Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “protocol optimization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 469 records · Page 26Linked to original sources
PubMed1976

A comparative study of different experimental protocols for mutagenesis assays with the 9-azaguanine resistance system in cultured Chinese hamster cells.

Both spontaneous and EMS-induced mutant frequencies were determined in cultured cells from V79 Chinese hamsters using three different experimental protocols. After optimal expression time was attained, mutation frequencies only remained constant when a protocol was used in whcih the cell density was maintained below critical values both before and during mutant selection. The identification of such a plateau allows, besides more reliable and reproducible estimates of mutation frequency, reduction in the size of experiments for quantitative evaluation of mutagenicity. Determination of mutation frequencies over a wide range of expression times becomes in fact unnecessary.

Azaguanine↗
PubMedDate not supplied

Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy.

Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.

Adenoviridae↗
PubMed1993

Dynamic contrast-enhanced CT of the liver: comparison of contrast medium injection rates and uniphasic and biphasic injection protocols.

To determine the effect of contrast medium injection rate and biphasic versus uniphasic bolus on hepatic parenchymal enhancement at computed tomography (CT), 105 patients were randomized into four protocols. Hepatic and aortic enhancement curves were measured for each protocol. The optimal scanning interval (the duration between onset of a desired hepatic enhancement threshold and decline of enhancement below the threshold or onset of the equilibrium phase) and contrast enhancement index (CEI) (area under the hepatic enhancement curve above a desired threshold during the nonequilibrium phase) were the primary determinants of merit. Measurements were performed at hepatic enhancement thresholds between 10 and 60 HU. At most thresholds of hepatic enhancement, optimal scanning intervals were significantly longer and CEIs were significantly higher for the biphasic protocols than for the uniphasic protocols (P < .03). A biphasic injection with a high initial flow rate produces high peak contrast enhancement but delayed onset of equilibrium and thus provides a longer optimal scanning interval.

Adult↗
PubMedDate not supplied

An MR protocol for presurgical evaluation of patients with complex partial seizures of temporal lobe origin.

PURPOSE: To find an optimal diagnostic protocol for the presurgical MR evaluation of patients with temporal lobe epilepsy. METHODS: MR imaging in 14 healthy subjects and 25 consecutive patients with temporal lobe epilepsy was performed in paracoronal sections perpendicular to the hippocampi with T1-weighted inversion recovery and T2 weighting. Volume measurements of the hippocampus/amygdala complex were performed and a multiecho sequence yielded T2-calculated images. RESULTS: Hippocampal disease was seen in 22 of 25 temporal lobe epilepsy patients on paracoronal T1-weighted inversion recovery images. Four had bilateral abnormalities. Characteristic for hippocampal disease were features such as volume loss, decreased signal, and loss of internal morphology. Only 17 of 25 patients demonstrated hippocampal pathology on T2-weighted images, and in one patient this was bilateral. Patients with only minimal structural loss on T1-weighted inversion recovery had normal T2-weighted images. T2 calculation was no more sensitive than visual assessment on the T2-weighted images. Volume measurements were normal in one patient and misleading in two patients. Lateralization, as compared with clinical and electroencephalographic findings, was most confidently done with paracoronal T1-weighted inversion recovery images and volume measurements. CONCLUSIONS: An optimum MR protocol for temporal lobe epilepsy patients is proposed. Its essential feature is that the hippocampus be evaluated by paracoronal T1-weighted inversion recovery images and volume measurements. T2-weighted imaging can be omitted.

Adolescent↗
PubMed2002

The need for standardisation of cardiac FDG PET imaging in the evaluation of myocardial viability in patients with chronic ischaemic left ventricular dysfunction.

The evaluation of myocardial glucose utilisation with fluorine-18 fluorodeoxyglucose (FDG) and positron emission tomography is currently considered the most reliable tool for the identification of myocardial viability. However, the investigations using FDG imaging to predict improvement in left ventricular (LV) function after revascularisation have reported wide ranges for sensitivity (71%-100%) and, in particular, for specificity (33%-91%). The variable results may be related to differences in study populations but also to differences in the imaging protocols employed. Detailed analysis of the published studies has revealed differences in study populations, patient selection criteria, the methods for assessing changes in LV function post revascularisation and the timing of these assessments. Even more importantly, protocols have varied substantially with regard to imaging equipment, perfusion tracers, metabolic conditions, data analysis and interpretation of results. In addition, evaluation of patients with insulin resistance appears to represent a specific challenge. This review examines the different study protocols and methodologies used for myocardial FDG imaging in order to draw conclusions concerning optimal imaging protocols. It appears that the optimisation and standardisation of study protocols and analysis of FDG images for the assessment of myocardial viability are critical. In addition, multi-centre trials seem warranted on prediction of long-term function, congestive heart failure symptoms, survival and quality of life.

Fluorodeoxyglucose F18↗
PubMed2002

Experimental colonic phantom for the evaluation of the optimal scanning technique for CT colonography using a multidetector spiral CT equipment.

Our objective was to optimize a scanning protocol for CT colonography using a multidetector scanner with an adaptive-array matrix in a phantom study. A colonic phantom with 27 simulated lesions was examined using a multidetector CT scanner (Somatom Plus 4 Volume Zoom, Siemens, Erlangen, Germany). Three scanning protocols were tested: (a) collimation 1.0 mm, slice thickness 1.0 mm; (b) collimation 2.5 mm, slice thickness 3.0 mm; and (c) collimation 2.5 mm, slice thickness 5.0 mm. Image analysis was performed by two radiologists blinded to the construction of the phantom and to imaging parameters. Sensitivity for lesion detection and image quality was assessed. Computed tomographic colonography detected 27 of 27 lesions with protocol 1 (sensitivity 100%); 26 of 27 lesions with protocol 2 (sensitivity 96.3%); and 23 of 27 lesions with protocol 3 (sensitivity 85.2%). Image quality was graded as optimal for protocol 1, good for protocol 2, and poor for protocol 3. When multidetector CT equipment with an adaptive-array matrix is used, protocols with 1.0- or 3.0-mm slice thickness have a sensitivity for the detection of lesions higher than that obtained with a 5.0-mm slice thickness. Image quality decreases progressively when the slice thickness is increased; therefore, in a clinical setting, we recommend the use of a high-resolution protocol (1.0-mm effective slice thickness) with the highest pitch value in order to reduce scanning time. Dose exposure for such a protocol if combined with 80 mAs is within the range limits recommended by the ICRP for abdominal CT scanning.

Chi-Square Distribution↗
PubMed2001

Optimized 3-D CT scan protocol for longitudinal morphological estimation in craniofacial surgery.

Frequent three-dimensional computed tomography scanning may cause deterioration of the lenses of the eye, which are susceptible to x-rays. The authors performed an experimental study using a phantom head to establish an optimized three-dimensional computed tomography scan protocol for longitudinal morphological estimation in craniofacial surgery. Volume computed tomography scans were performed using a Hi-Speed Advantage SG CT scanner (GE Medical Systems, Milwaukee, WI) in the axial plane with a combination of scan parameters of varied values. The radiation doses induced by each scanning were measured using thermoluminescent dosimeter chips attached to the position of the lenses in the phantom. Two-dimensional images in the coronal plane and three-dimensional images of the osseous surface were generated from each accumulated data set. For each scan parameter, the images generated from data accumulated using different values were compared. The study showed that lens radiation dose increased with tube potential and was almost directly proportional to tube current and 1/pitch. The slice thickness did not affect lens radiation doses significantly. Images with good contrast resolution and low artifact level sufficient for estimating morphological changes were obtained using a low tube potential of 100 kVp and a tube current of 100 mA. In regard to z-axis spatial resolution, a slice thickness of less than 3 mm was required for precisely pointing out bony edges in the two-dimensional reformation images. These results led us to conclude that volume computed tomography for longitudinal examination in craniofacial surgery should be kept to a minimum frequency and performed using a low-dose technique, small slice thickness, and large pitch.

Artifacts↗
PubMed2000

Considerations preliminary to the application of early and immediate loading protocols in dental implantology.

In oral implantology, a 3-6 month stress-free healing period is presently accepted as a prerequisite to achieve bone apposition without interposition of a fibrous scar tissue. This protocol was introduced by Brånemark and co-workers in 1977. The aim of the present paper is to review the reasons that led Brånemark and collaborators to require long delayed loading periods. It is shown that the requirement for long delayed loading periods was drawn from the initiation and development periods of their original clinical trial. Demanding conditions were met involving simultaneously: 1) patients with poor bone quality and quantity, 2) non-optimized implant design, 3) short implants, 4) non-optimized surgical placement, 5) non-optimized surgical protocol and 6) biomechanically non-optimized prosthesis. Extrapolation of the requirement for long healing periods from these particular conditions to more standard situations involving refined surgical protocols and careful patient selection might be questioned. Albeit premature loading has been interpreted as inducing fibrous tissue interposition, immediate loading per se is not responsible for fibrous encapsulation. It is the excess of micromotion during the healing phase that interferes with bone repair. A threshold of tolerated micromotion exists, that is somewhere between 50 microns and 150 microns. It is suggested that loading protocols might be shortened through 2 different approaches. The first way would be to decrease stepwise the delayed loading period for free-standing implants below the presently accepted 3-6 months of healing. The second way would be to identify immediate loading protocols that are capable of keeping the amount of micromotion beneath the threshold of deleterious micromotion. Immediate loading protocols for implants-retained overdentures and fixed bridges are reviewed. It is shown that successful premature loading protocols require a careful and strict patient selection aimed to achieve the best primary stability. These various protocols need to be further documented in order to assess their predictability.

Clinical Protocols↗
PubMed1995

Rapid and effective detection of mutations in the p53 gene using nonradioactive single-strand conformation polymorphism (SSCP) technique applied on PhastSystem.

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method is a powerful tool for the screening of genetic alterations, including single-base substitutions. In the present study, the conventional SSCP technique was modified on the semiautomated electrophoresis system (PhastSystem) for the detection of mutations in the p53 tumor suppressor gene. The SSCP running conditions were optimized for three PCR-amplified DNA fragments, spanning exons 5 through 9 of the p53 gene, using the PCR-products derived from the CaSki and HaCaT cells as the normal and mutant controls, respectively. The optimized SSCP protocols were tested on nine human vulvar and vaginal carcinoma-derived cell lines. The optimizing experiments indicated that the running temperature and gel density can affect significantly the electrophoretic mobility and resolution of single-stranded DNA molecules. Because the gel temperature is the most important parameter affecting the conformation and thus electrophoretic mobility of single strands, one of the most important advantages of the SSCP technique on the PhastSystem is that the running temperature is controlled precisely. In addition to the fast electrophoretic separation, the PhastSystem also offers the use of a silver staining method allowing direct visualization of DNA with high detection sensitivity. Thus, the important advantage of this modified SSCP technique is the short time required for analysis, including electrophoresis and DNA detection. It is concluded that the SSCP method applied on the PhastSystem has the advantages of simplicity, efficiency, speed and reproducibility, and is suitable for clinical diagnostic purposes.

Base Sequence↗
PubMed1995

The use of L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals.

Guidelines have been proposed to assess the potential of chemicals to affect human health. Written into these guidelines is the requirement that information be submitted on mutagenic activity. Although regulatory agencies accept mutagenicity data from both the hprt and tk loci in mammalian cells, many studies suggest that the L5178Y mouse lymphoma assay at the thymidine kinase locus is likely to detect a greater spectrum of mutagenic lesions. Thus, there is increasing emphasis being placed on this assay in many proposed and published guidelines. The L5178Y mouse lymphoma suspension protocol produces both small and large colonies which are the products of mutants growing at different rates. There is a reduction in the proportion of slowly growing mutants with respect to the total population of cells when expression is carried out in suspension. This potentially leads to quantitatively inaccurate assessments of the mutagenic activity of chemicals. Therefore an in situ procedure was developed that more accurately assesses the mutagenic activity of chemicals by maximizing the detection of small colonies. Many guidelines recommend tests that assess the clastogenic activity of chemicals. Some regulatory agencies accept data from the mouse lymphoma mutation assay to detect clastogens if the protocol is optimized for the detection of small colonies or if colony sizing data are submitted. The conventional suspension assay protocol is not sufficiently validated for this purpose. The in situ protocol has greater potential to meet these requirements.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed2000

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature.

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.

Animals↗
PubMed1992

Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions.

Assay conditions and results of cytochrome P-450 dependent 7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) O-dealkylation (OD) by rat liver microsomes were compared by four laboratories in the Netherlands. Microsomal mixtures were prepared from control, 3-methylcholanthrene and phenobarbital pretreated animals, resulting in different levels of cytochrome P-450 isozymes. EROD and PROD activities were determined in each laboratory according to their own protocols. Considerable variability was found both between and within laboratories. Further studies demonstrated that protocol differences are important factors causing this interlaboratory variation. Main factors of influence were buffer type, batch of resorufin used for calibration, substrate solvent and substrate concentration. Based on the results obtained, standardized protocols for optimized measurement of microsomal EROD and PROD activities were developed. Additional experiments demonstrated that the use of these standardized protocols reduced intralaboratory variation in both the EROD and the PROD assay, whereas it also reduced the interlaboratory variability for the PROD determinations. The interlaboratory variation for measurement of microsomal EROD activities was only reduced for the laboratories using a Cobas-Bio analyzer. The results of the present study demonstrate clearly that data obtained with EROD and PROD activity measurements are highly sensitive to factors frequently varying from one laboratory to another. In addition, they demonstrate the necessity to be careful with absolute values presented in the literature for these activities, unless well characterized assay conditions are applied.

Animals↗
PubMed2000

Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments.

Permafrost represents a unique ecosystem that has allowed the prolonged survival of certain bacterial lineages at subzero temperatures. To better understand the permafrost microbial community, it is important to identify isolation protocols that optimize the recovery of genetically diverse bacterial lineages. We have investigated the impact of different low-temperature isolation protocols on recovery of aerobic bacteria from northeast Siberian permafrost of variable geologic origin and frozen for 5000 to 3 million years. Low-nutrient media enhanced the quantitative recovery of bacteria, whereas the isolation of diverse morphotypes was maximized on rich media. Cold enrichments done directly in natural, undisturbed permafrost led not only to recovery of increased numbers of bacteria but also to isolation of genotypes not recovered by means of liquid low-temperature enrichments. On the other hand, direct plating and growth at 4 degrees C also led to recovery of diverse genotypes, some of which were not recovered following enrichment. Strains recovered from different permafrost samples were predominantly oligotrophic and non-spore-forming but were otherwise variable from each other in terms of a number of bacteriological characteristics. Our data suggest that a combination of isolation protocols from different permafrost samples should be used to establish a culture-based survey of the different bacterial lineages in permafrost.

Bacteria↗
PubMed1990

The biophysical profile. A literature review and reassessments of its usefulness in the evaluation of fetal well-being.

Antenatal monitoring studies to assess fetal well-being and to identify the compromised fetus are in widespread use. This literature review analyzes the theoretical basis for fetal monitoring and the clinical research that has defined its utility, and it discusses the variations in protocols and scoring systems for the biophysical profile. While recognizing that there, is as yet, no consensus on the optimal monitoring protocol, the authors espouse one approach in which the cardiac nonstress test and the amniotic fluid volume are the initial tests, with full biophysical profile reserved for abnormal test outcomes. This approach to monitoring should be expeditious without diminishing its predictive value.

Embryonic and Fetal Development↗
PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗
PubMed2003

Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris.

Heat shock proteins with a molecular mass of 70000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p-Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.

Chromatography, Ion Exchange↗
PubMed1999

Identification and optimization of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach.

A general regeneration, identification, and optimization (RO) protocol for Biacore systems was developed. The RO protocol uses six multi-ingredient stock solutions that represent the six most common chemical properties employed as regeneration agents. The regeneration effect of different regeneration cocktails of these six stock solutions were tested iteratively until a satisfactory result was obtained. The RO protocol was designed with an ease-of-use and multivariate approach. The RO protocol was tested on 13 different antibody-antigen systems. For 10 of these, only the first screening session was tested. For 9 of the 13 systems, the RO-protocol screening session identified cocktails that removed more than 90% of the bound analyte in a 30 s pulse. For 5 systems, the RO protocol identified cocktails that regenerated the surface completely and that were more gentle than previously used regeneration conditions. Furthermore, the regeneration optimization results can be interpreted as a characterization of the interacting molecules. The relevance of testing cocktails was justified by the fact that at least one cocktail was significantly better than all diluted stock solutions for all tested model systems. By using the multivariate approach, the risk of missing relevant combinations of stock solutions was minimized. This resulted in an unexpected discovery of excellent properties of EDTA as an additive in regeneration cocktails containing chaotropic agents and ions in high concentration.

Biosensing Techniques↗
PubMed1991

A qualitative approach to cell growth modeling and simulation for cancer chemotherapy.

A qualitative model of cell growth, based on qualitative process theory, is presented. The model can be used to analyze the effects of the interaction of antiproliferative drugs on cells when the effects of each specific drug are known, mainly when designing multi-drug protocols for optimal cancer treatment.

Antineoplastic Combined Chemotherapy Protocols↗