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At least 739 records · Page 41Linked to original sources
PubMed2001

Simultaneous optimization of cryoprobe placement and thermal protocol for cryosurgery.

We demonstrate that it is possible to simultaneously optimize multiple cryoprobe placements and their thermal protocol for one freeze-thaw cycle. A numerical optimization algorithm is used and three different forms of objective function are examined in terms of algorithm convergence rate, minimum value of the chosen objective function, temperature-volume histograms and isotherm distributions. The optimization results depend on the initial values of the variables, the form of the objective function, optimization goals and the mathematical method adopted for gradient calculation. The proposed optimization model offers significant advantages over the previously reported semi-empirical approach to conformal cryotherapy, such as the ability to handle an unlimited number of variables and eliminating the need for the user input between iterations, thereby reducing, if not removing, the subjectivity of cryosurgery treatment planning.

Algorithms↗
PubMed2004

Scientific approach to the optimization of protein crystallization conditions for microgravity experiments.

The National Space Development Agency of Japan (NASDA) developed a practical protocol to optimize protein crystallization conditions for microgravity experiments. This protocol focuses on the vapor diffusion method using high density protein crystal growth (HDPCG)--hardware developed by the University of Alabama, Birmingham--that flew on the STS-107 mission. The objective of this development was to increase the success rate of microgravity experiments by setting crystallization conditions based on knowledge of crystal growth and fluid dynamics. The protocol consists of four steps: (1) phase diagram preparation, (2) estimation of condensation rate in the vapor diffusion method, (3) fluid dynamic property measurement, and (4) fluid dynamic simulation. First, a phase diagram was constructed. Crystallization characteristics were investigated by a microbatch method. The data were recalculated based on classical nucleation theory and the crystallization boundary was determined as a function of time. The second step was to develop a practical model to estimate the condensation rate of the crystallizing solution, including protein and precipitant, as a function of the precipitant concentration and solution volume. By considering the crystallization map and the vapor diffusion condensation model we were able to optimize the crystallization conditions that generate crystals in the desired time. This was particularly important in a shuttle mission whose mission duration is limited. The third step was fluid dynamic property measurement necessary for fluid dynamics simulation and crystal growth study. The last step was to estimate the mass transport in space on the basis of the fluid dynamics simulation transport model. It turned out that neither the vapor phase nor the solution phase was seriously affected by gravity until nucleation provided the hardware was set in a normal direction. Therefore, we concluded that the optimized crystallization conditions could be directly applied to microgravity experiments. By completing the approach, we were able to control the time for nucleation in the vapor diffusion method.

Animals↗
PubMed1981

A visual field screening protocol for glaucoma.

In this prospective evaluation of the reliability and efficiency of an optimized visual field screening protocol for glaucoma, we tested 145 eyes (73 patients) with increased intraocular pressures to determine the location of early glaucomatous visual field defects. The field examination, which was used as a control, and protocol testing were performed on a manual Goldmann perimeter using kinetic and suprathreshold static techniques, respectively. Of the 145 eyes tested, kinetic Goldmann perimetry detected 43 eyes with glaucomatous defects. The screening protocol detected 39 defective eyes. There were four false-negative tests and one false-positive test. The false-negative rate of just under 10% was within the range predicted from earlier data. The computer-generated optimal protocols performed as predicted and provided an excellent screening technique for the detection of early glaucomatous visual field defects.

Clinical Trials as Topic↗
PubMed2006

Optimized adaptor polymerase chain reaction method for efficient genomic walking.

Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.

Animals↗
PubMed2005

In vitro characterization of atherosclerotic carotid plaque with multidetector computed tomography and histopathological correlation.

This in vitro study evaluated the performance of 16-slice multidetector computed tomography (MDCT) in the assessment of carotid plaque components, with histology as the gold standard. Twenty-one specimens (n=21) were scanned and reconstructed after optimization of the protocol. Three corresponding MDCT images and histologic sections were selected from each specimen. The Hounsfield values (HV) of the major plaque components (calcifications, fibrous tissue and lipid) were assessed. Plaque areas (mm2) assessed with MDCT were compared with the results from histologic analysis. A value of 140 kVp and an intermediate reconstruction algorithm was the optimal protocol. In 15 out of 21 specimens it was possible to match MDCT images with histology. The HV of calcifications, fibrous tissue and lipid were 45+/-21, 79+/-20 and 960+/-491 HU (P<0.001), respectively. Plaque areas were compared in 27 matched levels. The calcified and lipid areas on MDCT and histology correlate well (R2=0.83 and R2=0.68, respectively). The mean difference in lipid area was 0.1 mm2 (95% CI=-2.1-2.3 mm2). This in vitro study showed that MDCT is capable of characterizing and quantifying the lipid rich portion of the atherosclerotic plaque.

Adult↗
PubMed1992

Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

Actinobacillus Infections↗
PubMed1998

Effect of hyperbaric oxygen on a rat transverse rectus abdominis myocutaneous flap model.

The single-pedicle transverse rectus abdominis myocutaneous (TRAM) flap is frequently associated with partial flap necrosis. Hyperbaric oxygen has previously been shown to increase the survival of skin flaps, although there has been no investigation of possible beneficial effects of hyperbaric oxygen on survival of the TRAM flap. The present study compares the effectiveness of hyperbaric oxygen therapy, normobaric 100% oxygen, a hyperbaric air-equivalent mixture, and no treatment at all (control group), in the prevention of TRAM flap necrosis in a rat model. Forty-eight animals were randomly assigned to one of the four above-mentioned groups. The surviving area of the flap was evaluated 7 days after surgery. The hyperbaric oxygen treatment protocol consisted of five 9-minute sessions breathing 100% oxygen at a pressure of 2.5 atmospheres absolute during the first 48 hours, starting within 1 hour of surgery. The areas of surviving skin paddles ranged from 38.5 percent in the control group to 52.5 percent in the group treated with hyperbaric oxygen. One-way analysis of variance indicated that flap area survival was significantly greater in the hyperbaric oxygen group (F = 2.69, p = 0.05). Tukey's pairwise comparison and the two-sample t test indicated that the group treated with hyperbaric oxygen differed significantly from the control group (Tukey's critical value = 3.8, rejection level = 0.05, t test p = 0.01). Our results suggest that the hyperbaric oxygen treatment protocol used improves survival in the rat TRAM flap. However, the optimal treatment protocol to achieve this objective even in the rat seems to be variable, and further studies are required before extrapolating these data to human applications.

Animals↗
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↗
PubMed2006

High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison.

We report the development of a heteroduplex-based mutation detection method using multicapillary automated sequencers, known as conformation-sensitive capillary electrophoresis (CSCE). Our optimized CSCE protocol detected 93 of 95 known base substitution sequence variants. Since the optimization of the method, we have analyzed 215 Mb of DNA and identified 3397 unique variants. An analysis of this data set indicates that the sensitivity of CSCE is above 95% in the central 56% of the average PCR product. To fully exploit the mutation detection capacity of this method, we have developed software, canplot, which automatically compares normal and test results to prioritize samples that are most likely to contain variants. Using multiple fluorescent dyes, CSCE has the capacity to screen over 2.2 Mb on one ABI3730 each day. Therefore this technique is suitable for projects where a rapid and sensitive DNA mutation detection system is required.

Algorithms↗
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↗
PubMed2006

Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function.

BACKGROUND: Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome. RESULTS: Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted. CONCLUSION: This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.

Animals↗
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↗
PubMed2006

Multiphasic contrast-enhanced multidetector-row CT of liver: contrast-enhancement theory and practical scan protocol with a combination of fixed injection duration and patients' body-weight-tailored dose of contrast material.

The introduction of multidetector-row CT (MDCT) has provided the abdominal radiologists with dramatic changes for the imaging of the liver. At just present, establishment of new and optimal injection protocol in multiphasic contrast-enhanced MDCT of the liver has been yearned because the difficulty and the importance to optimize the scan timing for each phase imaging have obviously increased because of the extremely rapid scan speed with MDCT scanners. Among technical factors employed in an injection protocol for multiphasic contrast-enhanced MDCT of the liver, to make an injection duration of contrast material fixed is the most important for the precise estimation of the scan timing for each phase imaging. With the fixed injection duration, the peak enhancement time of the aorta, portal vein, and liver constantly appear approximately 10, 20, and 30s after the any fixed injection durations (completion of injection of contrast material), respectively. Moreover, using a combination of the fixed injection duration and patients' body-weight-tailored dose of contrast material, similarity of time-density curves of each organ in shape and pattern can be achieved for individual patient with different body weight. In this issue, we will attempt to determine an ultimately optimal, but practical injection/scan protocol to successfully realize multiphasic contrast-enhanced MDCT of the liver based on the contrast-enhancement theory.

Body Weight↗
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↗
PubMed2006

Analysis of high throughput protein expression in Escherichia coli.

The ability to efficiently produce hundreds of proteins in parallel is the most basic requirement of many aspects of proteomics. Overcoming the technical and financial barriers associated with high throughput protein production is essential for the development of an experimental platform to query and browse the protein content of a cell (e.g. protein and antibody arrays). Proteins are inherently different one from another in their physicochemical properties; therefore, no single protocol can be expected to successfully express most of the proteins. Instead of optimizing a protocol to express a specific protein, we used sequence analysis tools to estimate the probability of a specific protein to be expressed successfully using a given protocol, thereby avoiding a priori proteins with a low success probability. A set of 547 proteins, to be used for antibody production and selection, was expressed in Escherichia coli using a high throughput protein production pipeline. Protein properties derived from sequence alone were correlated to successful expression, and general guidelines are given to increase the efficiency of similar pipelines. A second set of 68 proteins was expressed to investigate the link between successful protein expression and inclusion body formation. More proteins were expressed in inclusion bodies; however, the formation of inclusion bodies was not a requirement for successful expression.

Cloning, Molecular↗
PubMed2006

The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter.

BACKGROUND: The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the beta-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree. RESULTS: The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The beta-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the beta-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system. CONCLUSION: The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce recombinant yields.

Journal Article↗
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↗