01-01

The employment of carriers to enhance drug selectivity is one of the strategies to increase the efficacy and reduce the side effects of antitumor therapy. The concept of a modular carrier system (MCS) was developed to construct a complex drug having a high efficacy and selectivity. An MCS employs diverse units or modules: beside the therapeutic unit, an addressing unit (e.g., an antibody) serves to direct the drug to its target, and a multiplying unit has the role of increasing the number of biological active moieties the system can carry. In this paper, we report on the synthesis of a modular carrier system in which the role of multiplying unit is given to a [5:1]fullerene hexakis adduct. This fullerene hexaadduct has five malonate spacers which can bind two therapeutic units (the photosensitizer pyropheophorbide-a) each, for a total of ten, and a longer malonate spacer which serves for the conjugation to the addressing unit, the monoclonal antibody rituximab. Confocal microscopy studies using Epstein-Barr virus-transformed B-lymphocytes and Jurkat cells showed that the antibody conjugate conserves the affinity for its receptor (CD20) and its selectivity toward CD20 positive B-lymphocytes. On the contrary, the antibody-free complex did not show any bounding or intracellular uptake.

Boronic acid-based chemical sensors for saccharides.

PubMed

Zhang, Xiao-Tai; Liu, Guang-Jian; Ning, Zhang-Wei; Xing, Guo-Wen

2017-11-27

During the past decades, the interaction between boronic acids-functionalized sensors and saccharides is of great interest in the frontier domain of the interdiscipline concerning both biology and chemistry. Various boronic acid-based sensing systems have been developed to detect saccharides and corresponding derivatives in vitro as well as in vivo, which embrace unimolecular sensors, two-component sensing ensembles, functional assemblies, and boronic acid-loaded nanomaterials or surfaces. New sensing strategies emerge in endlessly with excellent selectivity and sensitivity. In this review, several typical sensing systems were introduced and some promising examples were highlighted to enable the deep insight of saccharides sensing on the basis of boronic acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Delivery of risperidone from gels across porcine skin in vitro and in vivo in rabbits.

PubMed

Ning, Yuming; Chen, Xiaojin; Yu, Zhenwei; Liang, Wenquan; Li, Fanzhu

2018-05-01

The purpose of this study was to develop and evaluate a transdermal delivery system for RIS using hydrogels. First, the effects of different concentrations of hydroxypropyl methylcellulose and Carbomer 934 (CBR) on RIS permeation were investigated in porcine skin. The optimized formulation was chosen as the base gel to screen for penetration enhancers. The pharmacokinetics of the optimized RIS formulation was then studied in vitro in rabbits. A formulation with 0.5% CBR showed the highest RIS permeation and was selected as the base gel. RIS permeation was further increased by incorporation of Azone, lauryl alcohol, or menthol, and the enhancing effects of the three were dose-dependent. When each enhancer combined with propylene glycol (PG) a synergistic effect was found. A combination of 6% menthol and 6% PG exhibited highest RIS in vitro penetration rate and showed a high efficiency in vivo, with a relative bioavailability of 131.53% compared with intragastric administration. These findings showed that 1% RIS in 0.5% CBR, containing a combination of 6% menthol and 6% PG, can deliver doses of RIS that are therapeutically relevant for treating patients with schizophrenia.

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.

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

[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.

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

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.

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.

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-silico Leishmania target selectivity of antiparasitic terpenoids.

PubMed

Ogungbe, Ifedayo Victor; Setzer, William N

2013-07-03

Neglected Tropical Diseases (NTDs), like leishmaniasis, are major causes of mortality in resource-limited countries. The mortality associated with these diseases is largely due to fragile healthcare systems, lack of access to medicines, and resistance by the parasites to the few available drugs. Many antiparasitic plant-derived isoprenoids have been reported, and many of them have good in vitro activity against various forms of Leishmania spp. In this work, potential Leishmania biochemical targets of antiparasitic isoprenoids were studied in silico. Antiparasitic monoterpenoids selectively docked to L. infantum nicotinamidase, L. major uridine diphosphate-glucose pyrophosphorylase and methionyl t-RNA synthetase. The two protein targets selectively targeted by germacranolide sesquiterpenoids were L. major methionyl t-RNA synthetase and dihydroorotate dehydrogenase. Diterpenoids generally favored docking to L. mexicana glycerol-3-phosphate dehydrogenase. Limonoids also showed some selectivity for L. mexicana glycerol-3-phosphate dehydrogenase and L. major dihydroorotate dehydrogenase while withanolides docked more selectively with L. major uridine diphosphate-glucose pyrophosphorylase. The selectivity of the different classes of antiparasitic compounds for the protein targets considered in this work can be explored in fragment- and/or structure-based drug design towards the development of leads for new antileishmanial drugs.

Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

PubMed

Jonnalagadda, Mahesh; Brown, Christine E; Chang, Wen-Chung; Ostberg, Julie R; Forman, Stephen J; Jensen, Michael C

2013-01-01

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

Optimization of in vitro inhibition of HT-29 colon cancer cell cultures by Solanum tuberosum L. extracts.

PubMed

Zuber, T; Holm, D; Byrne, P; Ducreux, L; Taylor, M; Kaiser, M; Stushnoff, C

2015-01-01

Secondary metabolites in potato have been reported to possess bioactive properties, including growth inhibition of cancer cells. Because potatoes are widely consumed globally, potential health benefits may have broad application. Thus we investigated growth inhibition of HT-29 colon cancer cell cultures by extracts from 13 diverse genetic breeding clones. Extracts from three pigmented selections (CO97226-2R/R, CO97216-1P/P, CO04058-3RW/RW) inhibited growth of in vitro HT-29 cell cultures more effectively than other clones tested. While inhibition was highest from pigmented selections and pigmented tuber tissue sectors, not all pigmented breeding lines tested had appreciable inhibitory properties. Thus, inhibition was not uniquely linked to pigmentation. Immature tubers had the highest inhibitory properties, and in most cases mature tubers retained very low inhibition properties. Flowers and skins inhibited strongly at lower extract concentrations. An extract consisting of 7.2 mg mL⁻¹ cell culture medium was the lowest effective concentration. While raw tuber extracts inhibited most effectively, a few clones at higher concentrations retained inhibition after cooking. Heated whole tubers retained higher inhibition than heated aqueous extracts. While all aqueous extracts from the two tuber selections (CO97216-1P/P and CO97226-2R/R) inhibited HT-29 cell cultures, inhibition was significantly enhanced in purple pigmented tubers of CO97216-1P/P prepared cryogenically as liquid nitrogen powders compared to extracts from freeze dried samples. Upregulation of caspase-3 protease activity, indicative of apoptosis, was highest among the most inhibitory clone samples. The unique sectorial red pigment expressing selection (CO04058-3RW/RW) provided a model system that isolated expression in pigmented sectors, and thus eliminated developmental, environmental and genetic confounding.

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.

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.

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

Induction of human embryonic and induced pluripotent stem cells into urothelium.

PubMed

Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

2014-05-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

PubMed Central

Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

2014-01-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

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.

A targeted illumination optical fiber probe for high resolution fluorescence imaging and optical switching

NASA Astrophysics Data System (ADS)

Shinde, Anant; Perinchery, Sandeep Menon; Murukeshan, Vadakke Matham

2017-04-01

An optical imaging probe with targeted multispectral and spatiotemporal illumination features has applications in many diagnostic biomedical studies. However, these systems are mostly adapted in conventional microscopes, limiting their use for in vitro applications. We present a variable resolution imaging probe using a digital micromirror device (DMD) with an achievable maximum lateral resolution of 2.7 μm and an axial resolution of 5.5 μm, along with precise shape selective targeted illumination ability. We have demonstrated switching of different wavelengths to image multiple regions in the field of view. Moreover, the targeted illumination feature allows enhanced image contrast by time averaged imaging of selected regions with different optical exposure. The region specific multidirectional scanning feature of this probe has facilitated high speed targeted confocal imaging.

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.

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

In vitro digestive stability of complexes between gliadin and synthetic blocking peptides.

PubMed

Hoffmann, Karolina; Carlsson, Nils-Gunnar; Alminger, Marie; Chen, Tingsu; Wold, Agnes; Olsson, Olof; Sandberg, Ann-Sofie

2011-05-01

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

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.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

PubMed Central

Fitzmaurice, Arthur G.; Rhodes, Shannon L.; Lulla, Aaron; Murphy, Niall P.; Lam, Hoa A.; O’Donnell, Kelley C.; Barnhill, Lisa; Casida, John E.; Cockburn, Myles; Sagasti, Alvaro; Stahl, Mark C.; Maidment, Nigel T.; Ritz, Beate; Bronstein, Jeff M.

2013-01-01

Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), preferential degeneration of dopaminergic neurons, and development of PD. This hypothesis is supported by multiple lines of evidence. (i) We previously showed in mice the metabolism of benomyl to S-methyl N-butylthiocarbamate sulfoxide, which inhibits ALDH at nanomolar levels. We report here that benomyl exposure in primary mesencephalic neurons (ii) inhibits ALDH and (iii) alters dopamine homeostasis. It induces selective dopaminergic neuronal damage (iv) in vitro in primary mesencephalic cultures and (v) in vivo in a zebrafish system. (vi) In vitro cell loss was attenuated by reducing DOPAL formation. (vii) In our epidemiology study, higher exposure to benomyl was associated with increased PD risk. This ALDH model for PD etiology may help explain the selective vulnerability of dopaminergic neurons in PD and provide a potential mechanism through which environmental toxicants contribute to PD pathogenesis. PMID:23267077

-A curated transcriptomic dataset collection relevant to embryonic development associated with in vitro fertilization in healthy individuals and patients with polycystic ovary syndrome.

PubMed

Mackeh, Rafah; Boughorbel, Sabri; Chaussabel, Damien; Kino, Tomoshige

2017-01-01

The collection of large-scale datasets available in public repositories is rapidly growing and providing opportunities to identify and fill gaps in different fields of biomedical research. However, users of these datasets should be able to selectively browse datasets related to their field of interest. Here we made available a collection of transcriptome datasets related to human follicular cells from normal individuals or patients with polycystic ovary syndrome, in the process of their development, during in vitro fertilization. After RNA-seq dataset exclusion and careful selection based on study description and sample information, 12 datasets, encompassing a total of 85 unique transcriptome profiles, were identified in NCBI Gene Expression Omnibus and uploaded to the Gene Expression Browser (GXB), a web application specifically designed for interactive query and visualization of integrated large-scale data. Once annotated in GXB, multiple sample grouping has been made in order to create rank lists to allow easy data interpretation and comparison. The GXB tool also allows the users to browse a single gene across multiple projects to evaluate its expression profiles in multiple biological systems/conditions in a web-based customized graphical views. The curated dataset is accessible at the following link: http://ivf.gxbsidra.org/dm3/landing.gsp.

­A curated transcriptomic dataset collection relevant to embryonic development associated with in vitro fertilization in healthy individuals and patients with polycystic ovary syndrome

PubMed Central

Mackeh, Rafah; Boughorbel, Sabri; Chaussabel, Damien; Kino, Tomoshige

2017-01-01

The collection of large-scale datasets available in public repositories is rapidly growing and providing opportunities to identify and fill gaps in different fields of biomedical research. However, users of these datasets should be able to selectively browse datasets related to their field of interest. Here we made available a collection of transcriptome datasets related to human follicular cells from normal individuals or patients with polycystic ovary syndrome, in the process of their development, during in vitro fertilization. After RNA-seq dataset exclusion and careful selection based on study description and sample information, 12 datasets, encompassing a total of 85 unique transcriptome profiles, were identified in NCBI Gene Expression Omnibus and uploaded to the Gene Expression Browser (GXB), a web application specifically designed for interactive query and visualization of integrated large-scale data. Once annotated in GXB, multiple sample grouping has been made in order to create rank lists to allow easy data interpretation and comparison. The GXB tool also allows the users to browse a single gene across multiple projects to evaluate its expression profiles in multiple biological systems/conditions in a web-based customized graphical views. The curated dataset is accessible at the following link: http://ivf.gxbsidra.org/dm3/landing.gsp. PMID:28413616

Decreased nitric oxide levels stimulate osteoclastogenesis and bone resorption both in vitro and in vivo on the chick chorioallantoic membrane in association with neoangiogenesis.

PubMed

Collin-Osdoby, P; Rothe, L; Bekker, S; Anderson, F; Osdoby, P

2000-03-01

High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.

A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

PubMed Central

Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

2017-01-01

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

Local delivery of hormonal therapy with silastic tubing for prevention and treatment of breast cancer.

PubMed

Park, Jeenah; Thomas, Scott; Zhong, Allison Y; Wolfe, Alan R; Krings, Gregor; Terranova-Barberio, Manuela; Pawlowska, Nela; Benet, Leslie Z; Munster, Pamela N

2018-01-08

Broad use of germline testing has identified an increasing number of women at risk for breast cancer with a need for effective chemoprevention. We report a novel method to selectively deliver various anti-estrogens at high drug levels to the breast tissue by implanting a device comprised of silastic tubing. Optimized tubing properties allow elution of otherwise poorly bioavailable anti-estrogens, such as fulvestrant, into mammary tissue in vitro and in vivo with levels sufficient to inhibit estrogen receptor activation and tumor cell proliferation. Implantable silastic tubing delivers fulvestrant selectively to mouse mammary fat tissue for one year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Furthermore, local delivery of fulvestrant significantly decreases cell proliferation, as assessed by Ki67 expression, most effectively in tumor sections adjacent to tubing. This approach may thereby introduce a potential paradigm shift and offer a promising alternative to systemic therapy for prevention and early interception of breast cancer.

A device for emulating cuff recordings of action potentials propagating along peripheral nerves.

PubMed

Rieger, Robert; Schuettler, Martin; Chuang, Sheng-Chih

2014-09-01

This paper describes a device that emulates propagation of action potentials along a peripheral nerve, suitable for reproducible testing of bio-potential recording systems using nerve cuff electrodes. The system is a microcontroller-based stand-alone instrument which uses established nerve and electrode models to represent neural activity of real nerves recorded with a nerve cuff interface, taking into consideration electrode impedance, voltages picked up by the electrodes, and action potential propagation characteristics. The system emulates different scenarios including compound action potentials with selectable propagation velocities and naturally occurring nerve traffic from different velocity fiber populations. Measured results from a prototype implementation are reported and compared with in vitro recordings from Xenopus Laevis frog sciatic nerve, demonstrating that the electrophysiological setting is represented to a satisfactory degree, useful for the development, optimization and characterization of future recording systems.

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

Integrated biocircuits: engineering functional multicellular circuits and devices.

PubMed

Prox, Jordan; Smith, Tory; Holl, Chad; Chehade, Nick; Guo, Liang

2018-04-01

Implantable neurotechnologies have revolutionized neuromodulatory medicine for treating the dysfunction of diseased neural circuitry. However, challenges with biocompatibility and lack of full control over neural network communication and function limits the potential to create more stable and robust neuromodulation devices. Thus, we propose a platform technology of implantable and programmable cellular systems, namely Integrated Biocircuits, which use only cells as the functional components of the device. We envision the foundational principles for this concept begins with novel in vitro platforms used for the study and reconstruction of cellular circuitry. Additionally, recent advancements in organoid and 3D culture systems account for microenvironment factors of cytoarchitecture to construct multicellular circuits as they are normally formed in the brain. We explore the current state of the art of these platforms to provide knowledge of their advancements in circuit fabrication and identify the current biological principles that could be applied in designing integrated biocircuit devices. We have highlighted the exemplary methodologies and techniques of in vitro circuit fabrication and propose the integration of selected controllable parameters, which would be required in creating suitable biodevices. We provide our perspective and propose new insights into the future of neuromodulaion devices within the scope of living cellular systems that can be applied in designing more reliable and biocompatible stimulation-based neuroprosthetics.

Integrated biocircuits: engineering functional multicellular circuits and devices

NASA Astrophysics Data System (ADS)

Prox, Jordan; Smith, Tory; Holl, Chad; Chehade, Nick; Guo, Liang

2018-04-01

Objective. Implantable neurotechnologies have revolutionized neuromodulatory medicine for treating the dysfunction of diseased neural circuitry. However, challenges with biocompatibility and lack of full control over neural network communication and function limits the potential to create more stable and robust neuromodulation devices. Thus, we propose a platform technology of implantable and programmable cellular systems, namely Integrated Biocircuits, which use only cells as the functional components of the device. Approach. We envision the foundational principles for this concept begins with novel in vitro platforms used for the study and reconstruction of cellular circuitry. Additionally, recent advancements in organoid and 3D culture systems account for microenvironment factors of cytoarchitecture to construct multicellular circuits as they are normally formed in the brain. We explore the current state of the art of these platforms to provide knowledge of their advancements in circuit fabrication and identify the current biological principles that could be applied in designing integrated biocircuit devices. Main results. We have highlighted the exemplary methodologies and techniques of in vitro circuit fabrication and propose the integration of selected controllable parameters, which would be required in creating suitable biodevices. Significance. We provide our perspective and propose new insights into the future of neuromodulaion devices within the scope of living cellular systems that can be applied in designing more reliable and biocompatible stimulation-based neuroprosthetics.

Inhibiting the immunoproteasome exacerbates the pathogenesis of systemic Candida albicans infection in mice

PubMed Central

Mundt, Sarah; Basler, Michael; Buerger, Stefanie; Engler, Harald; Groettrup, Marcus

2016-01-01

Apart from its role in MHC class I antigen processing, the immunoproteasome has recently been implicated in the modulation of T helper cell differentiation under polarizing conditions in vitro and in the pathogenesis of autoimmune diseases in vivo. In this study, we investigated the influence of LMP7 on T helper cell differentiation in response to the fungus Candida albicans. We observed a strong effect of ONX 0914, an LMP7-selective inhibitor of the immunoproteasome, on IFN-γ and IL-17A production by murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated with C. albicans in vitro. Using a murine model of systemic candidiasis, we could confirm reduced generation of IFN-γ- and IL-17A-producing cells in ONX 0914 treated mice in vivo. Interestingly, ONX 0914 treatment resulted in increased susceptibility to systemic candidiasis, which manifested at very early stages of infection. Mice treated with ONX 0914 showed markedly increased kidney and brain fungal burden which resulted in enhanced neutrophil recruitment and immunopathology. Together, these results strongly suggest a role of the immunoproteasome in promoting proinflammatory T helper cells in response to C. albicans but also in affecting the innate antifungal immunity in a T helper cell-independent manner. PMID:26776888

A modified parallel artificial membrane permeability assay for evaluating the bioconcentration of highly hydrophobic chemicals in fish.

PubMed

Kwon, Jung-Hwan; Escher, Beate I

2008-03-01

Low cost in vitro tools are needed at the screening stage of assessment of bioaccumulation potential of new and existing chemicals because the number of chemical substances that needs to be tested highly exceeds the capacity of in vivo bioconcentration tests. Thus, the parallel artificial membrane permeability assay (PAMPA) system was modified to predict passive uptake/ elimination rate in fish. To overcome the difficulties associated with low aqueous solubility and high membrane affinity of highly hydrophobic chemicals, we measured the rate of permeation from the donor poly(dimethylsiloxane)(PDMS) disk to the acceptor PDMS disk through aqueous and PDMS membrane boundary layers and term the modified PAMPA system "PDMS-PAMPA". Twenty chemicals were selected for validation of PDMS-PAMPA. The measured permeability is proportional to the passive elimination rate constant in fish and was used to predict the "minimum" in vivo elimination rate constant. The in vivo data were very close to predicted values except for a few polar chemicals and metabolically active chemicals, such as pyrene and benzo[a]pyrene. Thus, PDMS-PAMPA can be an appropriate in vitro system for nonmetabolizable chemicals. Combination with metabolic clearance rates using a battery of metabolic degradation assays would enhance the applicability for metabolizable chemicals.

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…

The osteogenic response of mesenchymal stem cells to an injectable PLGA bone regeneration system.

PubMed

Curran, Judith M; Fawcett, Sandra; Hamilton, Lloyd; Rhodes, Nicholas P; Rahman, Cheryl V; Alexander, Morgan; Shakesheff, Kevin; Hunt, John A

2013-12-01

The enrichment of substrates/surfaces with selected functional groups, methyl (-CH3), allyl amine (-NH2), allyl alcohol (-OH) and acrylic acid (-COOH), can be used to trigger mesenchymal stem (MSC) cell differentiation into specified lineages, minimising the need for exogenous biological supplementation. We present the successful translation of this research phenomenon to an injectable two phase injectable PLGA system, utilising plasma techniques, for the repair of bone defects. Modified microspheres were characterised using water contact angel (WCA), X-ray Photon Spectroscopy (XPS) and scanning electron microscopy (SEM). When cultured in contact with MSCs in vitro, the ability of the modified particles, within the 2 phase system, to induce differentiation was characterised using quantitative assays for cell viability and histological analysis for key markers of differentiation throughout the entirety of the three dimensional scaffold. Biological analysis proved that selected modified microspheres have the ability to induce MSC osteogenic (-NH2 modified scaffolds) and chondrogenic (-OH modified scaffolds) differentiation throughout the entirety of the formed scaffold. Therefore optimised plasma modification of microspheres is an effective tool for the production of injectable systems for the repair of bone and cartilage defects. Copyright © 2013 Elsevier Ltd. All rights reserved.

System-level multi-target drug discovery from natural products with applications to cardiovascular diseases.

PubMed

Zheng, Chunli; Wang, Jinan; Liu, Jianling; Pei, Mengjie; Huang, Chao; Wang, Yonghua

2014-08-01

The term systems pharmacology describes a field of study that uses computational and experimental approaches to broaden the view of drug actions rooted in molecular interactions and advance the process of drug discovery. The aim of this work is to stick out the role that the systems pharmacology plays across the multi-target drug discovery from natural products for cardiovascular diseases (CVDs). Firstly, based on network pharmacology methods, we reconstructed the drug-target and target-target networks to determine the putative protein target set of multi-target drugs for CVDs treatment. Secondly, we reintegrated a compound dataset of natural products and then obtained a multi-target compounds subset by virtual-screening process. Thirdly, a drug-likeness evaluation was applied to find the ADME-favorable compounds in this subset. Finally, we conducted in vitro experiments to evaluate the reliability of the selected chemicals and targets. We found that four of the five randomly selected natural molecules can effectively act on the target set for CVDs, indicating the reasonability of our systems-based method. This strategy may serve as a new model for multi-target drug discovery of complex diseases.

Improved oral bioavailability of poorly water-soluble indirubin by a supersaturatable self-microemulsifying drug delivery system.

PubMed

Chen, Zhi-Qiang; Liu, Ying; Zhao, Ji-Hui; Wang, Lan; Feng, Nian-Ping

2012-01-01

Indirubin, isolated from the leaves of the Chinese herb Isatis tinctoria L, is a protein kinase inhibitor and promising antitumor agent. However, the poor water solubility of indirubin has limited its application. In this study, a supersaturatable self-microemulsifying drug delivery system (S-SMEDDS) was developed to improve the oral bioavailability of indirubin. A prototype S-SMEDDS was designed using solubility studies and phase diagram construction. Precipitation inhibitors were selected from hydrophilic polymers according to their crystallization-inhibiting capacity through in vitro precipitation tests. In vitro release of indirubin from S-SMEDDS was examined to investigate its likely release behavior in vivo. The in vivo bioavailability of indirubin from S-SMEDDS and from SMEDDS was compared in rats. The prototype formulation of S-SMEDDS comprised Maisine™ 35-1:Cremophor(®) EL:Transcutol(®) P (15:40:45, w/w/w). Polyvinylpyrrolidone K17, a hydrophilic polymer, was used as a precipitation inhibitor based on its better crystallization-inhibiting capacity compared with polyethylene glycol 4000 and hydroxypropyl methylcellulose. In vitro release analysis showed more rapid drug release from S-SMEDDS than from SMEDDS. In vivo bioavailability analysis in rats indicated that improved oral absorption was achieved and that the relative bioavailability of S-SMEDDS was 129.5% compared with SMEDDS. The novel S-SMEDDS developed in this study increased the dissolution rate and improved the oral bioavailability of indirubin in rats. The results suggest that S-SMEDDS is a superior means of oral delivery of indirubin.

Modeling the Blood-Brain Barrier in a 3D triple co-culture microfluidic system.

PubMed

Adriani, G; Ma, D; Pavesi, A; Goh, E L K; Kamm, R D

2015-01-01

The need for a blood-brain barrier (BBB) model that accurately mimics the physiological characteristics of the in-vivo situation is well-recognized by researchers in academia and industry. However, there is currently no in-vitro model allowing studies of neuronal growth and/or function influenced by factors from the blood that cross through the BBB. Therefore, we established a 3D triple co-culture microfluidic system using human umbilical vein endothelial cells (HUVEC) together with primary rat astrocytes and neurons. Immunostaining confirmed the successful triple co-culture system consisting of an intact BBB with tight intercellular junctions in the endothelial monolayer. The BBB selective permeability was determined by a fluorescent-based assay using dextrans of different molecular weights. Finally, neuron functionality was demonstrated by calcium imaging.

Cowpea and Groundnut Haulms Fodder Trading and Its Lessons for Multidimensional Cowpea Improvement for Mixed Crop Livestock Systems in West Africa

PubMed Central

Samireddypalle, Anandan; Boukar, Ousmane; Grings, Elaine; Fatokun, Christian A.; Kodukula, Prasad; Devulapalli, Ravi; Okike, Iheanacho; Blümmel, Michael

2017-01-01

Cowpea is an important legume crop in Africa, valued highly for its grain and also haulms, which are a tradable commodity in fodder markets. Fodder market surveys in Northern Nigeria showed that groundnut haulms were priced higher than cowpea haulms, probably because of their superior nutritive value. The economic value of haulms has prompted cowpea breeders and livestock nutritionists to explore haulm fodder traits as additional selection and breeding criteria. Fifty cowpea genotypes cultivated across five locations in Nigeria in 2013 and 2014 were evaluated for food fodder traits. Significant (P < 0.05) genotypic dependent variations were observed in yields (kg/ha) of grains (537–1082) and haulms (1173–3368), though significant (P < 0.05) effects of location and year were observed. Grain and fodder yield had a tendency to be positively correlated (r = 0.26, P = 0.07). Haulms were analyzed for nitrogen (N), fiber fractions, in vitro digestibility, and metabolizable energy content. Highly significant variations were observed in all genotypic and livestock nutrition traits, although location and year had significant effects. Trade-offs between grain yield and haulm fodder quality traits were largely absent and haulm acid detergent lignin and grain yield were even inversely correlated (r = -0.28, P = 0.05), that is high grain yielders had decreased haulm lignin. However, haulm N and grain yield also tended to be negatively associated (r = -0.26, P = 0.07). Haulm fodder quality traits and haulm yield were mostly positively correlated (P < 0.05). Broad sense heritabilities for grain and fodder yield were 0.50 and 0.29, respectively, while heritability for haulm fodder quality traits ranged from 0.61 to 0.67, providing opportunities for concomitant increase in grain yield and haulm fodder quality traits. Selection of the 10 highest ranking genotypes for grain yield, haulm yield, haulm N, and haulm in vitro organic matter digestibility showed selection groups overlapping, suggesting that multi-trait selection is feasible. Economical evaluation showed that choice of primary traits is context specific, highlighting the need for identifying and targeting appropriate genotypes to fit different production systems. Considering haulm quantity and quality as traits of economic value can increase overall plant value in mixed crop-livestock systems. PMID:28197154

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

Comparative evaluation of the efficacy of a herbal mouthwash and chlorhexidine mouthwash on select periodontal pathogens: An in vitro and ex vivo study

PubMed Central

Pathan, Multazim Muradkhan; Bhat, Kishore Gajanan; Joshi, Vinayak Mahableshwar

2017-01-01

Background: Several herbal mouthwash and herbal extracts have been tested in vitro and in vivo in search of a suitable adjunct to mechanical therapy for long-term use. In this study, we aimed to look at the antimicrobial effect of the herbal mouthwash and chlorhexidine (CHX) mouthwash on select organisms in in vitro test and an ex vivo model. Materials and Methods: The antimicrobial effects were determined against standard strains of bacteria that are involved in different stages of periodontal diseases. The in vitro tests included determination of minimum inhibitory concentration (MIC) using broth dilution and agar diffusion. In the ex vivo part of the study supragingival dental plaque were obtained from 20 periodontally healthy adult volunteers. Descriptive analysis was done for the entire quantitative and qualitative variable recorded. Results: The MIC by broth dilution method found no statistically significant difference between the mouthwashes. The agar dilution method showed CHX was more effective as compared to the herbal mouthwash against standard strains of Streptococcus mutans, Streptococcus sanguinis, and Aggregatibacter actinomycetemcomitans. However, no difference was observed between the mouthwashes for Porphyromonas, Pseudomonas aeruginosa, and Fusobacterium nucleatum. The ex vivo results conclude that none of the selected mouthwashes were statistically significantly different from each other. Conclusion: In the present study, CHX showed higher levels of antimicrobial action than the herbal mouthwash against bacterial species. The results reinforce the earlier findings that the in vitro testing is sensitive to methods and due diligence is needed when extrapolating the data for further use. However, long-term use and in vivo effectiveness against the periopathogens need to be tested in well-planned clinical trials. PMID:29456300

Risperidone mucoadhesive buccal tablets: formulation design, optimization and evaluation

PubMed Central

Çelik, Burak

2017-01-01

The aim of this study was to design and optimize risperidone (RIS) mucoadhesive buccal tablets for systemic delivery as an alternative route. Direct compression method was used for the preparation of buccal tablets, and screening studies were conducted with different polymers to determine their effects on tablet characteristics. Carbopol® (CP) and sodium alginate (SA) were selected as two polymer types for further optimization studies by applying response surface methodology. Tablet hardness (TH), ex vivo residence time (RT), and peak detachment force (DF) from buccal mucosa were selected as three important responses. Physicochemical compatibility of formulation excipients and RIS was evaluated by using Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) analysis. In vitro drug release profiles and release kinetics were investigated; swelling index and matrix erosion studies were conducted. Optimum formulation consisted of 16.4% CP and 20.3% SA, which provided 7.67±0.29 hour ex vivo RT, 45.52±4.85 N TH, and 2.12±0.17 N DF. FT-IR spectroscopy and DSC analysis revealed that there was no chemical interaction present between tablet ingredients. Cumulative RIS release of >90% was achieved after 8 hours of in vitro dissolution studies, which was supported by swelling and matrix erosion analysis. Mechanism of RIS release was fitted best to zero-order model, while release exponent (n) value of 0.77 demonstrated an anomalous (non-Fickian) release, indicating combined erosion and swelling mechanism. The results suggested that optimized buccal tablets of RIS would be a promising and alternative delivery system for the treatment of schizophrenia. PMID:29225461

Preparation, characterization and in vitro evaluation of microemulsion of raloxifene hydrochloride.

PubMed

Golmohammadzadeh, Shiva; Farhadian, Nafiseh; Biriaee, Amir; Dehghani, Faranak; Khameneh, Bahman

2017-10-01

Raloxifene hydrochloride (RLX) is a selective estrogen receptor modulator which is orally used for treatment of osteoporosis and prevention of breast cancer. The drug has low aqueous solubility and bioavailability. The aim of the present study is to formulate and characterize oil-in-water microemulsion systems for oral delivery of RLX. To enhance the drug aqueous solubility, microemulsion based on sesame oil was prepared. Sesame oil and Tween 80 were selected as the drug solvent oil and surfactant, respectively. In the first and second formulations, Edible glycerin and Span 80 were applied as co-surfactant, respectively. Pseudo-ternary phase diagrams showed that the best surfactant/co-surfactant ratios in the first and second formulations were 4:1 and 9:1, respectively. The particle size of all free drug-loaded and drug loaded samples were in the range of 31.25 ± 0.3 nm and 60.9 ± 0.1 nm, respectively. Electrical conductivity coefficient and refractive index of all microemulsion samples confirmed the formation of oil-in-water type of microemulsion. In vitro drug release profile showed that after 24 hours, 46% and 63% of the drug released through the first formulation in 0.1% (w/v) Tween 80 in distilled water as a release medium and phosphate buffer solution (PBS) at pH = 5.5, respectively. These values were changed to 57% and 98% for the second formulation. Results confirmed that the proposed microemulsion system containing RLX could improve and control the drug release profile in comparison to conventional dosage form.

Apoptin towards safe and efficient anticancer therapies.

PubMed

Backendorf, Claude; Noteborn, Mathieu H M

2014-01-01

The chicken anemia virus derived protein apoptin harbors cancer-selective cell killing characteristics, essentially based on phosphorylation-mediated nuclear transfer in cancer cells and efficient cytoplasmic degradation in normal cells. Here, we describe a growing set of preclinical experiments underlying the promises of the anti-cancer potential of apoptin. Various non-replicative oncolytic viral vector systems have revealed the safety and efficacy of apoptin. In addition, apoptin enhanced the oncolytic potential of adenovirus, parvovirus and Newcastle disease virus vectors. Intratumoral injection of attenuated Salmonella typhimurium bacterial strains and plasmid-based systems expressing apoptin resulted in significant tumor regression. In-vitro and in-vivo experiments showed that recombinant membrane-transferring PTD4- or TAT-apoptin proteins have potential as a future anticancer therapeutics. In xenografted hepatoma and melanoma mouse models PTD4-apoptin protein entered both cancer and normal cells, but only killed cancer cells. Combinatorial treatment of PTD4-apoptin with various (chemo)therapeutic compounds revealed an additive or even synergistic effect, reducing the side effects of the single (chemo)therapeutic treatment. Degradable polymeric nanocapsules harboring MBP-apoptin fusion-protein induced tumor-selective cell killing in-vitro and in-vivo and revealed the potential of polymer-apoptin protein vehicles as an anticancer agent.Besides its direct use as an anticancer therapeutic, apoptin research has also generated novel possibilities for drug design. The nuclear location domains of apoptin are attractive tools for targeting therapeutic compounds into the nucleus of cancer cells. Identification of cancer-related processes targeted by apoptin can potentially generate novel drug targets. Recent breakthroughs important for clinical applications are reported inferring apoptin-based clinical trials as a feasible reality.

Drug delivery to macrophages for the therapy of cancer and infectious diseases.

PubMed

Kirsh, R; Bugelski, P J; Poste, G

1987-01-01

The mechanisms by which mononuclear phagocytes discriminate between self and nonself, recognize foreign materials, senescent, damaged, old, or effete cells, and tumor cells are unknown. However, regardless of the mechanism(s) involved, once activated by the appropriate signal(s), macrophages are able to selectively recognize and destroy neoplastic cells in vitro and in vivo. Liposomes injected intravenously, in common with other particulate or polymeric matrices, localize preferentially in organs with high mononuclear phagocyte activity and in circulating blood monocytes. This behavior allows microparticulates to serve as a convenient system for the selective delivery of encapsulated drugs to cells of the mononuclear phagocyte series in vivo. Liposomes are a particularly attractive experimental system because of their capacity to incorporate a wide variety of water-soluble and lipid-soluble drugs. At this time, however, there is no reason to assume that a liposome-based drug delivery system will offer any significant therapeutic advantage compared to other microparticulate drug delivery systems. As in commercial development of any pharmaceutical preparation, considerations of cost-of-goods, shelf life, and acceptance of the formulation and dosing regimen by both physicians and patients will be of major importance in determining success and widespread clinical use. Liposomes containing macrophage-activating agents are highly effective at augmenting macrophage-mediated tumoricidal activity in vitro eradicating tumor metastasis in vivo, as well as protecting animals from a wide variety of microbial and viral infections. Although the demands of solving the scientific and technical problems associated with liposome development are substantial, the rapid rate of progress in biology and in pharmaceutical sciences enhances the prospect of success for at least several aspects of liposome-mediated drug delivery. The next few years will be crucial in determining whether the commercial development of liposomes is feasible or whether they will join the ranks of other drug carrier designs that have failed to fulfill their initial promise.

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

In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells.

PubMed

Jung, Kwang Bo; Lee, Hana; Son, Ye Seul; Lee, Ji Hye; Cho, Hyun-Soo; Lee, Mi-Ok; Oh, Jung-Hwa; Lee, Jaemin; Kim, Seokho; Jung, Cho-Rok; Kim, Janghwan; Son, Mi-Young

2018-01-01

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5 mCherry ) and intestine-specific homeobox enhanced green fluorescence protein (ISX eGFP ). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. © FASEB.

Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos.

PubMed

Chang, T Arthur; Bondarenko, Gennadiy I; Gerami-Naini, Behzad; Drenzek, Jessica G; Durning, Maureen; Garthwaite, Mark A; Schmidt, Jenna Kropp; Golos, Thaddeus G

2018-03-16

The initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system. Blastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation. Signs of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding. This 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.

Toward toxicity testing of nanomaterials in the 21st century: a paradigm for moving forward.

PubMed

Lai, David Y

2012-01-01

A challenge-facing hazard identification and safety evaluation of engineered nanomaterials being introduced to market is the diversity and complexity of the types of materials with varying physicochemical properties, many of which can affect their toxicity by different mechanisms. In general, in vitro test systems have limited usefulness for hazard identification of nanoparticles due to various issues. Meanwhile, conducting chronic toxicity/carcinogenicity studies in rodents for every new nanomaterial introduced into the commerce is impractical if not impossible. New toxicity testing systems which rely on predictive, high-throughput technologies may be the ultimate goal of evaluating the potential hazard of nanomaterials. However, at present, this approach alone is unlikely to succeed in evaluating the toxicity of the wide array of nanomaterials and requires validation from in vivo studies. This article proposes a paradigm for toxicity testing and elucidation of the molecular mechanisms of reference materials for specific nanomaterial classes/subclasses using short-term in vivo animal studies in conjunction with high-throughput screenings and mechanism-based short-term in vitro assays. The hazard potential of a particular nanomaterial can be evaluated by conducting only in vitro high-throughput assays and mechanistic studies and comparing the data with those of the reference materials in the specific class/subclass-an approach in line with the vision for 'Toxicity Testing in the 21st Century' of chemicals. With well-designed experiments, testing nanomaterials of varying/selected physicochemical parameters may be able to identify the physicochemical parameters contributing to toxicity. The data so derived could be used for the development of computer model systems to predict the hazard potential of specific nanoparticles based on property-activity relationships. Copyright © 2011 John Wiley & Sons, Inc.

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...

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB(1) receptor antagonist for the management of obesity.

PubMed

Hadcock, John R; Griffith, David A; Iredale, Phillip A; Carpino, Phillip A; Dow, Robert L; Black, Shawn C; O'Connor, Rebecca; Gautreau, Denise; Lizano, Jeffrey S; Ward, Karen; Hargrove, Diane M; Kelly-Sullivan, Dawn; Scott, Dennis O

2010-04-02

Cannabinoid CB(1) receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB(1) receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB(1) receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB(1) receptors in both binding (K(i)=0.7 nM) and functional assays (K(i)=0.2 nM). The compound has low affinity (K(i)=7600 nM) for human CB(2) receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB(1) receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system. 2010 Elsevier Inc. All rights reserved.

ATP Mediates Neuroprotective and Neuroproliferative Effects in Mouse Olfactory Epithelium following Exposure to Satratoxin G In Vitro and In Vivo

PubMed Central

Jia, Cuihong; Sangsiri, Sutheera; Belock, Bethany; Iqbal, Tania R.; Pestka, James J.; Hegg, Colleen C.

2011-01-01

Intranasal aspiration of satratoxin G (SG), a mycotoxin produced by the black mold Stachybotrys chartarum, selectively induces apoptosis in olfactory sensory neurons (OSNs) in mouse olfactory epithelium (OE) through unknown mechanisms. Here, we show a dose-dependent induction of apoptosis 24 h post-SG exposure in vitro as measured by increased activated caspases in the OP6 olfactory placodal cell line and increased propidium iodide staining in primary OE cell cultures. Intranasal aspiration of SG increased TUNEL (Terminal dUTP Nick End Labeling) staining in the neuronal layer of the OE and significantly increased the latency to find a buried food pellet, confirming that SG selectively induces neuronal apoptosis and demonstrating that SG impairs the sense of smell. Next, we investigated whether ATP can prevent SG-induced OE toxicity. ATP did not decrease apoptosis under physiological conditions but significantly reduced SG-induced OSN apoptosis in vivo and in vitro. Furthermore, purinergic receptor inhibition significantly increased apoptosis in OE primary cell culture and in vivo. These data indicate that ATP is neuroprotective against SG-induced OE toxicity. The number of cells that incorporated 5′-bromodeoxyuridine, a measure of proliferation, was significantly increased 3 and 6 days post-SG aspiration. Treatment with purinergic receptor antagonists significantly reduced SG-induced cell proliferation, whereas post-treatment with ATP significantly potentiated SG-induced cell proliferation. These data indicate that ATP is released and promotes cell proliferation via activation of purinergic receptors in SG-induced OE injury. Thus, the purinergic system is a therapeutic target to alleviate or restore the loss of OSNs. PMID:21865290

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays.

PubMed

Le Fol, Vincent; Aït-Aïssa, Selim; Sonavane, Manoj; Porcher, Jean-Marc; Balaguer, Patrick; Cravedi, Jean-Pierre; Zalko, Daniel; Brion, François

2017-08-01

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolism and disposition of MM-433593, a selective FAAH-1 inhibitor, in monkeys

PubMed Central

Banijamali, Ali R; Wakefield, James D; Mermerian, Ara H; Busby, Robert W

2014-01-01

MM-433593 is a highly potent and selective inhibitor of fatty acid amide hydrolase-1 (FAAH-1) with potential utility as an orally administered treatment of pain, inflammation, and other disorders. In this study, we investigated the metabolism and pharmacokinetics of MM-433593 in monkeys, and compared plasma and urine metabolites of this compound to the in vitro metabolites produced by monkey hepatocytes. Intravenous administration of MM-433593 to cynomolgus monkeys produced a rapid distribution phase and slower elimination phase with a mean systemic clearance rate of 8–11 mL/min/kg. Absolute oral bioavailability was determined to be 14–21% with maximum plasma concentrations reached ∼3 h (Tmax) following a 10 mg/kg oral dose. The average terminal half-life of MM-433593 was 17–20 h, and there were no qualitative sex differences in the metabolite profile of MM-433593. The major site of metabolism was oxidation of the methyl group at the five position of the indole ring, which was confirmed by chromatography and mass spectrometry comparison to a synthesized authentic standard. This metabolite was further oxidized to the corresponding carboxylic acid and/or conjugated with sulfate, glucuronide, or glutathione. In all, 18 metabolites were found in plasma and urine. In vitro incubations of MM-433593 with monkey hepatocytes yielded 13 metabolites, all of which were found in vivo, indicating a good correlation between the in vitro and in vivo metabolism data. A comprehensive pathway for the metabolism of MM-433593 is proposed, including a plausible, five-step biotransformation for the formation of N-acetylcysteine conjugate metabolite (M18) from the hydroxylated parent (M5). PMID:25505606

Clinical Confirmation that the Selective JAK1 Inhibitor Filgotinib (GLPG0634) has a Low Liability for Drug-drug Interactions.

PubMed

Namour, Florence; Desrivot, Julie; Van der Aa, Annegret; Harrison, Pille; Tasset, Chantal; van't Klooster, Gerben

2016-01-01

The selective Janus kinase 1 inhibitor filgotinib (GLPG0634), which is currently in clinical development for the treatment of rheumatoid arthritis (RA) and Crohn's disease, demonstrated encouraging safety and efficacy profiles in RA patients after 4 weeks of daily dosing. As RA patients might be treated with multiple medications simultaneously, possible drug-drug interactions of filgotinib with cytochrome P450 enzymes and with key drug transporters were evaluated in vitro and in clinical studies. The enzymes involved in filgotinib's metabolism and the potential interactions of the parent and its active major metabolite with drug-metabolizing enzymes and drug transporters, were identified using recombinant enzymes, human microsomes, and cell systems. Furthermore, filgotinib's interaction potential with CYP3A4 was examined in an open-label study in healthy volunteers, which evaluated the impact of filgotinib co-administration on the CYP3A4-sensitive substrate midazolam. The potential interaction with the common RA drug methotrexate was investigated in a clinical study in RA patients. In vitro, filgotinib and its active metabolite at clinically relevant concentrations did not interact with cytochrome P450 enzymes and uridine 5'-diphospho-glucuronosyltransferases, and did not inhibit key drug transporters. In the clinic, a lack of relevant pharmacokinetic drug interactions by filgotinib and its active metabolite with substrates of CYP3A4, as well as with organic anion transporters involved in methotrexate elimination were found. the collective in vivo and in vitro data on drug-metabolizing enzymes and on key drug transporters, support co-administration of filgotinib with commonly used RA drugs to patients without the need for dose adjustments.

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.

Clinically significant borderline resistance of sequential clinical isolates of Klebsiella pneumoniae.

PubMed

Wagenlehner, F M E; Heisig, P; Irtenkauf, C; Notka, F; Decker, J; Lehn, N; Linde, H

2003-10-01

Two sequential clinical isolates of Klebsiella pneumoniae (Kpn) were isolated from bronchoalveolar lavage fluid (Kpn#1) and sputum (Kpn#2) of a patient with pneumonia, complicated by anatomical and immunosuppressive problems due to Wegener's granulomatosis. Despite 4 weeks of systemic treatment with ciprofloxacin (CIP) Kpn#2 was isolated thereafter. A fluoroquinolone-resistant mutant (Kpn#1-SEL) was derived from Kpn#1 in vitro by selecting on agar plates supplemented with ofloxacin. Kpn#1, Kpn#1-SEL and Kpn#2 had an identical pattern in PFGE. CIP MICs were 0.25, 2 and 4 mg/l for Kpn#1, Kpn#2 and Kpn#1-SEL, respectively. Kpn ATCC 10031 (CIP MIC 0.002 mg/l) served as control. We analyzed mechanisms of fluoroquinolone resistance by determining antibiotic susceptibility, organic solvent tolerance, accumulation of fluoroquinolones, dominance testing with wild-type topoisomerase genes (gyrA/B, parC/E), sequencing of the quinolone resistance determining regions of gyrA/B, parC/E and marR and Northern blotting of marR and acrAB genes. Compared with Kpn ATCC 10031, elevated MICs to fluoroquinolones and unrelated antibiotics in Kpn#1 was presumably due to a primary efflux pump other than AcrAB and increased the CIP MIC 125-fold. Although Kpn#1 tested sensitive according to NCCLS breakpoints, the elevated CIP MIC of 0.25 mg/l presumably rendered this isolate clinically resistant and lead to therapeutic failure in this case. Further increase of MIC to fluoroquinolones in vivo and in vitro was distinct. Kpn#1-SEL, selected in vitro, acquired a GyrA target mutation, whereas in Kpn#2 no known resistance mechanism could be detected.

Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial

PubMed Central

2012-01-01

The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a “stop effect” of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial. PMID:23176151

Novel orally available salvinorin A analog PR-38 inhibits gastrointestinal motility and reduces abdominal pain in mouse models mimicking irritable bowel syndrome.

PubMed

Sałaga, M; Polepally, P R; Sobczak, M; Grzywacz, D; Kamysz, W; Sibaev, A; Storr, M; Do Rego, J C; Zjawiony, J K; Fichna, J

2014-07-01

The opioid and cannabinoid systems play a crucial role in multiple physiological processes in the central nervous system and in the periphery. Selective opioid as well as cannabinoid (CB) receptor agonists exert a potent inhibitory action on gastrointestinal (GI) motility and pain. In this study, we examined (in vitro and in vivo) whether PR-38 (2-O-cinnamoylsalvinorin B), a novel analog of salvinorin A, can interact with both systems and demonstrate therapeutic effects. We used mouse models of hypermotility, diarrhea, and abdominal pain. We also assessed the influence of PR-38 on the central nervous system by measurement of motoric parameters and exploratory behaviors in mice. Subsequently, we investigated the pharmacokinetics of PR-38 in mouse blood samples after intraperitoneal and oral administration. PR-38 significantly inhibited mouse colonic motility in vitro and in vivo. Administration of PR-38 significantly prolonged the whole GI transit time, and this effect was mediated by µ- and κ-opioid receptors and the CB1 receptor. PR-38 reversed hypermotility and reduced pain in mouse models mimicking functional GI disorders. These data expand our understanding of the interactions between opioid and cannabinoid systems and their functions in the GI tract. We also provide a novel framework for the development of future potential treatments of functional GI disorders. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

PubMed

Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

2009-01-01

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

Photodynamic characterization of amino acid conjugated 15(1)-hydroxypurpurin-7-lactone for cancer treatment.

PubMed

Lim, Siang Hui; Yam, Mun Li; Lam, May Lynn; Kamarulzaman, Fadzly Azhar; Samat, Norazwana; Kiew, Lik Voon; Chung, Lip Yong; Lee, Hong Boon

2014-09-02

This study aims to improve the photodynamic properties and biological effectiveness of 15(1)-hydroxypurpurin-7-lactone dimethyl ester (G2), a semisynthetic photosensitizer, for the PDT treatment of cancer. The strategy we undertook was by conjugating G2 with aspartic acid and lysine amino acid moieties. The photophysical properties, singlet oxygen generation, distribution coefficiency (Log D in octanol/PBS pH 7.4), and photostability of these analogues and their in vitro bioactivities such as cellular uptake, intracellular localization, and photoinduced cytotoxicity were evaluated. In addition, selected analogues were also investigated for their PDT-induced vasculature occlusion in the chick chorioallantoic membrane model and for their antitumor efficacies in Balb/C mice bearing 4T1 mouse mammary tumor. From the study, conjugation with aspartic acid improved the aqueous solubility of G2 without affecting its photophysical characteristics. G2-Asp showed similar in vitro and in vivo antitumor efficacies compared to the parent compound. Given the hydrophilic nature of G2-Asp, the photosensitizer is a pharmaceutically advantageous candidate as it can be formulated easily for systemic administration and has reduced risk of aggregation in vascular system.

Nanotechnology Enhanced Functional Assays of Actomyosin Motility - Potentials and Challenges

NASA Astrophysics Data System (ADS)

Månsson, A.; Nicholls, I. A.; Omling, P.; Tågerud, S.; Montelius, L.

Muscle contraction occurs as a result of force-producing interactions between the contractile proteins myosin II and actin with the two proteins highly ordered in the filament lattice of the muscle sarcomere. In contrast to this wellordered structure, most in vitro studies are performed with the contractile proteins in a disordered arrangement. Here we first review the existing in vitro motility assays and then consider how they can be improved by the use of nanotechnology. As a basis for such improvement we describe our recent work where we used chemically and topographically patterned surfaces to achieve selective localization of actomyosin motor function to predetermined areas of sub-micrometer dimensions. We also describe guidance and unidirectional actin filament sliding on nanosized tracks and suggest how such tracks can be combined with 1. microfluidics-based rapid solution exchange and 2. application of electromagnetic forces of well-defined orientation, thus simulating the lifting of a weight by actomyosin. As a related issue we discuss the usefulness of nanotechnology based assay systems for miniaturized highthroughput drug screening systems with molecular motors as drug targets. Finally, we consider the potentials and challenges in using nanotechnology to reconstruct the most essential aspects of cellular order within the muscle sarcomere.

Bio-based topical system for enhanced salicylic acid delivery: preparation and performance of gels.

PubMed

Langasco, Rita; Spada, Gianpiera; Tanriverdi, Sakine Tuncay; Rassu, Giovanna; Giunchedi, Paolo; Özer, Özgen; Gavini, Elisabetta

2016-08-01

New salicylic acid (SA)-loaded gels were developed using excipients made from renewable materials, and our goal was to improve drug permeation in the topical treatment of acne vulgaris. We studied the preparation parameters to obtain suitable gel formulations. Only naturally occurring polymers were used as gelling agents. Two hydrogels and three lipogels were selected and characterized in terms of drug loading, pH, viability cells, rheology, mechanical properties and in vitro permeation; these hydrogels and lipogels were compared with the traditional ointment. We also evaluated skin parameters before and after gel application. The formulations that we studied are non-Newtonian fluids; they have high drug loading and suitable mechanical properties. Lipogels exhibit a slower and more linear in vitro permeation profile compared with hydrogels. The different vehicles that we used affected drug permeation and improve patient compliance. Cytotoxicity studies suggest that all of the formulations are non-toxic. Lipogels demonstrate appropriate technological features and improved performance compared with the traditional ointment with regard to their composition. Lipogels may represent a new bio-based topical system for SA delivery. The use of 'green' excipients leads to 'skin-friendly' formulations that are able to satisfy environmental safety. © 2016 Royal Pharmaceutical Society.

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.

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

[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.

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.

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.

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.

A coated-wire ion-selective electrode for ionic calcium measurements

NASA Technical Reports Server (NTRS)

Hines, John W.; Arnaud, Sara; Madou, Marc; Joseph, Jose; Jina, Arvind

1991-01-01

A coated-wire ion-selective electrode for measuring ionic calcium was developed, in collaboration with Teknektron Sensor Development Corporation (TSDC). This coated wire electrode sensor makes use of advanced, ion-responsive polyvinyl chloride (PVC) membrane technology, whereby the electroactive agent is incorporated into a polymeric film. The technology greatly simplifies conventional ion-selective electrode measurement technology, and is envisioned to be used for real-time measurement of physiological and environment ionic constituents, initially calcium. A primary target biomedical application is the real-time measurement of urinary and blood calcium changes during extended exposure to microgravity, during prolonged hospital or fracture immobilization, and for osteoporosis research. Potential advanced life support applications include monitoring of calcium and other ions, heavy metals, and related parameters in closed-loop water processing and management systems. This technology provides a much simplified ionic calcium measurement capability, suitable for both automated in-vitro, in-vivo, and in-situ measurement applications, which should be of great interest to the medical, scientific, chemical, and space life sciences communities.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

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.

Trispecific antibodies for CD16A-directed NK cell engagement and dual-targeting of tumor cells.

PubMed

Gantke, Thorsten; Weichel, Michael; Herbrecht, Carmen; Reusch, Uwe; Ellwanger, Kristina; Fucek, Ivica; Eser, Markus; Müller, Thomas; Griep, Remko; Molkenthin, Vera; Zhukovsky, Eugene A; Treder, Martin

2017-09-01

Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Lysosomotropic properties of weakly basic anticancer agents promote cancer cell selectivity in vitro.

PubMed

Ndolo, Rosemary A; Luan, Yepeng; Duan, Shaofeng; Forrest, M Laird; Krise, Jeffrey P

2012-01-01

Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC(50) values of the inhibitors in normal fibroblasts to the IC(50) values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity.

Lysosomotropic Properties of Weakly Basic Anticancer Agents Promote Cancer Cell Selectivity In Vitro

PubMed Central

Ndolo, Rosemary A.; Luan, Yepeng; Duan, Shaofeng; Forrest, M. Laird; Krise, Jeffrey P.

2012-01-01

Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC50 values of the inhibitors in normal fibroblasts to the IC50 values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity. PMID:23145164

Towards the engineering of in vitro systems.

PubMed

Hold, Christoph; Panke, Sven

2009-08-06

Synthetic biology aims at rationally implementing biological systems from scratch. Given the complexity of living systems and our current lack of understanding of many aspects of living cells, this is a major undertaking. The design of in vitro systems can be considerably easier, because they can consist of fewer constituents, are quasi time invariant, their parameter space can be better accessed and they can be much more easily perturbed and then analysed chemically and mathematically. However, even for simplified in vitro systems, following a comprehensively rational design procedure is still difficult. When looking at a comparatively simple system, such as a medium-sized enzymatic reaction network as it is represented by glycolysis, major issues such as a lack of comprehensive enzyme kinetics and of suitable knowledge on crucial design parameters remain. Nevertheless, in vitro systems are very suitable to overcome these obstacles and therefore well placed to act as a stepping stone to engineering living systems.

Molecularly Imprinted Microrods via Mesophase Polymerization.

PubMed

Parisi, Ortensia Ilaria; Scrivano, Luca; Candamano, Sebastiano; Ruffo, Mariarosa; Vattimo, Anna Francesca; Spanedda, Maria Vittoria; Puoci, Francesco

2017-12-28

The aim of the present research work was the synthesis of molecularly imprinted polymers (MIPs) with a rod-like geometry via "mesophase polymerization". The ternary lyotropic system consisting of sodium dodecyl sulfate (SDS), water, and decanol was chosen to prepare a hexagonal mesophase to direct the morphology of the synthesized imprinted polymers using theophylline, methacrylic acid, and ethylene glycol dimethacrylate as a drug model template, a functional monomer, and a crosslinker, respectively. The obtained molecularly imprinted microrods (MIMs) were assessed by performing binding experiments and in vitro release studies, and the obtained results highlighted good selective recognition abilities and sustained release properties. In conclusion, the adopted synthetic strategy involving a lyotropic mesophase system allows for the preparation of effective MIPs characterized by a rod-like morphology.

Breakdown of Immune Tolerance in Systemic Lupus Erythematosus by Dendritic Cells

PubMed Central

Reihl, Alec M.

2016-01-01

Dendritic cells (DC) play an important role in the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disease with multiple tissue manifestations. In this review, we summarize recent studies on the roles of conventional DC and plasmacytoid DC in the development of both murine lupus and human SLE. In the past decade, studies using selective DC depletions have demonstrated critical roles of DC in lupus progression. Comprehensive in vitro and in vivo studies suggest activation of DC by self-antigens in lupus pathogenesis, followed by breakdown of immune tolerance to self. Potential treatment strategies targeting DC have been developed. However, many questions remain regarding the mechanisms by which DC modulate lupus pathogenesis that require further investigations. PMID:27034965

Active systems based on silver-montmorillonite nanoparticles embedded into bio-based polymer matrices for packaging applications.

PubMed

Incoronato, A L; Buonocore, G G; Conte, A; Lavorgna, M; Nobile, M A Del

2010-12-01

Silver-montmorillonite (Ag-MMT) antimicrobial nanoparticles were obtained by allowing silver ions from nitrate solutions to replace the Na(+) of natural montmorillonite and to be reduced by thermal treatment. The Ag-MMT nanoparticles were embedded in agar, zein, and poly(ε-caprolactone) polymer matrices. These nanocomposites were tested in vitro with a three-strain cocktail of Pseudomonas spp. to assess antimicrobial effectiveness. The results indicate that Ag-MMT nanoparticles embedded into agar may have antimicrobial activity against selected spoilage microorganisms. No antimicrobial effects were recorded with active zein and poly(ε-caprolactone). The water content of the polymeric matrix was the key parameter associated with antimicrobial effectiveness of this active system intended for food packaging applications.

In vitro model to evaluate reliability and accuracy of a dental shade-matching instrument.

PubMed

Kim-Pusateri, Seungyee; Brewer, Jane D; Dunford, Robert G; Wee, Alvin G

2007-11-01

There are several electronic shade-matching instruments available for clinical use; unfortunately, there are limited acceptable in vitro models to evaluate their reliability and accuracy. The purpose of this in vitro study was to evaluate the reliability and accuracy of a dental clinical shade-matching instrument. Using the shade-matching instrument (ShadeScan), color measurements were made of 3 commercial shade guides (VITA Classical, VITA 3D-Master, and Chromascop). Shade tabs were selected and placed in the middle of a gingival matrix (Shofu Gummy), with tabs of the same nominal shade from additional shade guides placed on both sides. Measurements were made of the central region of the shade tab inside a black box. For the reliability assessment, each shade tab from each of the 3 shade guide types was measured 10 times. For the accuracy assessment, each shade tab from 10 guides of each of the 3 types evaluated was measured once. Reliability, accuracy, and 95% confidence intervals were calculated for each shade tab. Differences were determined by 1-way ANOVA followed by the Bonferroni multiple comparison procedure. Reliability of ShadeScan was as follows: VITA Classical = 95.0%, VITA 3D-Master = 91.2%, and Chromascop = 76.5%. Accuracy of ShadeScan was as follows: VITA Classical = 65.0%, VITA 3D-Master = 54.2%, Chromascop = 84.5%. This in vitro study showed a varying degree of reliability and accuracy for ShadeScan, depending on the type of shade guide system used.

TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

2012-06-01

Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

Analogues of Marine Guanidine Alkaloids Are in Vitro Effective against Trypanosoma cruzi and Selectively Eliminate Leishmania (L.) infantum Intracellular Amastigotes.

PubMed

Martins, Ligia F; Mesquita, Juliana T; Pinto, Erika G; Costa-Silva, Thais A; Borborema, Samanta E T; Galisteo Junior, Andres J; Neves, Bruno J; Andrade, Carolina H; Shuhaib, Zainab Al; Bennett, Elliot L; Black, Gregory P; Harper, Philip M; Evans, Daniel M; Fituri, Hisham S; Leyland, John P; Martin, Claire; Roberts, Terence D; Thornhill, Andrew J; Vale, Stephen A; Howard-Jones, Andrew; Thomas, Dafydd A; Williams, Harri L; Overman, Larry E; Berlinck, Roberto G S; Murphy, Patrick J; Tempone, Andre G

2016-09-23

Synthetic analogues of marine sponge guanidine alkaloids showed in vitro antiparasitic activity against Leishmania (L.) infantum and Trypanosoma cruzi. Guanidines 10 and 11 presented the highest selectivity index when tested against Leishmania. The antiparasitic activity of 10 and 11 was investigated in host cells and in parasites. Both compounds induced depolarization of mitochondrial membrane potential, upregulation of reactive oxygen species levels, and increased plasma membrane permeability in Leishmania parasites. Immunomodulatory assays suggested an NO-independent effect of guanidines 10 and 11 on macrophages. The same compounds also promoted anti-inflammatory activity in L. (L.) infantum-infected macrophages cocultived with splenocytes, reducing the production of cytokines MCP-1 and IFN-γ. Guanidines 10 and 11 affect the bioenergetic metabolism of Leishmania, with selective elimination of parasites via a host-independent mechanism.

The Impact of Polymeric Nanoencapsulation on the Bioavailability of Lutein

NASA Astrophysics Data System (ADS)

Kamil, Alison

Lutein, a fat-soluble xanthophyll, contributes partially to the health benefits from consuming plant foods. Like all dietary carotenoids, lutein has a low bioavailability. In addition to increasing the intake of lutein-rich foods to enhance lutein status, delivery of lutein in polymeric nanoparticles (NP) presents a novel approach to enhancing lutein bioavailability. The overall research objective of this project was to investigate, in rats, the impact of nanoencapsulation using poly(lactic-co-glycolic acid) (PLGA) on the pharmacokinetics of lutein. We also used an in vitro cell culture approach utilizing human epithelial colorectal adenocarcinoma (Caco-2) cells grown in both conventional (CONV) and permeable support (PS) systems to investigate the impact of PLGA-NP on the absorption of lutein in intestinal cells. In chapter one, we compared the efficacy of lutein absorption in vitro using Caco-2 cells grown in both CONV and PS systems. We further examined the role of the micelle, the physiological vehicle for lutein within the small intestine, on its intestinal absorption in vitro compared to an organic solvent, ethanol, which is safe and consumed by humans. The finding from this study demonstrated that the CONV system displayed a larger efficacy of lutein uptake by Caco-2 cells. Further, in the PS system, micelle components appeared to facilitate more effective intestinal secretion of lutein. These findings suggest that lutein uptake by Caco-2 cells is subject to the influence of culturing system (CONV vs. PS) and delivery vehicle (ethanol vs. micelle). Chapter two examined the impact of PLGA-NP in rats on lutein pharmacokinetics in plasma and distribution in selected tissues as compared to free lutein. We also investigated the effect of nanoencapsulation on the absorption of lutein in intestinal cells compared to a more physiological vehicle, the micelle, using the PS method. In addition, we explored the need of additional micelles for the ultimate absorption of lutein loaded in a water soluble NP. The findings of the rat study indicated that, compared to free lutein, PLGA-NP improved the pharmacokinetics (Cmax and AUC) of lutein in the plasma of rats and in general promoted lutein accumulation in mesenteric adipose tissue and spleen but not liver. Yet, compared to micellized lutein, although NP improved the maximal concentration of lutein in the plasma of rats as well as in selected tissues it decreased the cell uptake and secretion of lutein in Caco-2 cells. The negative effect of the NP on cell uptake and secretion was partially remedied by the addition of micelle components. These findings suggest that the delivery of lutein within polymeric NP appears to be a promising approach to improving the bioavailability of lutein in rats. The inconsistent results between the rat and cell culture models warrant further investigations to determine which approach better predicts responses in humans. Further, bile salts and phospholipids, which are necessary to stimulate synthesis and secretion of chylomicrons, appear to facilitate more effective intestinal secretion of PLGA-NP lutein. In summary, with Caco-2 cells cultured in the PS system reliably grown to display phenotypes and functions of enterocytes in the small intestine, this in vitro platform enables the generation of information that is closer to the physiology of the absorptive enterocytes. However, although the CONV system has the physiological attributes of colonic tissue, it appeared to display a greater efficacy of lutein uptake by Caco-2 cells which can provide a rapid preliminary tool for methodology development for nutrient absorption studies. Further, the delivery of lutein in polymeric NP appears to be a promising approach to improve the bioavailability of lutein in vivo but raises issues with regard to the comparability and the predictive value of in vitro models to in vivo responses.

Effects of implant system, impression technique, and impression material on accuracy of the working cast.

PubMed

Wegner, Kerstin; Weskott, Katharina; Zenginel, Martha; Rehmann, Peter; Wöstmann, Bernd

2013-01-01

This in vitro study aimed to identify the effects of the implant system, impression technique, and impression material on the transfer accuracy of implant impressions. The null hypothesis tested was that, in vitro and within the parameters of the experiment, the spatial relationship of a working cast to the placement of implants is not related to (1) the implant system, (2) the impression technique, or (3) the impression material. A steel maxilla was used as a reference model. Six implants of two different implant systems (Standard Plus, Straumann; Semados, Bego) were fixed in the reference model. The target variables were: three-dimensional (3D) shift in all directions, implant axis direction, and rotation. The target variables were assessed using a 3D coordinate measuring machine, and the respective deviations of the plaster models from the nominal values of the reference model were calculated. Two different impression techniques (reposition/pickup) and four impression materials (Aquasil Ultra, Flexitime, Impregum Penta, P2 Magnum 360) were investigated. In all, 80 implant impressions for each implant system were taken. Statistical analysis was performed using multivariate analysis of variance. The implant system significantly influenced the transfer accuracy for most spatial dimensions, including the overall 3D shift and implant axis direction. There was no significant difference between the two implant systems with regard to rotation. Multivariate analysis of variance showed a significant effect on transfer accuracy only for the implant system. Within the limits of the present study, it can be concluded that the transfer accuracy of the intraoral implant position on the working cast is far more dependent on the implant system than on the selection of a specific impression technique or material.

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%.

Discovery and Validation of a Series of Aryl Sulfonamides as Selective Inhibitors of Tissue-Nonspecific Alkaline Phosphatase (TNAP)

PubMed Central

Dahl, Russell; Sergienko, Eduard A.; Mostofi, Yalda S.; Yang, Li; Su, Ying; Simao, Ana Maria; Narisawa, Sonoko; Brown, Brock; Mangravita-Novo, Arianna; Vicchiarelli, Michael; Smith, Layton H.; O’Neill, W. Charles; Millán, José Luis; Cosford, Nicholas D. P.

2009-01-01

We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural requirements for activity were determined, and the compounds were subsequently profiled for in vitro activity and bioavailability parameters including metabolic stability and permeability. The plasma levels following subcutaneous administration of a member of the lead series in rat was determined, demonstrating the potential of these TNAP inhibitors as systemically active therapeutic agents to target various diseases involving soft tissue calcification. A representative member of the series was also characterized in mechanistic and kinetic studies. PMID:19821572

Pharmacokinetics and tissue distribution of parenterally administered human alpha-lymphotoxin in normal and meth-A tumor-bearing BALB/c mice

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

Averbook, B.J.; Jeffes, E.B.; Yamamoto, R.S.

1989-08-01

These in vivo studies examine the pharmacokinetics of parenterally administered purified, native human alpha-lymphotoxin (LT) in normal and Meth-A bearing BALB/c mice. We found that the lytic activity of alpha-LT was inactivated within 5 h in the blood of both normal and tumor-bearing mice in vivo. However, LT bioactivity in vitro was not affected by incubation with fresh serum. Radioiodinated LT was rapidly sequestered in the kidneys of both normal and tumor-bearing animals. Systemically administered, radioiodinated LT did not selectively localize in tumor tissues.

Discovery of a Potent, Dual Serotonin and Norepinephrine Reuptake Inhibitor

PubMed Central

2013-01-01

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug–drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions. PMID:24900709

Saw palmetto ethanol extract inhibits adipocyte differentiation.

PubMed

Villaverde, Nicole; Galvis, Adriana; Marcano, Adriana; Priestap, Horacio A; Bennett, Bradley C; Barbieri, M Alejandro

2013-07-01

The fruits of saw palmetto have been used for the treatment of a variety of urinary and reproductive system problems. In this study we investigated whether the fruit extracts affect in vitro adipogenesis. Saw palmetto ethanol extract inhibited the lipid droplet accumulation by induction media in a dose-dependent manner, and it also attenuated the protein expressions of C-EBPα and PPARγ. Phosphorylation of Erk1/2 and Akt1 were also decreased by saw palmetto ethanol extract. This report suggests that saw palmetto extracts selectively affect the adipocyte differentiation through the modulation of several key factors that play a critical role during adipogenesis.

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.

Formulation and evaluation of once-a-day transdermal gels of diclofenac diethylamine.

PubMed

Baboota, S; Shakeel, F; Kohli, K

2006-03-01

The present study was undertaken to prepare and evaluate transdermal gels of diclofenac diethylamine (DDEA) containing penetration enhancers such as olesan oil and dimethyl sulfoxide (DMSO). Transdermal gels were prepared using different polymers such as carbopol-940, polyvinyl alcohol (PVA), hydroxy propyl methyl cellulose-K(4) M, hydroxy propyl cellulose-M, and sodium carboxy methyl cellulose. The formulated gels were subjected to physicochemical studies, in vitro release studies and in vitro skin permeations studies and were evaluated for drug content, viscosity, extrudability, spreadability, and pH. The in vitro release studies of prepared gels were performed using specially designed Fites cell and in vitro skin permeation studies were performed using keshary-chien diffusion cell through rat skin. Selected formulations were evaluated for their antiinflammatory activity using the carrageenan-induced paw edema in rats. The carbopol-940 and PVA gels containing 10% DMSO showed best in vitro skin permeation of DDEA. In vivo study for the selected formulation showed a sustained reduction in inflammation in the carrageenan induced paw edema in rats. The efficacies of carbopol-940 and PVA gels were also compared with that of the marketed Voveran gel,(R) and it was found that carbopol and PVA gels produced better results than the Voveran gel. (c) 2006 Prous Science. All rights reserved. (c) 2006 Prous Science. All rights reserved.

[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.

Improvement of banana cv. Rasthali (Silk, AAB) against Fusarium oxysporum f.sp. cubense (VCG 0124/5) through induced mutagenesis: Determination of LD50 specific to mutagen, explants, toxins and in vitro and in vivo screening for Fusarium wilt resistance.

PubMed

Saraswathi, M S; Kannan, G; Uma, S; Thangavelu, R; Backiyarani, S

2016-05-01

Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15% for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.

A microcomputed tomography evaluation of the marginal fit of cobalt-chromium alloy copings fabricated by new manufacturing techniques and alloy systems.

PubMed

Kim, Eun-Ha; Lee, Du-Hyeong; Kwon, Sung-Min; Kwon, Tae-Yub

2017-03-01

Although new digital manufacturing techniques are attracting interest in dentistry, few studies have comprehensively investigated the marginal fit of fixed dental prostheses fabricated with such techniques. The purpose of this in vitro microcomputed tomography (μCT) study was to evaluate the marginal fit of cobalt-chromium (Co-Cr) alloy copings fabricated by casting and 3 different computer-aided design and computer-aided manufacturing (CAD-CAM)-based processing techniques and alloy systems. Single Co-Cr metal crowns were fabricated using 4 different manufacturing techniques: casting (control), milling, selective laser melting, and milling/sintering. Two different commercial alloy systems were used for each fabrication technique (a total of 8 groups; n=10 for each group). The marginal discrepancy and absolute marginal discrepancy of the crowns were determined with μCT. For each specimen, the values were determined from 4 different regions (sagittal buccal, sagittal lingual, coronal mesial, and coronal distal) by using imaging software and recorded as the average of the 4 readings. For each parameter, the results were statistically compared with 2-way analysis of variance and appropriate post hoc analysis (using Tukey or Student t test) (α=.05). The milling and selective laser melting groups showed significantly larger marginal discrepancies than the control groups (70.4 ±12.0 and 65.3 ±10.1 μm, respectively; P<.001), whereas the milling/sintering groups exhibited significantly smaller values than the controls (P=.004). The milling groups showed significantly larger absolute marginal discrepancy than the control groups (137.4 ±29.0 and 139.2 ±18.9 μm, respectively; P<.05). In the selective laser melting and milling/sintering groups, the absolute marginal discrepancy values were material-specific (P<.05). Nonetheless, the milling/sintering groups yielded statistically comparable (P=.935) or smaller (P<.001) absolute marginal discrepancies to the control groups. The findings of this in vitro μCT study showed that the marginal fit values of the Co-Cr alloy greatly depended on the fabrication methods and, occasionally, the alloy systems. Fixed dental prostheses produced by using the milling/sintering technique can be considered clinically acceptable in terms of marginal fit. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo

PubMed Central

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M.; Brennan, Patrick J.; Banerjee, Pinaki P.; Wiener, Susan J.; Orange, Jordan S.; Brenner, Michael B.; Grupp, Stephan A.; Nichols, Kim E.

2013-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma. PMID:24563871

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

PubMed

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M; Brennan, Patrick J; Banerjee, Pinaki P; Wiener, Susan J; Orange, Jordan S; Brenner, Michael B; Grupp, Stephan A; Nichols, Kim E

2014-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. ©2013 AACR.

Ci8 short, a novel LPS-induced peptide from the ascidian Ciona intestinalis, modulates responses of the human immune system.

PubMed

Bonura, Angela; Vizzini, Aiti; Vlah, Sara; Gervasi, Francesco; Longo, Alessandra; Melis, Mario R; Schildberg, Frank A; Colombo, Paolo

2018-02-01

The selective modulation of immunity is an emerging concept driven by the vast advances in our understanding of this crucial host defense system. Invertebrates have raised researchers' interest as potential sources of new bioactive molecules owing to their antibacterial, anticancer and immunomodulatory activities. A LipoPolySaccharide (LPS) challenge in the ascidian Ciona intestinalis generates the transcript, Ci8 short, with cis-regulatory elements in the 3' UTR region that are essential for shaping innate immune responses. The derived amino acidic sequence in silico analysis showed specific binding to human Major Histocompatibility Complex (MHC) Class I and Class II alleles. The role of Ci8 short peptide was investigated in a more evolved immune system using human Peripheral Blood Mononuclear Cells (PBMCs) as in vitro model. The biological activities of this molecule include the activation of 70kDa TCR ζ chain Associated Protein kinase (ZAP-70) and T Cell Receptor (TCR) Vβ oligo clonal selection on CD4 + T lymphocytes as well as increased proliferation and IFN-γ secretion. Furthermore Ci8 short affects CD4 + /CD25 high induced regulatory T cells (iTreg) subset selection which co-expressed the functional markers TGF-β1/Latency Associated Protein (LAP) and CD39/CD73. This paper describes a new molecule that modulates important responses of the human adaptive immune system. Copyright © 2017 Elsevier GmbH. All rights reserved.

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

Methods and options in vitro dialyzability; benefits and limitations.

PubMed

Sandberg, Ann-Sofie

2005-11-01

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

Impact of pasteurization of human milk on preterm newborn in vitro digestion: Gastrointestinal disintegration, lipolysis and proteolysis.

PubMed

de Oliveira, Samira C; Bourlieu, Claire; Ménard, Olivia; Bellanger, Amandine; Henry, Gwénaële; Rousseau, Florence; Dirson, Emelyne; Carrière, Frédéric; Dupont, Didier; Deglaire, Amélie

2016-11-15

Human milk feeding is an important recommendation for preterm newborns considering their vulnerability and digestive immaturity. Holder pasteurization (62.5°C, 30min) applied in milk banks modifies its biological quality and its microstructure. We investigated the impact of pasteurization of preterm human milk on its gastrointestinal kinetics of lipolysis, proteolysis and structural disintegration. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of preterm newborns. A pool of preterm human milk was digested as raw or after Holder pasteurization. Pasteurization impacted the microstructure of undigested human milk, its gastrointestinal disintegration and tended to limit the intestinal lipolysis. Furthermore, the gastrointestinal bioaccessibility of some fatty acids was decreased by pasteurization, while the intestinal bioaccessibility of some amino acids was selectively modulated. The impact of pasteurization on the digestion of human milk may have nutritional relevance in vivo and potentially modulates preterm development and growth. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of simulated weightlessness on meiosis. Fertilization, and early development in mice

NASA Technical Reports Server (NTRS)

Wolgemuth, D. J.

1986-01-01

The initial goal was to construct a clinostat which could support mammalian cell culture. The clinostat was selected as a means by which to simulate microgravity conditions within the laboratory, by constant re-orientation of cells with respect to the gravity vector. The effects of this simulated microgravity on in-vitro meiotic maturation of oocytes, using mouse as the model system, was investigated. The effects of clinostat rotation on fertilization in-vitro was then examined. Specific endpoints included examining the timely appearance of male and female pronuclei (indicating fertilization) and the efficiency of extrusion of the second polar body. Particular attention was paid to detecting anomalies of fertilization, including parthenogenetic activation and multiple pronuclei. Finally, for the preliminary studies on mouse embryogenesis, a key feature of the clinostat was modified, that of the position of the cells during rotation. A means was found to immobilize the cells during the clinostat reotation, permitting the cells to remain at the axis of rotation yet not interfering with cellular development.

The influence of selected potential oncostatics of plant origin on the protein biosynthesis in vitro.

PubMed

Paszkiewicz-Gadek, A; Chlabicz, J; Gałasiński, W

1988-01-01

Five potential oncostatics of plant origin (reserpine, amphotericin B, rutoside, digoxin, dry aloe extract), and cyclic AMP were investigated for their effect on protein synthesis. The solutions of digoxin and dry aloe extract inhibited protein biosynthesis in vitro. The direct inhibiting effect of digoxin on the ribosomes suggests that this drug forms an inactive complex with this organelle. Therefore it can be concluded that ribosome is the target site of digoxin action. Aloin and aloeemodin are responsible for the inhibitory effect of the solution of dry aloe extract. They inhibit markedly [14C]-leucine incorporation into proteins. Aloin and aloeemodin do not influence directly the ribosomes, but they inhibit elongation factors and peptidyltransferase activities in the complete elongation system. Some preliminary experiments have shown that direct interaction between these substances and elongation factor EF-2 should be taken in account. This observation is the subject of further experiments, in which the characteristics of the inhibitory effect of the components isolated from dry aloe extract will be performed.

Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment.

PubMed

Murakami, Masahiro; Saito, Takashi; Tabata, Yasuhiko

2014-11-01

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by L-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A study of properties of "micelle-enhanced" polyelectrolyte capsules: Structure, encapsulation and in vitro release.

PubMed

Li, Xiaodong; Lu, Tian; Zhang, Jianxiang; Xu, Jiajie; Hu, Qiaoling; Zhao, Shifang; Shen, Jiacong

2009-07-01

"Micelle-enhanced" polyelectrolyte capsules were fabricated via a layer-by-layer technique, templated on hybrid calcium carbonate particles with built-in polymeric micelles based on polystyrene-b-poly(acrylic acid). Due to the presence of a large number of negatively charged micelles inside the polyelectrolyte capsule, which were liberated from templates, the capsule wall was reconstructed and had properties different to those of conventional polyelectrolyte capsules. This type of capsule could selectively entrap positively charged water-soluble substances. The encapsulation efficiency of positively charged substances was dependent on their molecular weight or size. For some positively charged compounds, such as rhodamine B and lysozyme, the concentration in the capsules was orders of magnitude higher than that in the incubation solution. In addition, in vitro release study suggested that the encapsulated compounds could be released through a sustained manner to a certain degree. All these results point to the fact that these capsules might be used as novel delivery systems for some water-soluble compounds.

Fractionation and antioxidant properties of rice bran protein hydrolysates stimulated by in vitro gastrointestinal digestion.

PubMed

Phongthai, Suphat; D'Amico, Stefano; Schoenlechner, Regine; Homthawornchoo, Wantida; Rawdkuen, Saroat

2018-02-01

Rice bran was used as a starting material to prepare protein concentrate through enzyme-assisted extraction. The hydrolysis of protein concentrate under in vitro gastrointestinal digestion (pepsin-trypsin system) greatly improved the antioxidant properties. Rice bran protein hydrolysate was further fractionated by membrane ultrafiltration (UF, F1: molecular weight (MW) <3kDa, F2: MW 3-5kDa, and F3: MW 5-10kDa). Peptides with smaller MW possessed higher antioxidant activities (P<0.05). UF showed a great efficacy to selectively separate the metal-chelating peptides. Tyrosine and phenylalanine had positive correlations with their DPPH & ABTS radicals scavenging activities and ferric reducing antioxidant power (r>0.831). A major peptide fragment was detected at m/z 1088 by a MALDI-TOF mass spectrometry. There is high potential that antioxidative peptides from rice bran might also be produced in the gastrointestinal tract of the human body. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

A Bacillus subtilis strain as probiotic in poultry: selection based on in vitro functional properties and enzymatic potentialities.

PubMed

Hmani, Houda; Daoud, Lobna; Jlidi, Mouna; Jalleli, Karim; Ben Ali, Manel; Hadj Brahim, Adel; Bargui, Mansour; Dammak, Alaeddine; Ben Ali, Mamdouh

2017-08-01

We have proposed and validate an in vitro probiotic selection, based on enzymatic potentialities associated to well-established probiotic functional properties. A new Bacillus subtilis HB2 isolate, selected based on its high extracellular enzyme production, was chosen as a probiotic candidate for application as animal feed supplement. The HB2 strain showed an excellent acid and bile salts tolerance, a strong adhesion to chick enterocytes and produced antimicrobials against pathogens. An in vivo trial in poultry farming was conducted to evaluate the HB2 probiotic performance. After 35 days, HB2 achieved the higher growth performance than the control groups. The mortality and the feed conversion ratio were significantly decreased. Finally, the HB2 treated group showed wet litter and less severe ammonia odor in the atmosphere. Our study provides new insights into the importance of enzymatic potentialities, associated with the common functional properties, as a novel approach for probiotic selection.

Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

PubMed Central

Lagaye, S.; Derrien, M.; Menu, E.; Coïto, C.; Tresoldi, E.; Mauclère, P.; Scarlatti, G.; Chaouat, G.; Barré-Sinoussi, F.; Bomsel, M.

2001-01-01

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies. PMID:11312350

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

Integrated microdroplet-based system for enzyme synthesis and sampling

NASA Astrophysics Data System (ADS)

Lapierre, Florian; Best, Michel; Stewart, Robert; Oakeshott, John; Peat, Thomas; Zhu, Yonggang

2013-12-01

Microdroplet-based microfluidic devices are emerging as powerful tools for a wide range of biochemical screenings and analyses. Monodispersed aqueous microdroplets from picoliters to nanoliters in volume are generated inside microfluidic channels within an immiscible oil phase. This results in the formation of emulsions which can contain various reagents for chemical reactions and can be considered as discrete bioreactors. In this paper an integrated microfluidic platform for the synthesis, screening and sorting of libraries of an organophosphate degrading enzyme is presented. The variants of the selected enzyme are synthesized from a DNA source using in-vitro transcription and translation method. The synthesis occurs inside water-in-oil emulsion droplets, acting as bioreactors. Through a fluorescence based detection system, only the most efficient enzymes are selected. All the necessary steps from the enzyme synthesis to selection of the best genes (producing the highest enzyme activity) are thus integrated inside a single and unique device. In the second part of the paper, an innovative design of the microfluidic platform is presented, integrating an electronic prototyping board for ensuring the communication between the various components of the platform (camera, syringe pumps and high voltage power supply), resulting in a future handheld, user-friendly, fully automated device for enzyme synthesis, screening and selection. An overview on the capabilities as well as future perspectives of this new microfluidic platform is provided.

Novel (188)Re multi-functional bone-seeking compounds: Synthesis, biological and radiotoxic effects in metastatic breast cancer cells.

PubMed

Fernandes, Célia; Monteiro, Sofia; Belchior, Ana; Marques, Fernanda; Gano, Lurdes; Correia, João D G; Santos, Isabel

2016-02-01

Radiolabeled bisphosphonates (BPs) have been used for bone imaging and delivery of β(-) emitting radionuclides for bone pain palliation. As a β(-) emitter, (188)Re has been considered particularly promising for bone metastases therapy. Aimed at finding innovative bone-seeking agents for systemic radiotherapy of bone metastases, we describe herein novel organometallic compounds of the type fac-[(188)Re(CO)3(k(3)-L)], (L=BP-containing chelator), their in vitro and in vivo stability, and their cellular damage in MDAMB231 cells, a metastatic breast cancer cell line. After synthesis and characterization of the novel organometallic compounds of the type fac-[(188)Re(CO)3(k(3)-L)] their radiochemical purity and in vitro stability was assessed by HPLC. In vivo stability and pharmacokinetic profile were evaluated in mice and the radiocytotoxic activity and DNA damage were assessed by MTT assay and by the cytokinesis-block micronucleus (CBMN) assay, respectively. Among all complexes, (188)Re3 was obtained with high radiochemical purity (>95%) and high specific activity and presented high in vitro and in vivo stability. Biodistribution studies of (188)Re3 in Balb/c mice showed fast blood clearance, high bone uptake (16.1 ± 3.3% IA/g organ, 1h p.i.) and high bone-to-blood and bone-to-muscle radioactivity ratios, indicating that it is able to deliver radiation to bone in a very selective way. The radiocytotoxic effect elicited by (188)Re3 in the MDAMB231 cells was dependent on its concentration, and was higher than that induced by identical concentrations of [(188)ReO4](-). Additionally, (188)Re3 elicited morphological changes in the cells and induced DNA damage by the increased number of MN observed. Altogether, our results demonstrate that (188)Re3 could be considered an attractive candidate for further preclinical evaluation for systemic radionuclide therapy of bone metastases considering its ability to deliver radiation to bone in a very selective way and to induce radiation damage. Copyright © 2015 Elsevier Inc. All rights reserved.

Model-Based Analysis of Biopharmaceutic Experiments To Improve Mechanistic Oral Absorption Modeling: An Integrated in Vitro in Vivo Extrapolation Perspective Using Ketoconazole as a Model Drug.

PubMed

Pathak, Shriram M; Ruff, Aaron; Kostewicz, Edmund S; Patel, Nikunjkumar; Turner, David B; Jamei, Masoud

2017-12-04

Mechanistic modeling of in vitro data generated from metabolic enzyme systems (viz., liver microsomes, hepatocytes, rCYP enzymes, etc.) facilitates in vitro-in vivo extrapolation (IVIV_E) of metabolic clearance which plays a key role in the successful prediction of clearance in vivo within physiologically-based pharmacokinetic (PBPK) modeling. A similar concept can be applied to solubility and dissolution experiments whereby mechanistic modeling can be used to estimate intrinsic parameters required for mechanistic oral absorption simulation in vivo. However, this approach has not widely been applied within an integrated workflow. We present a stepwise modeling approach where relevant biopharmaceutics parameters for ketoconazole (KTZ) are determined and/or confirmed from the modeling of in vitro experiments before being directly used within a PBPK model. Modeling was applied to various in vitro experiments, namely: (a) aqueous solubility profiles to determine intrinsic solubility, salt limiting solubility factors and to verify pK a ; (b) biorelevant solubility measurements to estimate bile-micelle partition coefficients; (c) fasted state simulated gastric fluid (FaSSGF) dissolution for formulation disintegration profiling; and (d) transfer experiments to estimate supersaturation and precipitation parameters. These parameters were then used within a PBPK model to predict the dissolved and total (i.e., including the precipitated fraction) concentrations of KTZ in the duodenum of a virtual population and compared against observed clinical data. The developed model well characterized the intraluminal dissolution, supersaturation, and precipitation behavior of KTZ. The mean simulated AUC 0-t of the total and dissolved concentrations of KTZ were comparable to (within 2-fold of) the corresponding observed profile. Moreover, the developed PBPK model of KTZ successfully described the impact of supersaturation and precipitation on the systemic plasma concentration profiles of KTZ for 200, 300, and 400 mg doses. These results demonstrate that IVIV_E applied to biopharmaceutical experiments can be used to understand and build confidence in the quality of the input parameters and mechanistic models used for mechanistic oral absorption simulations in vivo, thereby improving the prediction performance of PBPK models. Moreover, this approach can inform the selection and design of in vitro experiments, potentially eliminating redundant experiments and thus helping to reduce the cost and time of drug product development.

Development of Pyrazolone and Isoxazol-5-one Cambinol Analogues as Sirtuin Inhibitors

PubMed Central

2015-01-01

Sirtuins are a family of NAD+-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition. PMID:24697269

In Vitro and In Vivo Toxicity Profiling of Ammonium-Based Deep Eutectic Solvents

PubMed Central

Hayyan, Maan; Looi, Chung Yeng; Hayyan, Adeeb; Wong, Won Fen; Hashim, Mohd Ali

2015-01-01

The cytotoxic potential of ammonium-based deep eutectic solvents (DESs) with four hydrogen bond donors, namely glycerine (Gl), ethylene glycol (EG), triethylene glycol (TEG) and urea (U) were investigated. The toxicity of DESs was examined using In Vitro cell lines and In Vivo animal model. IC50 and selectivity index were determined for the DESs, their individual components and their combinations as aqueous solutions for comparison purposes. The cytotoxicity effect of DESs varied depending on cell lines. The IC50 for the GlDES, EGDES, UDES and TEGDES followed the sequence of TEGDES< GlDES< EGDES< UDES for OKF6, MCF-7, A375, HT29 and H413, respectively. GlDES was selective against MCF-7 and A375, EGDES was selective against MCF-7, PC3, HepG2 and HT29, UDES was selective against MCF-7, PC3, HepG2 and HT29, and TEGDES was selective against MCF-7 and A375. However, acute toxicity studies using ICR mice showed that these DESs were relatively toxic in comparison to their individual components. DES did not cause DNA damage, but it could enhance ROS production and induce apoptosis in treated cancer cells as evidenced by marked LDH release. Furthermore, the examined DESs showed less cytotoxicity compared with ionic liquids. To the best of our knowledge, this is the first time that combined In Vitro and In Vivo toxicity profiles of DESs were being demonstrated, raising the toxicity issue of these neoteric mixtures and their potential applicability to be used for therapeutic purposes. PMID:25679975

[High non-specific binding of the beta(1) -selective radioligand 2-(125)I-ICI-H].

PubMed

Riemann, B; Law, M P; Kopka, K; Wagner, St; Luthra, S; Pike, V W; Neumann, J; Kirchhefer, U; Schmitz, W; Schober, O; Schäfers, M

2003-08-01

As results of cardiac biopsies suggest, myocardial beta(1) -adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac beta(2)-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess beta-adrenoceptors non-invasively. Among the novel racemic analogues of the established beta(1)-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to beta(1)-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective beta(1)-adrenergic radioligand in cardiac imaging. Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac beta-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-(125)I-ICI-H from the silylated precursor 2-SiMe(3)-ICI-H was established. The specific activity was 80 GBq/ micro mol, the radiochemical yield ranged from 70 to 80%. The unlabelled compound 2-I-ICI-H showed high beta(1)-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac beta-adrenoceptors. The radiolabelled counterpart 2-(125)I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac beta(1)-adrenoceptors in vivo. Because of its high non-specific binding 2-(125)I-ICI-H is no suitable radiotracer for imaging in vivo.

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

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

NASA Astrophysics Data System (ADS)

Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

2014-08-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

Stereochemical preference toward oncotarget: Design, synthesis and in vitro anticancer evaluation of diastereomeric β-lactams.

PubMed

Olazarán-Santibáñez, Fabián; Bandyopadhyay, Debasish; Carranza-Rosales, Pilar; Rivera, Gildardo; Balderas-Rentería, Isaías

2017-06-06

In the battle against cancer discovery of new and novel chemotherapeutic agent demands extreme obligation. Development of anticancer compounds with higher potency and reduced side-effects is timely and challenging. A small series of fourteen diastereomeric β-lactams (seven pairs) were synthesized through multi-step process exploring [2+2] ketene-imine cycloaddition as the key step. Comparative stereochemical preferences were studied through computational docking and validated by in vitro evaluation. β-tubulin was considered as possible molecular target and in vitro anticancer evaluation was conducted against SiHa, B16F10, K562 and Chang cell lines. Caspase-3 activation assay and hematoxylin/eosin staining of the cells were also accomplished. Better docking scores of the cis- over the trans-β-lactams indicated favorable β-lactam-β-tubulin interactions in cis-geometry. In vitro (IC50) evaluation confirmed better anticancer activity of the cis-diastereoisomers. Apoptosis-induced cell death was supported by caspase-3 activation study. A cis-β-lactam [(±)-Cis-3-amino-1-phenyl-4-(p-tolyl) azetidin-2-one, 6C] was found to be more active (in vitro) than the marketed natural drug colchicine against SiHa and B16F10 (six times higher potency) cell lines. Reduced toxicity (compared to colchicine) in Chang cells confirmed better site-selectivity (accordingly less side-effects) of 6C than colchicine. Aside from 6C, most of the reported molecules demonstrated good to strong in vitro anticancer activity against SiHa and B16F10 cancer cell lines. Stereochemical preferences of the cis-β-lactams over their trans-counterparts, toward the molecular target β-tubulin, was confirmed by docking studies and in vitro anticancer evaluation. Apoptosis was identified as the cause of cell death. The lead 6C exhibited higher potency and selectivity than the marketed drug colchicine both in silico as well as in vitro.

Stereochemical preference toward oncotarget: Design, synthesis and in vitro anticancer evaluation of diastereomeric β-lactams

PubMed Central

Olazarán-Santibáñez, Fabián; Bandyopadhyay, Debasish; Carranza-Rosales, Pilar; Rivera, Gildardo; Balderas-Rentería, Isaías

2017-01-01

Purpose In the battle against cancer discovery of new and novel chemotherapeutic agent demands extreme obligation. Development of anticancer compounds with higher potency and reduced side-effects is timely and challenging. Experimental Design A small series of fourteen diastereomeric β-lactams (seven pairs) were synthesized through multi-step process exploring [2+2] ketene-imine cycloaddition as the key step. Comparative stereochemical preferences were studied through computational docking and validated by in vitro evaluation. β-tubulin was considered as possible molecular target and in vitro anticancer evaluation was conducted against SiHa, B16F10, K562 and Chang cell lines. Caspase-3 activation assay and hematoxylin/eosin staining of the cells were also accomplished. Results Better docking scores of the cis- over the trans-β-lactams indicated favorable β-lactam—β-tubulin interactions in cis-geometry. In vitro (IC50) evaluation confirmed better anticancer activity of the cis-diastereoisomers. Apoptosis-induced cell death was supported by caspase-3 activation study. A cis-β-lactam [(±)-Cis-3-amino-1-phenyl-4-(p-tolyl) azetidin-2-one, 6C] was found to be more active (in vitro) than the marketed natural drug colchicine against SiHa and B16F10 (six times higher potency) cell lines. Reduced toxicity (compared to colchicine) in Chang cells confirmed better site-selectivity (accordingly less side-effects) of 6C than colchicine. Aside from 6C, most of the reported molecules demonstrated good to strong in vitro anticancer activity against SiHa and B16F10 cancer cell lines. Conclusions Stereochemical preferences of the cis-β-lactams over their trans-counterparts, toward the molecular target β-tubulin, was confirmed by docking studies and in vitro anticancer evaluation. Apoptosis was identified as the cause of cell death. The lead 6C exhibited higher potency and selectivity than the marketed drug colchicine both in silico as well as in vitro. PMID:28562328

Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

PubMed Central

Kissmehl, R; Treptau, T; Hofer, H W; Plattner, H

1996-01-01

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells. PMID:8694788

Can in vitro systems capture the characteristic differences between the flexion-extension kinematics of the healthy and TKA knee?

PubMed

Varadarajan, Kartik M; Harry, Rubash E; Johnson, Todd; Li, Guoan

2009-10-01

In vitro systems provide a powerful means to evaluate the efficacy of total knee arthroplasty (TKA) in restoring normal knee kinematics. The Oxford knee rig (OKR) and the robotic knee testing system (RKTS) represent two systems that have been extensively used to study TKA biomechanics. Nonetheless, a frequently asked question is whether in vitro simulations can capture the in vivo behavior of the knee. Here, we compared the flexion-extension kinematics of intact knees and knees after TKA tested on the OKR and RKTS, to results of representative in vivo studies. The goal was to determine if the in vitro systems could capture the key kinematic features of knees in healthy subjects and TKA patients. Results showed that the RKTS and the OKR can replicate the femoral rollback and 'screw home' tibial rotation between 0 degrees and 30 degrees flexion seen in healthy subjects, and the reduced femoral rollback and absence of 'screw home' motion in TKA patients. The RKTS also replicated the overall internally rotated position of the tibia beyond 30 degrees flexion. However, ability of the OKR to replicate the internally rotated position of the knee beyond 30 degrees flexion was inconsistent. These data could aid in validation of new in vitro systems and physiologic interpretations of in vitro results.

Interaction of Antibiotics with Innate Host Defense Factors against Salmonella enterica Serotype Newport

PubMed Central

Kumaraswamy, Monika; Kousha, Armin; Nizet, Victor

2017-01-01

ABSTRACT This study examines the pharmacodynamics of antimicrobials that are used to treat Salmonella with each other and with key components of the innate immune system. Antimicrobial synergy was assessed using time-kill and checkerboard assays. Antimicrobial interactions with innate immunity were studied by employing cathelicidin LL-37, whole-blood, and neutrophil killing assays. Ceftriaxone and ciprofloxacin were found to be synergistic in vitro against Salmonella enterica serotype Newport. Ceftriaxone, ciprofloxacin, and azithromycin each demonstrated synergy with the human cathelicidin defense peptide LL-37 in killing Salmonella. Exposure of Salmonella to sub-MICs of ceftriaxone resulted in enhanced susceptibility to LL-37, whole blood, and neutrophil killing. The activity of antibiotics in vivo against Salmonella may be underestimated in bacteriologic media lacking components of innate immunity. The pharmacodynamic interactions of antibiotics used to treat Salmonella with each other and with components of innate immunity warrant further study in light of recent findings showing in vivo selection of antimicrobial resistance by single agents in this pathogen. IMPORTANCE It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo. PMID:29242830

In vitro sensitivity of granulo-monocytic progenitors as a new toxicological cell system and endpoint in the ACuteTox Project

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

Cerrato, Laura; Valeri, Antonio; Bueren, Juan A.

The ACuteTox Project (part of the EU 6th Framework Programme) was started up in January 2005. The aim of this project is to develop a simple and robust in vitro strategy for prediction of human acute systemic toxicity, which could replace animal tests used for regulatory purposes. Our group is responsible for the characterization of the effect of the reference chemicals on the hematopoietic tissue. CFU-GM assay based on the culture of human mononuclear cord blood cells has been used to characterize the effects of the selected compounds on the myeloid progenitors. Previous results have shown the relevance of themore » CFU-GM assay for the prediction of human acute neutropenia after treatment of antitumoral compounds, and this assay has been recently approved by the ECVAM's Scientific Advisory Committee. Among the compounds included in the study there were pharmaceuticals, environmental pollutants and industrial chemicals. Eleven out of 55 chemicals did not show any cytotoxic effect at the maximum concentration tested. The correlation coefficients of CFU-GM IC50, IC70 and IC90 values with human LC50 values (50% lethal concentration calculated from time-related sublethal and lethal human blood concentrations) were 0.4965, 0.5106 and 0.5142 respectively. Although this correlation is not improve respect to classical in vitro basal cytotoxicity tests such as 3T3 Neutral Red Uptake, chemicals which deviate substantially in the correlation with these assays (colchicine, digoxin, 5-Fluorouracil and thallium sulfate) fitted very well in the linear regression analysis of the CFU-GM progenitors. The results shown in the present study indicate that the sensitivity of CFU-GM progenitors correlates better than the sensitivity of HL-60 cells with human LC50 values and could help to refine the predictability for human acute systemic toxicity when a given chemical may affect to the hematopoietic myeloid system.« less

Comparative efficacies of candidate antibiotics against Yersinia pestis in an in vitro pharmacodynamic model.

PubMed

Louie, Arnold; Vanscoy, Brian; Liu, Weiguo; Kulawy, Robert; Brown, David; Heine, Henry S; Drusano, George L

2011-06-01

Yersinia pestis, the bacterium that causes plague, is a potential agent of bioterrorism. Streptomycin is the "gold standard" for the treatment of plague infections in humans, but the drug is not available in many countries, and resistance to this antibiotic occurs naturally and has been generated in the laboratory. Other antibiotics have been shown to be active against Y. pestis in vitro and in vivo. However, the relative efficacies of clinically prescribed regimens of these antibiotics with streptomycin and with each other for the killing of Yersinia pestis are unknown. The efficacies of simulated pharmacokinetic profiles for human 10-day clinical regimens of ampicillin, meropenem, moxifloxacin, ciprofloxacin, and gentamicin were compared with the gold standard, streptomycin, for killing of Yersinia pestis in an in vitro pharmacodynamic model. Resistance amplification with therapy was also assessed. Streptomycin killed the microbe in one trial but failed due to resistance amplification in the second trial. In two trials, the other antibiotics consistently reduced the bacterial densities within the pharmacodynamic systems from 10⁸ CFU/ml to undetectable levels (<10² CFU/ml) between 1 and 3 days of treatment. None of the comparator agents selected for resistance. The comparator antibiotics were superior to streptomycin against Y. pestis and deserve further evaluation.

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Natural material-decorated mesoporous silica nanoparticle container for multifunctional membrane-controlled targeted drug delivery

PubMed Central

Hu, Yan; Ke, Lei; Chen, Hao; Zhuo, Ma; Yang, Xinzhou; Zhao, Dan; Zeng, Suying; Xiao, Xincai

2017-01-01

To avoid the side effects caused by nonspecific targeting, premature release, weak selectivity, and poor therapeutic efficacy of current nanoparticle-based systems used for drug delivery, we fabricated natural material-decorated nanoparticles as a multifunctional, membrane-controlled targeted drug delivery system. The nanocomposite material coated with a membrane was biocompatible and integrated both specific tumor targeting and responsiveness to stimulation, which improved transmission efficacy and controlled drug release. Mesoporous silica nanoparticles (MSNs), which are known for their biocompatibility and high drug-loading capacity, were selected as a model drug container and carrier. The membrane was established by the polyelectrolyte composite method from chitosan (CS) which was sensitive to the acidic tumor microenvironment, folic acid-modified CS which recognizes the folate receptor expressed on the tumor cell surface, and a CD44 receptor-targeted polysaccharide hyaluronic acid. We characterized the structure of the nanocomposite as well as the drug release behavior under the control of the pH-sensitive membrane switch and evaluated the antitumor efficacy of the system in vitro. Our results provide a basis for the design and fabrication of novel membrane-controlled nanoparticles with improved tumor-targeting therapy. PMID:29200852

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

PubMed

Denoyer, Delphine; Pearson, Helen B; Clatworthy, Sharnel A S; Smith, Zoe M; Francis, Paul S; Llanos, Roxana M; Volitakis, Irene; Phillips, Wayne A; Meggyesy, Peter M; Masaldan, Shashank; Cater, Michael A

2016-06-14

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Non-animal photosafety assessment approaches for cosmetics based on the photochemical and photobiochemical properties.

PubMed

Onoue, Satomi; Suzuki, Gen; Kato, Masashi; Hirota, Morihiko; Nishida, Hayato; Kitagaki, Masato; Kouzuki, Hirokazu; Yamada, Shizuo

2013-12-01

The main purpose of the present study was to establish a non-animal photosafety assessment approach for cosmetics using in vitro photochemical and photobiochemical screening systems. Fifty-one cosmetics, pharmaceutics and other chemicals were selected as model chemicals on the basis of animal and/or clinical photosafety information. The model chemicals were assessed in terms of photochemical properties by UV/VIS spectral analysis, reactive oxygen species (ROS) assay and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Most phototoxins exhibited potent UV/VIS absorption with molar extinction coefficients of over 1000M(-1)cm(-1), although false-negative prediction occurred for 2 cosmetic phototoxins owing to weak UV/VIS absorption. Among all the cosmetic ingredients, ca. 42% of tested chemicals were non-testable in the ROS assay because of low water solubility; thereby, micellar ROS (mROS) assay using a solubilizing surfactant was employed for follow-up screening. Upon combination use of ROS and mROS assays, the individual specificity was 88.2%, and the positive and negative predictivities were estimated to be 94.4% and 100%, respectively. In the 3T3 NRU PT, 3 cosmetics and 4 drugs were incorrectly predicted not to be phototoxic, although some of them were typical photoallergens. Thus, these in vitro screening systems individually provide false predictions; however, a systematic tiered approach using these assays could provide reliable photosafety assessment without any false-negatives. The combined use of in vitro assays might enable simple and fast non-animal photosafety evaluation of cosmetic ingredients. Copyright © 2013 Elsevier Ltd. All rights reserved.

Isolated Lung Perfusion as an Adjuvant Treatment of Colorectal Cancer Lung Metastases: A Preclinical Study in a Pig Model

PubMed Central

Pagès, Pierre-Benoit; Facy, Olivier; Mordant, Pierre; Ladoire, Sylvain; Magnin, Guy; Lokiec, Francois; Ghiringhelli, Francois; Bernard, Alain

2013-01-01

Background The lung is a frequent site of colorectal cancer (CRC) metastases. After surgical resection, lung metastases recurrences have been related to the presence of micrometastases, potentially accessible to a high dose chemotherapy administered via adjuvant isolated lung perfusion (ILP). We sought to determine in vitro the most efficient drug when administered to CRC cell lines during a short exposure and in vivo its immediate and delayed tolerance when administered via ILP. Methods First, efficacy of various cytotoxic molecules against a panel of human CRC cell lines was tested in vitro using cytotoxic assay after a 30-minute exposure. Then, early (operative) and delayed (1 month) tolerance of two concentrations of the molecule administered via ILP was tested on 19 adult pigs using hemodynamic, biological and histological criteria. Results In vitro, gemcitabine (GEM) was the most efficient drug against selected CRC cell lines. In vivo, GEM was administered via ILP at regular (20 µg/ml) or high (100 µg/ml) concentrations. GEM administration was associated with transient and dose-dependant pulmonary vasoconstriction, leading to a voluntary decrease in pump inflow in order to maintain a stable pulmonary artery pressure. After this modulation, ILP using GEM was not associated with any systemic leak, systemic damage, and acute or delayed histological pulmonary toxicity. Pharmacokinetics studies revealed dose-dependant uptake associated with heterogenous distribution of the molecule into the lung parenchyma, and persistent cytotoxicity of venous effluent. Conclusions GEM is effective against CRC cells even after a short exposure. ILP with GEM is a safe and reproducible technique. PMID:23527205

Improved oral bioavailability of poorly water-soluble indirubin by a supersaturatable self-microemulsifying drug delivery system

PubMed Central

Chen, Zhi-Qiang; Liu, Ying; Zhao, Ji-Hui; Wang, Lan; Feng, Nian-Ping

2012-01-01

Background Indirubin, isolated from the leaves of the Chinese herb Isatis tinctoria L, is a protein kinase inhibitor and promising antitumor agent. However, the poor water solubility of indirubin has limited its application. In this study, a supersaturatable self-microemulsifying drug delivery system (S-SMEDDS) was developed to improve the oral bioavailability of indirubin. Methods A prototype S-SMEDDS was designed using solubility studies and phase diagram construction. Precipitation inhibitors were selected from hydrophilic polymers according to their crystallization-inhibiting capacity through in vitro precipitation tests. In vitro release of indirubin from S-SMEDDS was examined to investigate its likely release behavior in vivo. The in vivo bioavailability of indirubin from S-SMEDDS and from SMEDDS was compared in rats. Results The prototype formulation of S-SMEDDS comprised Maisine™ 35-1:Cremophor® EL:Transcutol® P (15:40:45, w/w/w). Polyvinylpyrrolidone K17, a hydrophilic polymer, was used as a precipitation inhibitor based on its better crystallization-inhibiting capacity compared with polyethylene glycol 4000 and hydroxypropyl methylcellulose. In vitro release analysis showed more rapid drug release from S-SMEDDS than from SMEDDS. In vivo bioavailability analysis in rats indicated that improved oral absorption was achieved and that the relative bioavailability of S-SMEDDS was 129.5% compared with SMEDDS. Conclusion The novel S-SMEDDS developed in this study increased the dissolution rate and improved the oral bioavailability of indirubin in rats. The results suggest that S-SMEDDS is a superior means of oral delivery of indirubin. PMID:22403491

Attachment and growth of human keratinocytes in a serum-free environment.

PubMed

Gilchrest, B A; Calhoun, J K; Maciag, T

1982-08-01

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single-cell suspensions of keratinocytes from near-confluent primary plates, plated on 5-10 microgram/cm2 HFN, showed approximately 30-40% attachment after 2-24 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-12 days at densities greater than or equal to 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30-35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30-40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured keratinocytes.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution

PubMed Central

Denoyer, Delphine; Pearson, Helen B.; Clatworthy, Sharnel A.S.; Smith, Zoe M.; Francis, Paul S.; Llanos, Roxana M.; Volitakis, Irene; Phillips, Wayne A.; Meggyesy, Peter M.; Masaldan, Shashank; Cater, Michael A.

2016-01-01

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed. PMID:27175597

Discovery of a novel class of potent coumarin monoamine oxidase B inhibitors: development and biopharmacological profiling of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate (NW-1772) as a highly potent, selective, reversible, and orally active monoamine oxidase B inhibitor.

PubMed

Pisani, Leonardo; Muncipinto, Giovanni; Miscioscia, Teresa Fabiola; Nicolotti, Orazio; Leonetti, Francesco; Catto, Marco; Caccia, Carla; Salvati, Patricia; Soto-Otero, Ramon; Mendez-Alvarez, Estefania; Passeleu, Celine; Carotti, Angelo

2009-11-12

In an effort to discover novel selective monoamine oxidase (MAO) B inhibitors with favorable physicochemical and pharmacokinetic profiles, 7-[(m-halogeno)benzyloxy]coumarins bearing properly selected polar substituents at position 4 were designed, synthesized, and evaluated as MAO inhibitors. Several compounds with MAO-B inhibitory activity in the nanomolar range and excellent MAO-B selectivity (selectivity index SI > 400) were identified. Structure-affinity relationships and docking simulations provided valuable insights into the enzyme-inhibitor binding interactions at position 4, which has been poorly explored. Furthermore, computational and experimental studies led to the identification and biopharmacological characterization of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate 22b (NW-1772) as an in vitro and in vivo potent and selective MAO-B inhibitor, with rapid blood-brain barrier penetration, short-acting and reversible inhibitory activity, slight inhibition of selected cytochrome P450s, and low in vitro toxicity. On the basis of this preliminary preclinical profile, inhibitor 22b might be viewed as a promising clinical candidate for the treatment of neurodegenerative diseases.

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

Electrochemistry-mass spectrometry for in-vitro determination of selected chemotherapeutics and their electrochemical products in comparison to in-vivo approach.

PubMed

Szultka-Mlynska, Malgorzata; Buszewski, Boguslaw

2016-11-01

Chemotherapeutics are among the most frequently prescribed medications in modern medicine. They are widely prescribed; however, problems with organisms developing resistance to these drugs means that their efficacy may be lost, so care should be taken to avoid unnecessary prescription. It is therefore of great interest to study the detailed metabolism of these biologically active compounds. This study aimed at developing an efficient analytical protocol for the determination of in-vitro electrochemical products of selected antibiotic drugs (amoxicillin, cefotaxime, fluconazole, linezolid, metronidazole and moxifloxacin). Combination of electrochemistry (EC) and mass spectrometry (MS) was applied for the in-vitro determination of the studied antibiotics and their electrochemical products. To identify the structure of the detected electrochemical products, MS/MS experiments were performed. This was one of the first applications of the EC system for generation of electrochemical products produced from antibiotic drugs. Adjustment of appropriate conditions and such parameters as the potential value, mobile phase (pH), working electrode and temperature had significant influence on electrochemical simulations and the creation of selected derivatives. Consequently, several working electrodes were evaluated for this purpose. In most of the studied cases, mainly two types of products were observed. One corresponded to an increase in mass by 14Da, which can be explained by a process consisting of oxidation (+16 m/z) and dehydrogenation (-2 m/z); The second in turn showed mass reduction by 14Da, which can be attributed to the loss of -CH2 as a result of N-demethylation. The performed experiments consisted of two stages: electrochemical oxidation of the analyzed samples (phase I of metabolic transformation), and addition of glutathione (GSH) for follow-up reactions (phase II conjunction). The electrochemical results were compared to in-vivo experiments by analyzing urine samples from patients after antibiotic drugs have been administered.. Overall, the comparison of electrochemistry to in-vivo experiments shows the high potential of EC-MS as a fast analytical tool in the prediction of electrochemical conversion that could be applied to therapeutic drug monitoring and pharmacokinetic studies as well. Copyright © 2016 Elsevier B.V. All rights reserved.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

PubMed

Speranza, Maria-Carmela; Nowicki, Michal O; Behera, Prajna; Cho, Choi-Fong; Chiocca, E Antonio; Lawler, Sean E

2016-02-05

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum

PubMed Central

Teichmann, Klaus; Kuliberda, Maxime; Schatzmayr, Gerd; Pacher, Thomas; Zitterl-Eglseer, Karin; Joachim, Anja; Hadacek, Franz

2016-01-01

Disposal of organic plant wastes and by-products from the food or pharmaceutical industries usually involves high costs. In the present study, 42 samples derived from such by-products were screened in vitro against Cryptosporidium parvum, a protozoan parasite that may contaminate drinking water and cause diarrhoea. The novel bioassay was previously established in the microtitre plate format. Human ileocaecal adenocarcinoma (HCT-8) cell cultures were seeded with C. parvum oocysts and parasite development was monitored by an indirect fluorescent antibody technique (IFAT) and microscopic assessment for clusters of secondary infection (CSI). Minimum inhibitory concentrations (MICs) and potential detrimental effects on the host cells were determined. An ethanolic extract from olive (Olea europaea) pomace, after oil pressing and phenol recovery, reproducibly inhibited C. parvum development (MIC = 250–500 μg mL−1, IC50 = 361 (279–438) μg mL−1, IC90 = 467 (398–615) μg mL−1). Accordingly, tyrosol, hydroxytyrosol, trans-coniferyl alcohol and oleuropein were selected as reference test compounds, but their contributions to the observed activity of the olive pomace extract were insignificant. The established test system proved to be a fast and efficient assay for identifying anti-cryptosporidial activities in biological waste material and comparison with selected reference compounds. PMID:27627637

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

PubMed

Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

2007-01-01

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

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.

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.

Lactoferricin-related peptides with inhibitory effects on ACE-dependent vasoconstriction.

PubMed

Centeno, José M; Burguete, María C; Castelló-Ruiz, María; Enrique, María; Vallés, Salvador; Salom, Juan B; Torregrosa, Germán; Marcos, José F; Alborch, Enrique; Manzanares, Paloma

2006-07-26

A selection of lactoferricin B (LfcinB)-related peptides with an angiotensin I-converting enzyme (ACE) inhibitory effect have been examined using in vitro and ex vivo functional assays. Peptides that were analyzed included a set of sequence-related antimicrobial hexapeptides previously reported and two representative LfcinB-derived peptides. In vitro assays using hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I as substrates allowed us to select two hexapeptides, PACEI32 (Ac-RKWHFW-NH2) and PACEI34 (Ac-RKWLFW-NH2), and also a LfcinB-derived peptide, LfcinB17-31 (Ac-FKCRRWQWRMKKLGA-NH2). Ex vivo functional assays using rabbit carotid arterial segments showed PACEI32 (both D- and L-enantiomers) and LfcinB17-31 have inhibitory effects on ACE-dependent angiotensin I-induced contraction. None of the peptides exhibited in vitro ACE inhibitory activity using bradykinin as the substrate. In conclusion, three bioactive lactoferricin-related peptides exhibit inhibitory effects on both ACE activity and ACE-dependent vasoconstriction with potential to modulate hypertension that deserves further investigation.

Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc, and vitamins B6, B12, D, and E

PubMed Central

Etcheverry, Paz; Grusak, Michael A.; Fleige, Lisa E.

2012-01-01

A review of in vitro bioaccessibility and bioavailability methods for polyphenols and selected nutrients is presented. The review focuses on in vitro solubility, dialyzability, the dynamic gastrointestinal model (TIM)™, and Caco-2 cell models, the latter primarily for uptake and transport, and a discussion of how these methods have been applied to generate data for a range of nutrients, carotenoids, and polyphenols. Recommendations are given regarding which methods are most justified for answering bioaccessibility or bioavailability related questions for specific nutrients. The need for more validation studies in which in vivo results are compared to in vitro results is also discussed. PMID:22934067

Cytarabine-AOT catanionic vesicle-loaded biodegradable thermosensitive hydrogel as an efficient cytarabine delivery system.

PubMed

Liu, Jing; Jiang, Yue; Cui, Yuting; Xu, Chuanshan; Ji, Xiaoqing; Luan, Yuxia

2014-10-01

Carrier with high drug loading content is one of the most important issues in drug delivery system. In the present work, an ion-pair amphiphilic molecule composed of anticancer drug cation and surfactant anion is used for straightforward fabricating vesicles for cancer therapy. Anticancer drug (cytarabine hydrochloride) and anionic surfactant (AOT) are selected for the fabrication of ion-pair amphiphilic molecule. One amphiphilic molecule contains one drug cation, thus the drug loading content is 50% (mol/mol) in theory. The in vitro drug release study shows that the release time of cytarabine is about 3 times of the pure cytarabine solution and the permeability of cytarabine has been improved about 160 times tested by parallel artificial membrane permeability assay model. However, the hemolytic toxicity is largely decreased in the studied concentration range. The in vitro cytotoxicity results show that cytarabine-AOT amphiphiles have a much lower IC50 (drug concentration resulting in 50% cell death) value and a higher cell inhibition rate comparing with their respective components, indicating its effective therapy for leukemic cells. To obtain a longer and a convenient drug release system, the prepared vesicles are further incorporated into the thermosensitive PLGA-PEG-PLGA hydrogel to prepare a subcutaneous administration. The in vivo drug release results indicate that cytarabine-AOT vesicle-loaded hydrogel is a good injectable delivery system for controlled release of cytarabine for cancer therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

A 1024-Channel CMOS Microelectrode Array With 26,400 Electrodes for Recording and Stimulation of Electrogenic Cells In Vitro

PubMed Central

Ballini, Marco; Müller, Jan; Livi, Paolo; Chen, Yihui; Frey, Urs; Stettler, Alexander; Shadmani, Amir; Viswam, Vijay; Jones, Ian Lloyd; Jäckel, David; Radivojevic, Milos; Lewandowska, Marta K.; Gong, Wei; Fiscella, Michele; Bakkum, Douglas J.; Heer, Flavio; Hierlemann, Andreas

2017-01-01

To advance our understanding of the functioning of neuronal ensembles, systems are needed to enable simultaneous recording from a large number of individual neurons at high spatiotemporal resolution and good signal-to-noise ratio. Moreover, stimulation capability is highly desirable for investigating, for example, plasticity and learning processes. Here, we present a microelectrode array (MEA) system on a single CMOS die for in vitro recording and stimulation. The system incorporates 26,400 platinum electrodes, fabricated by in-house post-processing, over a large sensing area (3.85 × 2.10 mm2) with sub-cellular spatial resolution (pitch of 17.5 μm). Owing to an area and power efficient implementation, we were able to integrate 1024 readout channels on chip to record extracellular signals from a user-specified selection of electrodes. These channels feature noise values of 2.4 μVrms in the action-potential band (300 Hz–10 kHz) and 5.4 μVrms in the local-field-potential band (1 Hz–300 Hz), and provide programmable gain (up to 78 dB) to accommodate various biological preparations. Amplified and filtered signals are digitized by 10 bit parallel single-slope ADCs at 20 kSamples/s. The system also includes 32 stimulation units, which can elicit neural spikes through either current or voltage pulses. The chip consumes only 75 mW in total, which obviates the need of active cooling even for sensitive cell cultures. PMID:28502989

Blue Laser Light Increases Perfusion of a Skin Flap Via Release of Nitric Oxide from Hemoglobin

PubMed Central

Mittermayr, Rainer; Osipov, Anatoly; Piskernik, Christina; Haindl, Susanne; Dungel, Peter; Weber, Carina; Vladimirov, Yuri A; Redl, Heinz; Kozlov, Andrey V

2007-01-01

It has recently been shown that nitrosyl complexes of hemoglobin (NO-Hb) are sensitive to low-level blue laser irradiation, suggesting that laser irradiation can facilitate the release of biologically active nitric oxide (NO), which can affect tissue perfusion. The aim of this study was to evaluate the therapeutic value of blue laser irradiation for local tissue perfusion after surgical intervention. Blood was withdrawn from a rat, exposed to NO and infused back to the same rat or used for in vitro experiments. In vitro, an increase of NO-Hb levels (electron paramagnetic resonance spectroscopy) up to 15 μM in rat blood did not result in the release of detectable amounts of NO (NO selective electrode). Blue laser irradiation of NO-Hb in blood caused decomposition of NO-Hb complexes and release of free NO. Systemic infusion of NO-Hb in rats affected neither systemic circulation (mean arterial pressure) nor local tissue perfusion (Doppler blood flow imaging system). In contrast, a clear enhancement of local tissue perfusion was observed in epigastric flap when elevated NO-Hb levels in blood were combined with local He-Cd laser irradiation focused on the left epigastric artery. The enhancement of regional tissue perfusion was not accompanied by any detectable changes in systemic circulation. This study demonstrates that blue laser irradiation improves local tissue perfusion in a controlled manner stimulating NO release from NO-Hb complexes. PMID:17515954

In vitro antiherpetic and antirotaviral activities of a sulfate prepared from Mimosa scabrella galactomannan.

PubMed

Chrestani, Francielli; Sierakowski, Maria Rita; de Andrade Uchoa, Daniel Esdras; Nozawa, Carlos; Sassaki, Guilherme Lanzi; Gorin, Philip Albert James; Ono, Lucy

2009-12-01

A chemically sulfated galactomannan (BRS) from seeds of Mimosa scabrella had in vitro antiviral activity against Herpes simplex virus 1 (HSV-1), but not against Simian rotavirus A/SA11 (SiRV-A/SA11). It was examined by (13)C NMR spectroscopy, which showed the sulfate groups to be mainly at C-6 of galactose residues. BRS had a selective inhibition against HSV-1 during its attachment step, having an IC(50) lower than 2.5microg/ml, determined by plaque reduction, and a selectivity index of greater than 181, suggesting that the antiviral effect is likely due to interactions between the virus and BRS, being influenced its overall surface charge.

The Effects of Ammonium Perchlorate on Reproduction and Development of Amphibians

DTIC Science & Technology

2008-01-01

Abstract: Ammonium perchlorate is a pervasive pollutant primarily from rocket fuel and fertilizers . It is know , among other things , to affect...females, their ovulated eggs collected, and in vitro fertilization conducted. Healthy ovulated eggs were selected and placed in Petri dishes...used and fertilization accomplished in vitro in the presence of perchlorate concentrations. *These tasks were not completed. Studies with Sodium

Effect of nerodilol and carvone on in vitro permeation of nicorandil across rat epidermal membrane.

PubMed

Krishnaiah, Yellela S R; Al-Saidan, Saleh M; Chandrasekhar, Dantam V; Rama, Bukka

2006-04-01

The objective of the study was to investigate the effect of nerodilol and carvone on the in vitro transdermal delivery of nicorandil so as to fabricate a membrane-moderated transdermal therapeutic system. The in vitro permeation studies were carried across the rat epidermal membrane from the hydroxypropyl methylcellulose (HPMC) gels (prepared with 70:30 v/v ethanol-water) containing selected concentrations of a terpene such as nerodilol (0%w/w, 4%w/w, 8%w/w, 10%w/w, or 12%w/w) or carvone (0%w/w, 4%w/w, 8%w/w, 12%w/w, or 16%w/w). The amount of nicorandil permeated (Q(24)) from HPMC gel drug reservoir without a terpene was 3424.6+/-51.4 microg/cm(2), and the corresponding flux of the drug was 145.5+/-2.2 microg/cm(2). h. The flux of nicorandil increased with an increase in terpene concentration in HPMC gel. It was increased ranging from 254.9+/-3.1 to 375.7+/-3.2 microg/cm(2).h or 207.6+/-4.7 to 356.7+/-15.3 microg/cm(2). h from HPMC gels containing nerodilol (4%w/w to 12%w/w) or carvone (4%w/w to 16%w/w), respectively. Nerodilol increased the flux of nicorandil by about 2.62-folds whereas carvone increased the flux of the drug by about 2.49-folds across the rat epidermal membrane. The results of the Fourier Transform Infrared (FT-IR) study indicated that the enhanced in vitro transdermal delivery of nicorandil might be due to the partial extraction of stratum corneum lipids by nerodilol or carvone. It was concluded that the terpenes, nerodilol and carvone, produced a marked penetration enhancing effect on the transdermal delivery of nicorandil that could be used in the fabrication of membrane-moderated transdermal therapeutic systems.

Alkyl Protocatechuate-Loaded Nanostructured Lipid Systems as a Treatment Strategy for Paracoccidioides brasiliensis and Paracoccidioides lutzii In Vitro

PubMed Central

Medina-Alarcón, Kaila P.; Singulani, Junya L.; Voltan, Aline R.; Sardi, Janaina C. O.; Petrônio, Maicon S.; Santos, Mariana B.; Polaquini, Carlos R.; Regasini, Luis O.; Bolzani, Vanderlan S.; da Silva, Dulce H. S.; Chorilli, Marlus; Mendes-Giannini, Maria J. S.; Fusco-Almeida, Ana M.

2017-01-01

Dodecyl protocatechuate (dodecyl) is a derivative of protocatechuic acid (3,4-dihydroxybenzoic acid) that possesses anti-oxidant and antifungal properties. Nanostructured lipid systems (NLS) can potentiate the action of many antifungal agents, reducing the required dose and side effects by improving their activity. This work aimed to evaluate dodecyl protocatechuate loaded into a NLS (NLS+dodecyl) as a strategy for the treatment of Paracoccidioides brasiliensis and P. lutzii in vitro. Antifungal activity against P. brasiliensis and P. lutzii was evaluated using the microdilution technique. NLS+dodecyl showed high antifungal activity with a minimum inhibitory concentration ranging from 0.06 to 0.03 μg/mL; 4- to 16-fold higher than that of free dodecyl. NLS+dodecyl was able to inhibit fungal adhesion of the extracellular artificial matrix proteins (laminin and fibronectin), resulting in 82.4 and 81% inhibition, respectively, an increase of 8–17% compared with free dodecyl. These findings corroborate previous results demonstrating 65 and 74% inhibition of fungal adhesion in pulmonary fibroblast cells by dodecyl and NLS+dodecyl, respectively, representing a 9% increase in inhibition for NLS+dodecyl. Subsequently, cytotoxicity was evaluated using the 0.4% sulforhodamine B assay. NLS+dodecyl did not exhibit cytotoxicity in MRC5 (human pneumocyte) and HepG2 (human hepatic carcinoma) cells, thus increasing the selectivity index for NLS+dodecyl. In addition, cytotoxicity was evaluated in vivo using the Caenorhabditis elegans model; neither dodecyl nor NLS+dodecyl exhibited any toxic effects. Taken together, these results suggest that NLS can be used as a strategy to improve the activity of dodecyl against P. brasiliensis and P. lutzii because it improves antifungal activity, increases the inhibition of fungal adhesion in lung cells and the extracellular matrix in vitro, and does not exhibit any toxicity both in vitro and in vivo. PMID:28659880

Testing for developmental neurotoxicity using a battery of in vitro assays for key cellular events in neurodevelopment.

PubMed

Harrill, Joshua A; Freudenrich, Theresa; Wallace, Kathleen; Ball, Kenneth; Shafer, Timothy J; Mundy, William R

2018-04-05

Medium- to high-throughput in vitro assays that recapitulate the critical processes of nervous system development have been proposed as a means to facilitate rapid testing and identification of chemicals which may affect brain development. In vivo neurodevelopment is a complex progression of distinct cellular processes. Therefore, batteries of in vitro assays that model and quantify effects on a variety of neurodevelopmental processes have the potential to identify chemicals which may affect brain development at different developmental stages. In the present study, the results of concentration-response screening of 67 reference chemicals in a battery of high content imaging and microplate reader-based assays that evaluate neural progenitor cell proliferation, neural proginitor cell apoptosis, neurite initiation/outgrowth, neurite maturation and synaptogenesis are summarized and compared. The assay battery had a high degree of combined sensitivity (87%) for categorizing chemicals known to affect neurodevelopment as active and a moderate degree of combined specificity (71%) for categorizing chemicals not associated with affects on neurodevelopment as inactive. The combined sensitivity of the assay battery was higher compared to any individual assay while the combined specificity of the assay battery was lower compared to any individual assay. When selectivity of effects for a neurodevelopmental endpoint as compared to general cytotoxicity was taken into account, the combined sensitivity of the assay battery decreased (68%) while the combined specificity increased (93%). The identity and potency of chemicals identified as active varied across the assay battery, underscoring the need for use of a combination of diverse in vitro models to comprehensively screen chemicals and identify those which potentially affect neurodevelopment. Overall, these data indicate that a battery of assays which address many different processes in nervous system development may be used to identify potential developmental neurotoxicants and to distinguish specific from generalized cytotoxic effects with a high degree of success. Published by Elsevier Inc.

Dendrimer-coupled sonophoresis-mediated transdermal drug-delivery system for diclofenac.

PubMed

Huang, Bin; Dong, Wei-Jiang; Yang, Gao-Yi; Wang, Wei; Ji, Cong-Hua; Zhou, Fei-Ni

2015-01-01

The purpose of the present study was to develop a novel transdermal drug-delivery system comprising a polyamidoamine dendrimer coupled with sonophoresis to enhance the permeation of diclofenac (DF) through the skin. The novel transdermal drug-delivery system was developed by using a statistical Plackett-Burman design. Hairless male Wistar rat skin was used for the DF-permeation study. Coupling media concentration, ultrasound-application time, duty cycle, distance from probe to skin, and a third-generation polyamidoamine-dendrimer concentration were selected as independent variables, while in vitro drug release was selected as a dependent variable. Independent variables were found to be statistically significant (P<0.05). DF gel without dendrimer and ultrasound treatment to skin (passive delivery, run 13) showed 56.69 µg/cm(2) cumulative drug permeated through the skin, while the DF-dendrimer gel without sonophoresis treatment (run 14) showed 257.3 µg/cm(2) cumulative drug permeated through the skin after 24 hours. However, when the same gel was applied to sonophoresis-treated skin, drastic permeation enhancement was observed. In the case of run 3, the cumulative drug that permeated through the skin was 935.21 µg/cm(2). It was concluded that dendrimer-coupled sonophoresis-mediated transdermal drug delivery system has the potential to enhance the permeation of DF through the skin.

Novel potato plants with enhanced cadmium resistance and antioxidative defence generated after in vitro cell line selection.

PubMed

Ashrafzadeh, Seyedardalan; Leung, David W M

2017-01-01

It is of interest to apply plant tissue culture to generate plants resistant to toxic effects of cadmium (Cd) on plant growth. Callus cultures were initiated from leaf explants of micropropagated potato plantlets (Solanum tuberosum L., cv. Iwa) for in vitro selection comprising 18 different Cd treatments varying in Cd exposure timing and duration. Plantlets regenerated from two different lines of Cd-selected calli, L9 and L11, were found to exhibit enhanced resistance to 218 μM Cd compared to control (source plantlets for leaf explants used to initiate callus cultures for Cd resistance). In response to 218 μM Cd, L11 plantlets had lower levels of lipid peroxidation and hydrogen peroxide than control and L9 plantlets. In addition, antioxidative enzyme activities in L11 were generally higher than control. L11 also had a higher level of proline than control.

Emergent biological properties of arrestin pathway-selective biased agonism.

PubMed

Appleton, Kathryn M; Luttrell, Louis M

2013-06-01

Our growing appreciation of the pluridimensionality of G protein-coupled receptor (GPCR) signaling, combined with the phenomenon of orthosteric ligand "bias", has created the possibility of drugs that selectively modulate different aspects of GPCR function for therapeutic benefit. When viewed from the short-term perspective, e.g. changes in receptor conformation, effector coupling or second messenger generation, biased ligands appear to activate a subset of the response profile produced by a conventional agonist. Yet when examined in vivo, the limited data available suggest that biased ligand effects can diverge from their conventional counterparts in ways that cannot be predicted from their in vitro efficacy profile. What is currently missing, at least with respect to G protein and arrestin pathway-selective ligands, is a rational framework for relating the in vitro efficacy of a "biased" agonist to its in vivo actions that will enable drug screening programs to identify ligands with the desired biological effects.

Selective propensity of bovine jugular vein material to bacterial adhesions: An in-vitro study.

PubMed

Jalal, Zakaria; Galmiche, Louise; Lebeaux, David; Villemain, Olivier; Brugada, Georgia; Patel, Mehul; Ghigo, Jean-Marc; Beloin, Christophe; Boudjemline, Younes

2015-11-01

Percutaneous pulmonary valve implantation (PPVI) using Melody valve made of bovine jugular vein is safe and effective. However, infective endocarditis has been reported for unclear reasons. We sought to assess the impact of valvular substrates on selective bacterial adhesion. Three valved stents (Melody valve, homemade stents with bovine and porcine pericardium) were tested in-vitro for bacterial adhesion using Staphylococcus aureus and Streptococcus sanguinis strains. Bacterial adhesion was higher on bovine jugular venous wall for S. aureus and on Melody valvular leaflets for S. sanguinis in control groups and significantly increased in traumatized Melody valvular leaflets with both bacteria (traumatized vs non traumatized: p=0.05). Bacterial adhesion was lower on bovine pericardial leaflets. Selective adhesion of S. aureus and S. sanguinis pathogenic strains to Melody valve tissue was noted on healthy tissue and increased after implantation procedural steps. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In vitro maturation of human oocytes for assisted reproduction.

PubMed

Jurema, Marcus W; Nogueira, Daniela

2006-11-01

To describe and evaluate the current practice of in vitro maturation of oocytes for assisted reproduction. Review of the available and relevant literature regarding in vitro maturation of oocytes. In vitro maturation of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies. This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of ovarian hyperstimulation syndrome, and simplicity of protocol. In vitro maturation of oocytes for assisted reproduction in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing in vitro maturation into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.

D-Serine and D-Cycloserine Reduce Compulsive Alcohol Intake in Rats

PubMed Central

Seif, Taban; Simms, Jeffrey A; Lei, Kelly; Wegner, Scott; Bonci, Antonello; Messing, Robert O; Hopf, F Woodward

2015-01-01

There is considerable interest in NMDAR modulators to enhance memory and treat neuropsychiatric disorders such as addiction, depression, and schizophrenia. D-serine and D-cycloserine, the NMDAR activators at the glycine site, are of particular interest because they have been used in humans without serious adverse effects. Interestingly, D-serine also inhibits some NMDARs active at hyperpolarized potentials (HA-NMDARs), and we previously found that HA-NMDARs within the nucleus accumbens core (NAcore) are critical for promoting compulsion-like alcohol drinking, where rats consume alcohol despite pairing with an aversive stimulus such as quinine, a paradigm considered to model compulsive aspects of human alcohol use disorders (AUDs). Here, we examined the impact of D-serine and D-cycloserine on this aversion-resistant alcohol intake (that persists despite adulteration with quinine) and consumption of quinine-free alcohol. Systemic D-serine reduced aversion-resistant alcohol drinking, without altering consumption of quinine-free alcohol or saccharin with or without quinine. Importantly, D-serine within the NAcore but not the dorsolateral striatum also selectively reduced aversion-resistant alcohol drinking. In addition, D-serine inhibited EPSCs evoked at −70 mV in vitro by optogenetic stimulation of mPFC–NAcore terminals in alcohol-drinking rats, similar to reported effects of the NMDAR blocker AP5. Further, D-serine preexposure occluded AP5 inhibition of mPFC-evoked EPSCs, suggesting that D-serine reduced EPSCs by inhibiting HA-NMDARs. Systemic D-cycloserine also selectively reduced intake of quinine-adulterated alcohol, and D-cycloserine inhibited NAcore HA-NMDARs in vitro. Our results indicate that HA-NMDAR modulators can reduce aversion-resistant alcohol drinking, and support testing of D-serine and D-cycloserine as immediately accessible, FDA-approved drugs to treat AUDs. PMID:25801502

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

PubMed

Escher, Beate I; Allinson, Mayumi; Altenburger, Rolf; Bain, Peter A; Balaguer, Patrick; Busch, Wibke; Crago, Jordan; Denslow, Nancy D; Dopp, Elke; Hilscherova, Klara; Humpage, Andrew R; Kumar, Anu; Grimaldi, Marina; Jayasinghe, B Sumith; Jarosova, Barbora; Jia, Ai; Makarov, Sergei; Maruya, Keith A; Medvedev, Alex; Mehinto, Alvine C; Mendez, Jamie E; Poulsen, Anita; Prochazka, Erik; Richard, Jessica; Schifferli, Andrea; Schlenk, Daniel; Scholz, Stefan; Shiraishi, Fujio; Snyder, Shane; Su, Guanyong; Tang, Janet Y M; van der Burg, Bart; van der Linden, Sander C; Werner, Inge; Westerheide, Sandy D; Wong, Chris K C; Yang, Min; Yeung, Bonnie H Y; Zhang, Xiaowei; Leusch, Frederic D L

2014-01-01

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

The Effect of Lactobacillus plantarum ATCC 8014 and Lactobacillus acidophilus NCFM Fermentation on Antioxidant Properties of Selected in Vitro Sprout Culture of Orthosiphon aristatus (Java Tea) as a Model Study

PubMed Central

Hunaefi, Dase; Akumo, Divine N.; Riedel, Heidi; Smetanska, Iryna

2012-01-01

High rosmarinic acid (RA) productivity has been achieved by applying jasmonic acid and yeast extract elicitors to the in vitro sprout culture of Orthosiphon aritatus (IOSC). The highest RA accumulation from three solvents was detected in IOSC after treatment with yeast extract (5 g/L). HPLC analysis clearly confirmed a drastic increase in RA subjected to yeast extract elicitation. Therefore, this yeast extract elicited IOSC was chosen for a lactic acid bacteria (LAB) fermentation study as a model system. This selected IOSC was subjected to different types of LAB fermentations (Lactobacillus plantarum ATCC 8014 and Lactobacillus acidophilus NCFM) for different periods of time 24, 48 and 72 h. The LAB fermentations consisted of solid state fermentations (SSF) and liquid state fermentations (LSF) in a Digital Control Unit (DCU) fermenter system. The aim was to determine the effect of fermentation on the antioxidant properties of the plant extract. Results indicated that all types of LAB fermentation decreased the level of RA and total phenolics, however, a slight increase in total flavonoids and flavonols was observed in SSF samples. HPLC results confirmed that the longer the fermentation, the greater the reduction in RA content. The highest reduction was obtained in the sample of LSF inoculated with L. plantarum for a period of 72 h. The temperature of fermentation (37 °C) was predicted as contributing to the declining level in RA content. The loss in RA was concomitant with a loss of total antioxidant activity (1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, Trolox Equivalent Antioxidant Capacity (TEAC), and Superoxide Dismutase (SOD)-like activity). These results indicate that RA is the major contributor to the antioxidant activity of this plant. PMID:26787613

Class I Histone Deacetylase Inhibition by Tianeptinaline Modulates Neuroplasticity and Enhances Memory.

PubMed

Zhao, Wen-Ning; Ghosh, Balaram; Tyler, Marshall; Lalonde, Jasmin; Joseph, Nadine F; Kosaric, Nina; Fass, Daniel M; Tsai, Li-Huei; Mazitschek, Ralph; Haggarty, Stephen J

2018-06-22

Through epigenetic and other regulatory functions, the histone deacetylase (HDAC) family of enzymes has emerged as a promising therapeutic target for central nervous system and other disorders. Here we report on the synthesis and functional characterization of new HDAC inhibitors based structurally on tianeptine, a drug used primarily to treat major depressive disorder (MDD) that has a poorly understood mechanism of action. Since the chemical structure of tianeptine resembles certain HDAC inhibitors, we profiled the in vitro HDAC inhibitory activity of tianeptine and demonstrated its ability to inhibit the lysine deacetylase activity of a subset of class I HDACs. Consistent with a model of active site Zn 2+ chelation by the carboxylic acid present in tianeptine, newly synthesized analogues containing either a hydroxamic acid or ortho-aminoanilide exhibited increased potency and selectivity among the HDAC family. This in vitro potency translated to improved efficacy in a panel of high-content imaging assays designed to assess HDAC target engagement and functional effects on critical pathways involved in neuroplasticity in both primary mouse neurons and, for the first time, human neurons differentiated from pluripotent stem cells. Most notably, tianeptinaline, a class I HDAC-selective analogue of tianeptine, but not tianeptine itself, increased histone acetylation, and enhanced CREB-mediated transcription and the expression of Arc (activity-regulated cytoskeleton-associated protein). Systemic in vivo administration of tianeptinaline to mice confirmed its brain penetration and was found to enhance contextual fear conditioning, a behavioral test of hippocampal-dependent memory. Tianeptinaline and its derivatives provide new pharmacological tools to dissect chromatin-mediated neuroplasticity underlying memory and other epigenetically related processes implicated in health and disease.

Glycyrrhetinic Acid-Mediated Polymeric Drug Delivery Targeting the Acidic Microenvironment of Hepatocellular Carcinoma.

PubMed

Zhang, Jinming; Zhang, Min; Ji, Juan; Fang, Xiefan; Pan, Xin; Wang, Yitao; Wu, Chuanbin; Chen, Meiwan

2015-10-01

The major hurdle of current drug carrier against hepatocellular carcinoma (HCC) is the lack of specific and selective drug delivery to HCC. In this study, a novel glycyrrhetinic acid (GA) and poly(L-Histidine) (PHIS) mediated polymeric drug delivery system was developed to target HCC that have GA binding receptors and release its encapsulated anticancer drug in the acidic microenvironment of HCC. Firstly, GA and PHIS were conjugated to form poly (ethylene glycol)-poly(lactic-co-glycolic acid) (GA-PEG-PHIS-PLGA, GA-PPP) micelles by grafting reaction between active terminal groups. Secondly, andrographolide (AGP) was encapsulated to GA-PPP to make AGP/GA-PPP using the solvent evaporation method. The pH-responsive property of AGP/GA-PPP micelles was validated by monitoring its stability and drug release behavior in different pH conditions. Furthermore, selective hepatocellular uptake of GA-PPP micelles in vitro, liver specific drug accumulation in vivo, as well as the enhanced antitumor effects of AGP/GA-PPP micelles confirmed the HCC targeting property of our novel drug delivery system. Average size of AGP/GA-PPP micelles increased significantly and the encapsulated AGP released faster in vitro at pH 5.0, while micelles keeping stable in pH 7.4. AGP/GA-PPP micelles were uptaken more efficiently by human Hep3B liver cells than that by human MDA-MB-231 breast cancer cells. GA-PPP micelles accumulated specifically in the liver and possessed long retention time in vivo. AGP/GA-PPP micelles significantly inhibited tumor growth and provided better therapeutic outcomes compared to free AGP and AGP/PEG-PLGA(AGP/PP) micelles without GA and PHIS decoration. This novel GA-PPP polymeric carrier is promising for targeted treatment of HCC.

Titanium wire implants with nanotube arrays: A study model for localized cancer treatment.

PubMed

Kaur, Gagandeep; Willsmore, Tamsyn; Gulati, Karan; Zinonos, Irene; Wang, Ye; Kurian, Mima; Hay, Shelley; Losic, Dusan; Evdokiou, Andreas

2016-09-01

Adverse complications associated with systemic administration of anti-cancer drugs are a major problem in cancer therapy in current clinical practice. To increase effectiveness and reduce side effects, localized drug delivery to tumour sites requiring therapy is essential. Direct delivery of potent anti-cancer drugs locally to the cancer site based on nanotechnology has been recognised as a promising alternative approach. Previously, we reported the design and fabrication of nano-engineered 3D titanium wire based implants with titania (TiO2) nanotube arrays (Ti-TNTs) for applications such as bone integration by using in-vitro culture systems. The aim of present study is to demonstrate the feasibility of using such Ti-TNTs loaded with anti-cancer agent for localized cancer therapy using pre-clinical cancer models and to test local drug delivery efficiency and anti-tumour efficacy within the tumour environment. TNF-related apoptosis-inducing ligand (TRAIL) which has proven anti-cancer properties was selected as the model drug for therapeutic delivery by Ti-TNTs. Our in-vitro 2D and 3D cell culture studies demonstrated a significant decrease in breast cancer cell viability upon incubation with TRAIL loaded Ti-TNT implants (TRAIL-TNTs). Subcutaneous tumour xenografts were established to test TRAIL-TNTs implant performance in the tumour environment by monitoring the changes in tumour burden over a selected time course. TRAIL-TNTs showed a significant regression in tumour burden within the first three days of implant insertion at the tumour site. Based on current experimental findings these Ti-TNTs wire implants have shown promising capacity to load and deliver anti-cancer agents maintaining their efficacy for cancer treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Vitro Exposure Systems and Dosimetry Assessment Tools ...

EPA Pesticide Factsheets

In 2009, the passing of The Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP) and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed “modified risk”. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference titled “In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products” to bring together stakeholders representing regulatory agencies, academia, and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapor exposure systems, as well as the various approaches and challenges to quantifying the complex exposures, in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were, 1) Tobacco Smoke And E-Cigarette Aerosols, 2) Air-Liquid Interface-In Vitro Exposure Systems, 3) Dosimetry Approaches For Particles And Vapors; In Vitro Dosimetry Determinations and 4) Exposure Microenvironment/Physiology Of Cells. The two and a half day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will re

Module assemblage technology for floating systems: in vitro flotation and in vivo gastro-retention.

PubMed

Strusi, Orazio Luca; Sonvico, Fabio; Bettini, Ruggero; Santi, Patrizia; Colombo, Gaia; Barata, Pedro; Oliveira, Ana; Santos, Delfim; Colombo, Paolo

2008-07-14

The aim of this research was to study, in vitro by resultant-weight measurement and in vivo by gamma-scintigraphy experiments in humans, the floatation behavior of systems obtained by modules assembled in void configuration. The assembled system technology allowed the manufacturing of a system characterized by the presence of an internal void space that provided an apparent density lower than water. The gastro-retention times of floating assembled systems were determined in comparison with non-floating systems having the same mass and composition. In vitro the floatation of the system started immediately after immersion in water and lasted for more than 5 h. The in vivo studies confirmed that the in vitro floating ability of void configuration was maintained also in the human stomach where the system stayed for periods of time ranging from 2.5 to 5.0 h, depending on the food regimen and the sex of the subject. Reiterate eating and drinking further prolonged the stomach residence time.

Discovery of novel aminobenzisoxazole derivatives as orally available factor IXa inhibitors

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

Sakurada, Isao; Endo, Toshiya; Hikita, Katsuyoshi

2017-06-01

Using structure based drug design, novel aminobenzisoxazoles as coagulation factor IXa inhibitors were designed and synthesized. Highly selective inhibition of FIXa over FXa was demonstrated. Anticoagulation profile of selected compounds was evaluated by aPTT and PT tests. In vitro ADMET and pharmacokinetic (PK) profiles were also evaluated.