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Results for “protocol optimization”

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At least 307 records · Page 17Linked to original sources

PubMed2006

Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells.

Recently, decellularized tissue has been reported to have the potential to regenerate a variety of tissues. However, the optimal protocol for a decellularized esophagus has not been studied. Here, we investigated the effect of different decellularization protocols on the histology and biocompatibility of decellularized esophagi in view of future applications to tissue engineering. The esophageal mucosal epithelium (EP) from 4-week-old Wistar rats was enzymatically dissociated and cultured with growth-arrested feeder cells. Two methods for decellularization using deoxycholic acid (DEOX) or Triton X-100 (TRITON) were compared on esophagi from adult Wistar rats. Those treated with DEOX showed superior mechanical properties, maintenance of extracellular matrix, and lower DNA content than those treated with TRITON. To evaluate the biocompatibility of the scaffold, cultured (passage 3) esophageal epithelial cells were seeded inside the decellularized esophagus and cultured for 7 days. The cells seeded onto the decellularized esophagus were examined histologically and immunocytochemically. Esophageal epithelial cells were stratified into three to four cellular layers in vitro inside the decellularized esophagus, to show polarity. The results from immunocytochemistry indicated that the seeded epithelial cells expressed characteristic marker proteins for native esophageal EP. Decellularized esophagus showed suitable compatibility as a scaffold material for esophageal tissue engineering.

Animals↗

PubMed2004

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy.

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.

Animals↗

PubMedDate not supplied

Contrast-to-noise ratio in functional MRI of relative cerebral blood volume with sprodiamide injection.

The purpose of this study was to investigate the dependence of contrast-to-noise ratio (CNR) on the dose and rate of sprodiamide injection in magnetic resonance relative cerebral blood volume (rCBV) imaging. rCBV maps for 35 normal volunteers were constructed from dynamic MR image sets acquired with echo-planar spin-echo imaging after intravenous injection of sprodiamide. Doses of .1, .2, and .3 mmol/kg, at rates of 2 ml/second and 5 ml/second, were tested. CNRs and blood/volume ratios of gray to white matter were computed. CNR depended on dose (P < .0001) but was independent of injection rate (P < .69). rCBV ratios of gray to white matter were dose independent (P < .38) and rate independent (P < .97). The dependence of CNR on dose, but not injection rate, has practical implications in optimal protocol design. The independence of gray/white ratios supports the theory underlying the generation of rCBV maps.

Adult↗

PubMed1992

Liver tissue characterization by in vitro NMR: tissue handling and biological variation.

The extraction of reliable and useful relaxation time data for tissue characterization by NMR requires strict protocols, optimized for each type of biological tissue, which include parameters like storage duration and temperature as well as measurement parameters. Spin-lattice relaxation times in liver tissue vary not only with NMR frequency but also with their "time-after-excision characteristics," while spin-spin relaxation times are almost independent of most parameters which influence T1 at 20 MHz in normal liver tissue (e.g., species, sex, circadian rythm, starvation). T2, however, being more sensitive to water content and pH changes, is well suited for detecting nonspecific tissue alterations (e.g., due to ischemia, chemical toxins). Following the suggestions outlined herein, investigation of at least 120 min of time-after-excision (storage) effects allows the significant distinguishing of various physiological differences in normal liver tissue as well as improvement of early detection of liver pathologies.

Animals↗

PubMed1992

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.

Animals↗

PubMed2006

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris.

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.

Cysteine↗

PubMed2001

Optimization of quartz tube pyrolysis atmospheric pressure ionization mass spectrometry for the generation of bacterial biomarkers.

Experimental procedures were investigated to improve the efflux of biomolecule pyrolyzates from quartz tube pyrolysis under atmospheric pressure ionization mass spectrometry conditions. Heating regimes, airflows, and ion focusing parameters were optimized to increase the informative mass spectral signals generated from the pyrolysis of Gram-positive bacterial spores and vegetative cells. Dipicolinic acid (DPA) is found in 5-15% by weight in Gram-positive Bacillus spores, and the parameter optimization procedures provided an intense mass spectral signature of the m/z 168 protonated DPA molecule with a minimization of pyrolytic and ionization fragments. Moreover, mass spectral information from the optimization protocols yielded peaks and mass patterns characteristic of DNA and RNA nitrogen bases, protein diketopiperazines, and amino sugars.

Bacillus↗

PubMed1993

Drug delivery optimization through Bayesian networks: an application to erythropoietin therapy in uremic anemia.

This paper describes how Bayesian networks can be used in combination with compartmental models to plan recombinant human erythropoietin delivery in the treatment of anemia of chronic uremic patients. Past measurements of hemoglobin concentration in a patient during the therapy can be exploited to adjust the parameters of a compartmental model of erythropoiesis. This adaptive process provides more accurate patient-specific predictions, and hence a more rational dosage planning. Inferences are performed by using a stochastic simulation algorithm called Gibbs sampling. We describe a drug delivery optimization protocol based on our approach. Some results obtained on real data are presented.

Adult↗

PubMed2000

Cryopreservation of seabream (Sparus aurata) spermatozoa.

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.

Animals↗

PubMed2001

Microarray-based analysis of early development in Xenopus laevis.

In order to examine transcriptional regulation globally, during early vertebrate embryonic development, we have prepared Xenopus laevis cDNA microarrays. These prototype embryonic arrays contain 864 sequenced gastrula cDNA. In order to analyze and store array data, a microarray analysis pipeline was developed and integrated with sequence analysis and annotation tools. In three independent experimental settings, we demonstrate the power of these global approaches and provide optimized protocols for their application to molecular embryology. In the first set, by comparing maternal versus zygotic transcription, we document groups of genes that are temporally regulated. This analytical approach resulted in the discovery of novel temporally regulated genes. In the second, we examine changes in gene expression spatially during development by comparing dorsal and ventral mesoderm dissected from early gastrula embryos. We have discovered novel genes with spatial enrichment from these experiments. Finally, we use the prototype microarray to examine transcriptional responses from embryonic explants treated with activin. We selected genes (two of which are novel) regulated by activin for further characterization. All results obtained by the arrays were independently tested by RT-PCR or by in situ hybridization to provide a direct assessment of the accuracy and reproducibility of these approaches in the context of molecular embryology.

Animals↗

PubMed2002

Validation of an ex vivo Xenopus liver slice bioassay for environmental estrogens and estrogen mimics.

A sensitive ex vivo bioassay for detecting environmental estrogens and estrogen mimics was developed using Xenopus liver slice culture. Vitellogenin synthesis by these cultures was used as biomarker for estrogenic activity of environmental water samples. Sensitization of the assay for estrogens and mimics was accomplished by employing tissue from animals after in vivo preexposure to estrogen. Effects of various tissue culture factors were investigated in order to obtain optimum conditions for the bioassay. It was found that endogenous vitellogenin and/or estrogen could be "washed out" of cultures and that not only uncontaminated males but also estrogen-pretreated males and females can successfully be used as bioindicators. Estrogenicity was detected, using the ex vivo Xenopus liver slice culture assay, in sewage effluent, lake water, and dam water. This article presents an optimized protocol for effective estrogen detection in environmental water samples.

Animals↗

PubMed2002

Analysis of shuffled gene libraries.

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.

Base Sequence↗

PubMed1995

Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation.

Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were purified by a subsequent Percoll centrifugation. The isolated PC had a viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were cryopreserved with an optimized protocol in a computer-controlled freezer. After thawing, the PC still retained a viability of 89%. The activities of representative xenobiotic metabolizing enzymes were compared between freshly isolated and cryopreserved PC after thawing. The cytochrome P450 content and the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of testosterone in intact cells, were not affected by the cryopreservation. The following phase II enzyme activities were also well maintained after cryopreservation: Phenol sulfotransferase (92%), 1-naphthol UDP-glucuronosyl transferase (95%), soluble epoxide hydrolase (87%), and glutathione S-transferase (88%), determined with broad spectrum substrate 1-chloro-2,4-dinitrobenzene. However, there was a significant decrease in plating efficiency between freshly isolated (86%) and cryopreserved (57%) PC when they were cultured. The initial quality of the freshly isolated PC is decisive for the success of cryopreservation. These results support the use of cryopreserved PC in pharmacology and toxicology with the aim to reduce the number of experimental animals used.

Animal Testing Alternatives↗

PubMed2002

Repeated cycles of alternate administration of fludarabine and Zidovudine plus Didanosine inhibits murine AIDS and reduces proviral DNA content in lymph nodes to undetectable levels.

The results of the combined use of Fludarabine, an anticancer agent that may be able to target latently infected cells, and conventional antiretroviral therapy (AZT+DDI) in a murine model of AIDS, i.e., LP-BM5 infection, are reported. Eighty percent of infected mice, treated with four cycles of alternate administration of Fludarabine and AZT+DDI, showed undetectable levels of proviral DNA in lymph nodes. After 8 weeks of treatment interruption, the infected/treated animals, although still alive at a time when all untreated animals had succumbed to the infection, showed disease progression and reappearance of proviral DNA in lymph nodes. The retrospective analysis of proviral DNA content in spleen and bone marrow at the end of the fourth cycle of treatment revealed a low but detectable amount of BM5d proviral DNA. We thus concluded that the spleen and bone marrow may be less sensitive to lympholitic drugs and therefore act as viral reservoirs in LP-BM5 infection. This study suggests that optimized protocols of alternate administration of cytolytic and antiretroviral drugs may represent a useful strategy to eradicate retroviral infections.

Animals↗

PubMed2007

Revealing the metabolome of animal tissues using 1H nuclear magnetic resonance spectroscopy.

The measurement of tissue-specific metabolic fingerprints can be of particular interest when investigating disease processes, mechanisms of toxicity, or when knowledge of the metabolic interactions between different organs is required. This chapter presents several optimized protocols for the extraction of metabolites from animal tissues, their analysis by 1H nuclear magnetic resonance (NMR) spectroscopy, and the subsequent spectral preprocessing required for an NMR-based metabolomics experiment. First, the three critical steps in the preparation of tissue extracts for NMR analysis are described, including both a perchloric acid protocol for the extraction of polar metabolites, and a methanol:chloroform protocol for extraction of polar and lipophilic metabolites. Then a series of NMR experiments are described including a standard one-dimensional (1D) 1H NMR study, a 1D 1H Carr-Purcell-Meiboom-Gill spin-echo experiment, and a two-dimensional 1H-1H J-resolved NMR experiment. The advantages and limitations of each experiment for metabolomics research are discussed. Analysis of the resulting NMR datasets is typically conducted in two phases comprising "low level" spectral preprocessing and "high level" multivariate analysis. NMR spectral preprocessing is a critical step that converts raw NMR spectra into an appropriate data format for multivariate analysis. A detailed protocol for preprocessing NMR data, using ProMetab software, is presented. Because a plethora of algorithms exist for multivariate analyses, which can be used to construct classification models or for biomarker discovery, this is beyond the scope of the current chapter.

Animals↗

PubMed1995

Measuring resistance to phagocytosis of group A and G streptococci: comparison of direct bactericidal assay and flow cytometry.

M protein is thought to contribute to the ability of non-opsonized group A and group G streptococci (GAS and GGS, respectively) to resist phagocytosis by polymorphonuclear leukocytes. In previous studies, correlation between M protein expression and phagocytosis was determined by incubating these pathogens in human blood and comparing colony-forming bacterial counts prior to and after exposure to blood (direct bactericidal assay; DBA). Here, we report the application of flow cytometry to measure GAS and GGS resistance to phagocytosis. The results of the assays were in complete agreement with those from DBAs. Nevertheless, flow cytometry was regarded as superior to DBA because of its speed and potential uses for quantitative studies. In addition, the use of anti-CD11b monoclonal antibody for granulocyte staining guaranteed a non-compromized granulocyte function. The optimized protocol for flow cytometry presented here could be utilized to directly measure the involvement of individual protein types in bacterial resistance to phagocytosis.

Blood Bactericidal Activity↗

PubMed1990

Measurement of cerebral blood flow with a bolus of oxygen-15-labelled water: comparison of dynamic and integral methods.

A method is presented for the measurement of cerebral blood flow (CBF) with a bolus of water labelled with oxygen 15. The method, which has been evaluated in normal volunteers, is based on Kety's model, with two additional parameters to account for the difference in the time of tracer arrival in the radial and carotid arteries ("delay") and for dispersion of the tracer in the body and/or blood counting systems. It combines the advantages of: (i) dynamic data collection for estimation of delay and dispersion; (ii) robustness and linearity of CBF estimates with an integral method; and (iii) simplicity of continuous external monitoring of arterial blood radioactivity, particularly with repeated measurements. An optimized protocol is proposed for routine applications in neurological and neurophysiological studies.

Adult↗

PubMed1983

The control issue in biofeedback training.

Well-delineated models and procedural descriptions are noticeably missing from the clinical biofeedback literature dealing with the training of physiological self-control. As a consequence, optimal protocols for the assessment and training of self-control of physiological functions have not eventuated. The present article reviews evidence suggesting that researchers have (a) confused self-control training, assessment, and measurement of tonic response levels, (b) neglected to determine the degree of acquired physiological self-control, and (c) failed to report the relationship between level of acquired physiological control and clinical outcome. The article also proposes an assessment and training paradigm that may be useful in reducing these methodological problems in future biofeedback research.

Arousal↗