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.

SourceResearch connectionFrom yearThrough year

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

At least 883 records · Page 49Linked to original sources

PubMed2005

Performance of male C57BL/6J mice and Wistar rats in the water maze following various schedules of phencyclidine treatment.

In order to establish an animal model of cognitive impairments relevant to schizophrenia, we set out to obtain an optimal treatment protocol with phencyclidine (PCP) that would lead to robust cognitive impairment with minimal PCP-related adverse effects. Effects of various doses (0.63-5 mg/kg), pre-treatment period (0, 3, 7 and 10 days before the beginning of acquisition) and treatment schedules (before the first or immediately after the last trial on each day) of PCP on the performance of male C57BL/6J mice and Wistar rats in the spatial version of the water maze were studied. In mice, a 10-day pre-treatment period was required to prevent PCP-induced motor impairments, whereas a 3-day pre-treatment was sufficient in rats. PCP impaired spatial learning in both rats and mice, if animals were administered PCP prior to the first trial. The optimal dose was 2.5 mg/kg. In contrast, animals given PCP immediately after the daily training sessions performed as well as controls. Thus, PCP impairs spatial learning in the water maze only when present in the organism. It can be concluded that PCP interferes with learning, and perhaps retrieval, but not consolidation of newly acquired information.

Animals↗

PubMedDate not supplied

Routine MR imaging protocol with breath-hold fast scans: diagnostic efficacy for focal liver lesions.

PURPOSE: To qualitatively and quantitatively evaluate the diagnostic efficacy of the breath-hold magnetic resonance (MR) imaging sequences in widespread clinical use for detection and characterization of focal hepatic lesions. MATERIALS AND METHODS: Fifty patients with 143 lesions [57 hepatocellular carcinomas (HCC), 10 borderline lesions, 18 metastatic tumors, 21 hemangiomas, and 37 cysts] underwent single-shot fast spin echo (SSFSE), fast spin echo (FSE), and gadolinium-enhanced dynamic fast spoiled gradient-recalled acquisition in steady state (FSPGR) breath-hold MR imaging of the liver. Alternative free receiver operating characteristic (AFROC) analysis was performed to independently and prospectively assess each sequence. RESULTS: For solid lesions, dynamic FSPGR allowed the most sensitive lesion detection and produced the highest lesion conspicuity and lesion-liver contrast-to-noise ratio (CNR). For non-solid benign lesions, SSFSE and FSE produced better results than dynamic FSPGR. SSFSE allowed the most sensitive detection and produced the best lesion conspicuity and lesion-liver CNR. CONCLUSION: SSFSE and dynamic FSPGR comprise the optimal imaging protocol for breath-hold MR assessment of focal hepatic lesions. This combination of sequences allows acquisition of critical diagnostic information about both inherent T2 and T1 lesion contrast and lesion vascularity.

Adult↗

PubMed2005

Automated purification of recombinant proteins: combining high-throughput with high yield.

Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.

Bacterial Proteins↗

PubMed2002

Heart failure management: multidisciplinary care has intrinsic benefit above the optimization of medical care.

PURPOSE: This work addresses the unanswered question of whether multidisciplinary care (MDC) of heart failure (HF) can reduce readmissions when optimal medical care is applied in both intervention and control groups. METHODS: In a randomized, controlled study, 98 patients (mean age, 70.8 +/- 10.5 years) admitted to hospital with left ventricular failure (New York Heart Association Class IV) were assigned to routine care (RC, n = 47) or MDC (n = 51). All patients received the same components of inpatient, optimal medical care of HF: specialist-led inpatient care; titration to maximum tolerated dose of angiotensin-converting enzyme inhibitor before discharge; attainment of predetermined discharge criteria (weight stable, off all intravenous therapy, and no change in oral regimen for 2 days). Only those in the MDC group received inpatient and outpatient education and close telephone and clinic follow-up. The primary study endpoint was rehospitalization or death for a HF-related issue at 3 months. MAIN FINDINGS: At 3 months, four people had events in the MDC group (7.8% rate over 3 months) compared with 12 people (25.5% rate over 3 months) in the RC group (P = 0.04). CONCLUSION: These data demonstrate for the first time the intrinsic benefit of MDC in the setting of protocol-driven, optimal medical management of HF. Moreover, the event rate of 7.8% at 3 months, as the lowest reported rate for such a high-risk group, underlines the value of this approach to the management of heart failure.

Aged↗

PubMed1999

Evaluation of transmyocardial laser revascularization (TMLR) by gated myocardial perfusion scintigraphy.

TMLR is a novel treatment for patients with coronary artery disease. It comprises the creation of transmyocardial channels thought to improve myocardial perfusion. Gated Tc-99m sestamibi scintigraphy was used to evaluate changes in myocardial perfusion after TMLR. Twelve patients underwent TMLR using a carbon dioxide laser. Sestamibi scans were carried out following a standard protocol prior to and 1, 3, 6, and 12 months after TMLR. Both visual and semi-quantitative assessment showed improvement in 4 patients, deterioration in 2 patients, and no change in the remaining 6 patients each. However, visual and semi-quantitative assessment were concordant in 6 patients and discordant in 6 patients. In 3 of these, semi-quantitative assessment suggested a better outcome, and in 3 patients visual assessment gave better results. Our findings in a small group of patients suggest that about a third benefited from TMLR. Gated myocardial perfusion scintigraphy using technetium-99m sestamibi is suitable for visual evaluation of changes in the lased area over time, but does not allow semi-quantitative evaluation in the patient population typically treated with TMLR. Further investigations using optimized imaging protocols, including positron emission tomography and three dimensional image presentation, are warranted.

Aged↗

PubMed2005

Cryopreservation of human embryonic stem cells without the use of a programmable freezer.

BACKGROUND: An effective freezing-thawing technique is crucial for the clinical application of human embryonic stem (ES) cells. The aim of this study was to find an optimal cryopreservation protocol for human ES cells using slow freezing-rapid thawing without a programmable freezer. METHODS: The human ES cell line, SNUhES-3, was cultured on an STO feeder layer in gelatin-coated tissue culture dishes. All cryopreservation steps were performed using a simple commercial freezing container. The survival rate of cryopreserved-thawed human ES cells was estimated by counting colony numbers under a stereomicroscope. Initially, we compared the survival rates of cryopreserved human ES cells using three cryoprotectants: dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol. In this experiment, 5% DMSO/95% fetal bovine serum (FBS) (vol/vol) showed the highest survival rate. We next tested the impact of various concentrations of FBS (95, 50 and 5%) with 5% DMSO, and then examined the effects of adding EG or glycerol to 5% DMSO + optimal FBS. RESULTS: No significant difference in survival rate was observed between 95 and 50% FBS in the presence of 5% DMSO. A significant improvement in survival rate was obtained by adding 10% EG to 5% DMSO+50% FBS. After thawing, surviving cells were found to maintain the inherent characteristics of human ES cells. CONCLUSION: 5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing-rapid thawing of human ES cells.

Base Sequence↗

PubMed2003

Optimal contrast application for cardiac 4-detector-row computed tomography.

RATIONALE AND OBJECTIVES: This study was designed to determine the optimal contrast protocol for 4-detector-row computed tomography angiography of the heart. METHODS: Sixty patients were randomly assigned to 1 of 4 groups with 300 and 400 mg/mL iodine concentrations and 2.5 and 3.5 mL/s flow rates. Contrast density was measured in the left ventricular cavity and coronary arteries. RESULTS: Low iodine concentration injected at slow flow rate (0.75 g iodine/s) resulted in acceptable contrast enhancement in only 53.8% of the patients. There was no significant difference between low contrast concentration injected at high flow rate and high contrast concentration injected at slow flow rate ( approximately 1 g iodine/s). High contrast concentration administered with high flow rates (1.4 g iodine/s) may result in an enhancement above 350 Hounsfield units (HU) and interfere with coronary calcifications. CONCLUSIONS: The injection of approximately 1 g iodine/s resulted in an optimal (250-300 HU) contrast enhancement for cardiac 4-detector-row computed tomography.

Adult↗

PubMed1997

The utilization of RAPD-PCR for identifying thermophilic and mesophilic Bacillus species.

A random amplified polymorphic DNA fingerprinting assay has been optimized that is able to discriminate between numerous thermophilic and mesophilic bacillus species and strains. Included in the analyses are thermophilic (able to grow at 55 degrees C) strains of Bacillus stearothermophilus, B. kaustophilus, B. coagulans, B. sphaericus, B. thermodenitrificans, B. thermocatenulatus, B. thermoleovorans, B. licheniformis, B. brevis, B. thermoglucosidasius, B. caldolyticus, B. caldotenax, B. caldovelox, B. thermocloacae and B. smithii. Mesophilic strains of B. pumilus, B. subtilis, B. megaterium, B. circulans, B. cereus and B. mycoides can also be used for fingerprinting with the assay. Increasing the concentration of primer from 0.2 to 2.0 microM is shown to have a significant effect on increasing the number of amplification products that can be used for the discrimination or identification of individual strains or species. It is suggested that this may be a general way of improving the resolution of a RAPD protocol. The optimized conditions have been used successfully to trace B. stearothermophilus, B. licheniformis and other bacillus species and strains in an industrial setting.

Bacillus↗

PubMed2005

Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Actins↗

PubMed2004

[Advances in cellular immunotherapy for malignant melanoma].

An alternative strategy for cancer treatment is the manipulation of the immune system, denominated cancer immunotherapy. The immunotherapeutical use of cells of the immune system, like dendritic cells (DC), is being explored in different clinical protocols. Recently, we finalized a clinical phase I protocol, for the treatment of malignant melanoma, using DCs loaded with tumor lysates. Our results indicate that the subcutaneous application of DCs do not produce adverse effects. We also observed an increase of tumor specific T lymphocytes precursors in the blood, associated to hypersensitivity reactions (DTH) in 60% of the treated patients. In most cases, an stability in the disease was observed, although without a significant association between vaccination and survival. Additionally, therapies based on Interleukin-2 (IL-2) have been used with relative success in the treatment of some kind of tumors since 1985. However, problems associated to the toxicity of IL-2 still restrict its massive use. Our direct experience with the use of IL-2, indicates that low doses and its subcutaneous application, maintains the beneficial effects for patients, eliminating the adverse effects. Based on the accumulated evidence during last the five years, we decided to implement an optimized clinical protocol, which alternatively combines dendritic cells vaccines with the use of low doses of IL-2 for the reinforcement of the immunological system.

Antigens, Neoplasm↗

PubMed1998

The treatment of late radiation effects with hyperbaric oxygenation (HBO).

BACKGROUND: Late radiation injuries may impose a negative influence on the quality of life in the affected patients. In several entities, standardized treatment protocols are lacking. Hyperbaric oxygenation (HBO) has been shown to have beneficial effects in the treatment of late radiation sequelae. MATERIAL AND METHODS: The basic principles of HBO are reviewed as well as clinical issues. Current study protocols are presented. RESULTS: During HBO-therapy the patient breathes pure oxygen at pressures above 100 kPa. The oxygen solubility within the fluid phase of the blood is largely increased. Biological effects include an increased oxygen diffusibility, improved collagen synthesis and neoangiogenesis as well as an enhancement of antimicrobial defenses. By decreasing the capillary filtration pressure a reduction of edema becomes possible. HBO has been shown to prevent complications following surgery in irradiated tissues. Its efficacy as an adjunct in the treatment of osteonecroses in radiation patients could be demonstrated. In addition, the loss of osseointegrated implants in the maxillofacial bones of these patients could be significantly reduced. Further indications include soft tissue necroses, hemorrhagic cystitis and proctitis in tumor patients that have been treated by radiotherapy as part of a multimodality approach. CONCLUSIONS: HBO in the treatment of late radiation effects is still subject of investigation, but remarkable results have been reported. Optimized treatment protocols need to be determined in various entities. The rate of side effects is acceptable low.

Humans↗

PubMedDate not supplied

Virtual bronchoscopy for three--dimensional pulmonary image assessment: state of the art and future needs.

Virtual bronchoscopy is emerging as a useful approach for assessment of three-dimensional (3D) computed tomographic (CT) pulmonary images. A protocol for virtual bronchoscopic assessment of a 3D CT pulmonary image would have two main stages: (a) preprocessing of image data, which involves extracting objects of interest, defining paths through major airways, and preparing the extracted objects for 3D rendering; and (b) interactive image assessment, which involves use of graphics-based software tools such as surface-rendered views, projection images, virtual endoscopic views, tube views, oblique section images, measurement data, global two-dimensional section images, and cross-sectional views. Although a virtual bronchoscope offers a unique opportunity for exploration and quantitation, it cannot replace a real bronchoscope. Limitations of current virtual endoscopy systems include high cost, lack of visual aids beyond simulated endoscopic views, difficulty in performing interactive anatomic exploration, lack of quantitative information, use of surface rendering instead of volume rendering, and need for substantial off-line display computation. Future needs include development of fully integrated user-friendly virtual bronchoscopes, development of optimal CT protocols for generating artifact-free data sets, and improvements in automated preprocessing of 3D CT images.

Bronchoscopy↗

PubMed1986

Current risks and protocols for operations for double-outlet right ventricle. Derivation from an 18 year experience.

Recent follow-up of 127 patients having repair of double-outlet right ventricle during an 18 year experience (1967 to July, 1984) indicated an overall actuarial survival rate at 12 years of 38%. However, multivariate analysis supported by contingency tables indicated that currently the early (2 week) survival rate after the intraventricular tunnel repair for double-outlet right ventricle with subaortic ventricular septal defect in 6-month-old infants is 99% and the 10 year survival rate 97%. Reoperation of the tunnel repair was rarely required (one of 56 patients), and the functional results were excellent. Results were similar in patients with doubly committed ventricular septal defect, except that two of 10 patients developed typical discrete localized subaortic stenosis late postoperatively. Early and late results in patients with double-outlet right ventricle and subpulmonary ventricular septal defect were poor when an atrial switch operation was part of the repair; when an intraventricular tunnel repair between the left ventricle and aorta was combined with a right ventricular-pulmonary arterial conduit, no early deaths occurred, but three patients died late postoperatively. Several techniques of repair of double-outlet right ventricle with noncommitted ventricular septal defect have provided only a 22% overall 10 year survival rate. These data are used to derive inferences as to optimal treatment protocols.

Actuarial Analysis↗

PubMed2005

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

DNA, Bacterial↗

PubMed1993

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

Animals↗

PubMed2005

Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles.

Controlled ovarian hyperstimulation (COH) used in IVF produces lower implantation rates per embryo transferred compared to natural cycles utilized in ovum donation, suggesting a suboptimal endometrial development. Endometrial receptivity has recently been investigated in natural menstrual cycles with the aid of microarray technology. The aim of this study is to investigate the impact of COH using urinary gonadotrophins with a long protocol with GnRH agonists without progesterone supplementation (similar to the natural cycle) on endometrial gene expression profiles during the window of implantation by comparing the profiles at day hCG + 7 of COH versus LH + 7 of a previous natural cycle in the same women. For this purpose we have used microarray technology by Affymetrix (GeneChip HG_U133A), which allows more than 22,000 genes to be tested simultaneously. Results were validated by semi-quantitative PCR and quantitative PCR experiments. We found that more than 200 genes showed a differential expression of more than 3-fold when COH and normal cycles were compared at hCG + 7 versus LH + 7. We simultaneously re-analysed the LH + 2 versus LH + 7 endometrial gene expression profiles in previous natural cycles in the same subject using this specific GeneChip, the results obtained were consistent with our own published results. This is the first time that gene expression profiles of the endometrium during COH are reported. The large degree of gene expression disturbance is surprising and highlights the need for further efforts to optimize COH protocols.

Embryo Implantation↗

PubMed2005

Coarctation of the aorta: pre and postoperative evaluation with MRI and MR angiography; correlation with echocardiography and surgery.

AIMS: To compare MRI and MRA with Doppler-echocardiography (DE) in native and postoperative aortic coarctation, define the best MR protocol for its evaluation, compare MR with surgical findings in native coarctation. MATERIALS AND METHODS: 136 MR studies were performed in 121 patients divided in two groups: Group I, 55 preoperative; group II, 81 postoperative. In group I, all had DE and surgery was performed in 35 cases. In group II, DE was available for comparison in 71 cases. MR study comprised: spin-echo, cine, velocity-encoded cine (VEC) sequences and 3D contrast-enhanced MRA. RESULTS: In group I, diagnosis of coarctation was made by DE in 33 cases and suspicion of coarctation and/or aortic arch hypoplasia in 18 cases. Aortic arch was not well demonstrated in 3 cases and DE missed one case. There was a close correlation between VEC MRI and Doppler gradient estimates across the coarctation, between MRI aortic arch diameters and surgery but a poor correlation in isthmic measurements. In group II, DE detected a normal isthmic region in 31 out of 35 cases. Postoperative anomalies (recoarctation, aortic arch hypoplasia, kinking, pseudoaneurysm) were not demonstrated with DE in 50% of cases. CONCLUSIONS: MRI is superior to DE for pre and post-treatment evaluation of aortic coarctation. An optimal MR protocol is proposed. Internal measurement of the narrowing does not correspond to the external aspect of the surgical narrowing.

Adolescent↗

PubMed2006

Cardiac 16-MDCT for anatomic and functional analysis: assessment of a biphasic contrast injection protocol.

OBJECTIVE: The purpose of this study was to determine the optimal contrast injection protocol for clear delineation of the endocardial and epicardial contours and coronary vessels in anatomic and functional imaging with cardiac 16-MDCT. SUBJECTS AND METHODS: Thirty-eight patients were allocated to three groups according to contrast injection protocol: a long-duration biphasic protocol in which diluted contrast material was used in the latter phase (protocol A, 13 patients); a uniphasic protocol with saline flush (protocol B, 12 patients); a uniphasic protocol without a flush (protocol C, 13 patients). Six regions of interest were drawn within the left ventricle (LV), right ventricle (RV), and interventricular septum along the z-axis. Mean ventricular attenuation, mean difference between maximum and minimum ventricular attenuation, and ventricular-myocardial contrast-to-noise ratio (CNR) were calculated. Attenuation and visualization of the coronary vessels also were compared. RESULTS: The difference between maximum and minimum RV attenuation was significantly smaller in group A (58.1 H) than in groups B (179.5 H) and C (157.0 H). RV-myocardial CNR was significantly higher in group A (9.0) than in group B (5.5). The mean LV attenuation, difference between maximum and minimum LV attenuation, and LV-myocardial CNR were not significantly different among three groups. In protocol A, both endocardial and epicardial contours were clearly delineated, and cardiac functional analysis was feasible in all cases. Average attenuation and visualization of the coronary vessels were not significantly different among groups. The diagnostic accuracies in detection of coronary stenosis were 92%, 93%, and 91%, respectively, for protocols A, B, and C. CONCLUSION: The long-duration contrast injection protocol with diluted contrast material is optimal for assessing the coronary vessels and cardiac function.

Aged↗