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PubMedDate not supplied

Optimized retroviral transduction protocol which preserves the primitive subpopulation of human hematopoietic cells.

Though both low-speed centrifugation and the use of fibronectin (Retronectin) fragments increase gene transduction efficiency, they still do not overcome the adverse effects of the presence of virus-containing medium (VCM). In this study, we improved transduction efficiency of primitive human hematopoietic cells by optimizing the conditions for preadsorbing culture dishes with retrovirus using a centrifugation protocol allowing subsequent infection to be carried out in the absence of VCM. We also demonstrate that preadsorbing tissue culture plates with retrovirus is dependent on the volume of VCM used for preadsorption and the length of centrifugation and the type of plasticware used but not on the temperature of centrifugation (4-33 degrees C). Direct exposure of CD34+ target cells to VCM depletes the primitive CD34+CD38neg subpopulation by more than 30%, whereas the optimized VCM-free infection protocol targets this population with equivalent efficiency but had no detrimental effects on CD34+CD38neg frequency. In summary, we demonstrate a high-frequency transduction protocol which preserves the therapeutically relevant primitive subpopulation of human hematopoietic cells.

Cell Culture Techniques↗

PubMed2003

A practical protocol for titrating "optimal" PEEP in acute lung injury: recruitment maneuver and PEEP decrement.

This study was conducted to evaluate the effectiveness and safety of a practical protocol for titrating positive end-expiratory pressure (PEEP) involving recruitment maneuver (RM) and decremental PEEP. Seventeen consecutive patients with acute lung injury who underwent PEEP titration were included in the analysis. After baseline ventilation, RM (continuous positive airway pressure, 35 cm H2O for 45 sec) was performed and PEEP was increased to 20 cmH2O or the highest PEEP guaranteeing the minimal tidal volume of 5 mL/kg. Then PEEP was decreased every 20 min in 2 cmH2O decrements. The "optimal" PEEP was defined as the lowest PEEP attainable without causing a significant drop (>10%) in PaO2. The "optimal PEEP" was 14.5 +/- 3.8 cmH2O. PaO2 /FI O2 ratio was 154.8 +/- 63.3 mmHg at baseline and improved to 290.0 +/- 96.4 mmHg at highest PEEP and 302.7 +/- 94.2 mmHg at "optimal PEEP", both significantly higher than baseline (p<0.05). Static compliance was significantly higher at "optimal" PEEP (27.2 +/- 10.4 mL/ cmH2O) compared to highest PEEP (22.3 +/- 7.7 mL/cmH2O) (p<0.05). Three patients experienced transient hypotension and one patient experienced atrial premature contractions. No patient had gross barotrauma. PEEP titration protocol involving RM and PEEP decrement was effective in improving oxygenation and was generally well-tolerated.

Adult↗

PubMed1990

Optimization of survey protocols for MRI.

A method for evaluating the sensitivity of MRI protocols to changes in an arbitrary number of tissue parameters over a broad range of parameter values is presented. This analysis is useful for choosing an optimal basis set of images for either a "survey" protocol or tissue segmentation algorithms. A survey protocol is required when searching for a lesion of unknown type or location. Segmentation requires unique signal signatures for tissues that may cover a broad range of tissue parameter values. Data acquisition is modeled as a mapping of a domain of tissue parameter values into a signal manifold in a signal strength space defined by the MRI protocol. The efficacy of the protocol is evaluated by investigating the characteristics of the signal manifold. A figure of merit which maximizes the probability of discriminating each point in the domain of tissue parameters from all others is developed.

Clinical Protocols↗

PubMed1995

[Optimization of a protocol for automatic solid phase synthesis of peptides using a variable volume flow reactor].

The protocol for solid phase peptide synthesis using automatic synthesizers MilliGen/Biosearch 9500 and 9600 with built-in variable volume flow-reactors has been elaborated. The data of swellographic monitoring were used for optimization of process parameters. The peptides involving from 10 to 21 amino acid residues were synthesized using developed protocol. Significant economy of reagents and solvents has been demonstrated for synthesis of good quality peptides.

Amino Acid Sequence↗

PubMed2006

Improved 2-nitrobenzenesulfenyl method: optimization of the protocol and improved enrichment for labeled peptides.

We have developed the NBS (2-nitrobenzenesulfenyl) method, a quantitative proteome analysis method utilizing stable isotope labeling followed by mass spectrometry. The potential of this method was reported previously, and the procedure has now been further optimized. Here, we describe a procedure utilizing urea or guanidine hydrochloride as a protein denaturant, in conjunction with an improved chromatographic enrichment method for the NBS-labeled peptides using a phenyl resin column. By using this new protocol, both sample loss throughout the protocol and the elution of unwanted unlabeled peptides can be minimized, improving the efficiency of the analysis significantly.

Animals↗

PubMed2006

Evaluation of early versus delayed lymphoscintigraphic imaging in detecting internal mammary sentinel lymph nodes in breast cancer: a multicenter study to establish an optimal lymphatic mapping protocol.

OBJECTIVE: Metastases in the internal mammary lymph nodes have an important prognostic value in breast cancer. Lymphatic mapping and sentinel node biopsy of internal mammary nodes improves staging and permits specific therapeutic strategies, thereby possibly improving final outcome. Therefore, optimal lymphoscintigraphic results are needed. Visualization of internal mammary lymph drainage, however, is influenced by several factors. We evaluated the effect of different time intervals between radioactive tracer injection and lymphoscintigraphy on visualization of internal mammary sentinel lymph nodes. METHODS: From February 1997 to August 2001 a total of 682 eligible breast cancer patients underwent sentinel lymph node mapping. The technique involved the injection of 370 MBq (10 mCi) (99m)Tc-nanocolloid peritumorally. In 470 patients (group A) the time interval between injection of the radiocolloid and lymphoscintigraphy was 16 h, compared to 2.5 h in 212 patients (group B). RESULTS: Patient characteristics showed no statistically significant difference between both groups for age and location of the tumour. Axillary hotspots were visualized in 97% in group A and 96% in group B. Lymphoscintigraphy showed internal mammary hotspots in 21% in group A, compared to 27% in group B. The mean number of internal mammary hotspots per patient was 1.9 in group A and 1.8 in group B. CONCLUSIONS: We found no significant differences between early and delayed lymphoscintigraphic imaging in visualizing internal mammary sentinel lymph nodes.

Adult↗

PubMed2005

Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols.

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo- DNA polymerase (Vent exo-) was developed. The optimal dosage of Vent exo- at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo- can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo- proves to be more efficient than Pyrococcus furiosus exo- (Pfu exo-) for this task. Vent exo- resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo-. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo- than for Pfu exo-, which is reflected by the sensibility of Vent exo- in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo- demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo- were established. Our results show that Vent exo- DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.

DNA↗

PubMed2003

Ruptured aneurysm of the infrarenal abdominal aorta: impact of age and postoperative complications on mortality.

Between 1985 and 2000, a total of 871 patients underwent surgical treatment for infrarenal abdominal aortic aneurysm (AAA), including 98 (11.2%) presenting with ruptured abdominal aortic aneurysms (RAAA). An optimized operative protocol was used to treat 77 RAAA starting in January 1989. The main features of the optimized protocol are routine use of intraoperative autotransfusion, revascularization by aortoaortic bypass, absence of systemic heparinization, and use of a collagen-impregnated prosthesis. Intraoperative mortality (IOM) was 3.8%. Postoperative mortality at 1 month (POM1) was 25.9% and postoperative mortality at 3 months (POM3) was 33.7%. Heart failure (p <0.001), hemodynamic shock (p <0.001), and hemorrhage (p = 0.04) were the only complications correlated with POM1. Pneumonia (p = 0.01) and sepsis (p = 0.01) were the only complications correlated with POM3. Isolated acute renal insufficiency was not a significant risk factor for postoperative mortality. Using a cutoff of 75 years, there was a significant age-related difference (p = 0.025) for POM1 but not for IOM and POM3. The findings of this study show that optimizing the operative protocol decreases mortality related to RAAA. The main predictor of POM1 was hemodynamic status while the main predictor of POM3 was infection. Isolated acute renal insufficiency was not a risk factor for mortality. Age should not be considered a contraindication for operative treatment.

Age Factors↗

PubMed2006

Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols.

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

Automation↗

PubMed2003

Enhanced scintigraphic protocol required for optimal preoperative localization before targeted minimally invasive parathyroidectomy.

At our tertiary care institution, a targeted minimally invasive parathyroidectomy (MIP) is the preferred surgical procedure for primary hyperparathyroidism. Similar to unilateral neck exploration (UNE), preoperative scintigraphic localization of the adenoma in relation to the midline is required. However, in contrast to the abbreviated standard incision for UNE, 2 distinct incision sites, 1 medial and 1 lateral, are available on each side with MIP. The incision site is ultimately chosen based on scintigraphic determination of the adenoma's vascular origin to facilitate ligation and removal. Unfortunately, the scintigraphic location of a parathyroid adenoma does not necessarily reflect the site of its vascular origin. We reviewed our database to identify factors that accurately predict the site of vascular origin of parathyroid adenomas. A retrospective chart review was performed on 125 patients who underwent Tc-99m sestamibi scintigraphy and parathyroidectomy. Scintigraphic localization, surgical findings, and histopathology were recorded. Preoperative image interpretations that were discordant with operative findings were independently reviewed. Scintigraphy identified the presence of an adenoma in 105 of 118 patients (89%) with primary hyperparathyroidism. In 17 of the 105 cases (16%), the scintigraphic interpretation did not accurately reflect the site of superior or inferior vascular origin seen at surgery. In many discordant cases, anterior images were insufficient for determining the vascular origin. The posterior displacement of an adenoma in relation to the thyroid on early lateral images was often critical in determining the superior or inferior vascular origin. Scintigraphic determination of the superior or inferior vascular origin of a parathyroid adenoma directs incision placement for MIP. Imaging protocols should include early lateral images when localizing parathyroid adenomas before minimally invasive parathyroidectomy.

Adenoma↗

PubMed2006

Generalizability of center of pressure measures of quiet standing.

Center of pressure (COP) measures are commonly used as indicators of balance and postural control. At present, there are no universally accepted standards in research investigating fluctuations in the COP with regard to the number of trials or the length of a given trial. The purpose of this study was to use the tools of Generalizability Theory (G-Theory) to investigate the reliability of COP measures of quiet standing and to establish an optimal measurement protocol. G-Theory provides a tool that allows us to break down the sources of variation, or facets, in a measurement procedure and ultimately design a protocol that provides optimal reliability. Fifteen participants completed 10 90-s trials with eyes open and closed. COP measures of anterior-posterior standard deviation (SD(AP)), medial-lateral SD (SD(ML)), average velocity (Vel), and 95% confidence ellipse area (Area) were calculated using the first 30, 60, and 90s of each trial. A G-study and follow-up D-studies were performed to estimate reliability coefficients (G-coefficients). The results of the G-Theory analysis suggest that these COP measures reached acceptable levels of reliability (G>or=0.70) with at least five 60s trials.

Adult↗

PubMed2005

Optimizing brain MRI protocols in the follow-up of patients with multiple sclerosis T2-weighted MRI of the brain after the administration of gadopentetate dimeglumine.

The aim of our study was to determine whether T2-weighted (T2w) MRI of the brain could be performed immediately after the administration of gadopentetate dimeglumine (gadolinium DTPA) in patients with multiple sclerosis (MS) without a loss in image quality or diagnostic reliability. Sixteen patients with clinically diagnosed MS were included in the study. Twenty-four patients with various cerebral pathologies (14 patients with multiple lacunar lesions) were examined in order to exclude masking of T2 hyperintense lesions other than MS lesions. Images of 10 patients without pathological changes served as a control condition for the qualitative analysis. In these 50 patients, T1w and T2w MRI was performed before and after the administration of gadolinium DTPA. Signal intensities were measured within T2 hyperintense cerebral lesions, in T1-enhancing lesions and in normal appearing brain tissue on T2w turbo spin-echo (TSE) sequences. Both quantitative and qualitative analysis did not show significant differences between T2w pre- and postcontrast series. T2w MRI performed prior to and after the administration of gadolinium DTPA provides similar information in patients with MS. With a TR of 3.2 s, not a single lesion was obscured on T2w postcontrast series. Acquisition of T2w MR images immediately after the administration of gadolinium DTPA allows for shorter examination time and assures sufficient time for contrast enhancement in cerebral lesions with a disrupted blood-brain barrier.

Adolescent↗

PubMed2006

[MRI of the prostate: optimization of imaging protocols].

This article details the imaging protocols for prostate MRI and the influence on image quality of each particular setting: type of coils to be used (endorectal or external phased-array coils?), patient preparation, type of sequences, spatial resolution parameters. The principle and technical constraints of dynamic contrast-enhanced MRI are also presented, as well as the predictable changes due to the introduction of high-field strength (3T) scanners.

Adenocarcinoma↗

PubMedDate not supplied

Comparison of conformation-sensitive gel electrophoresis and single-strand conformation polymorphism analysis for detection of mutations in the BRCA1 gene using optimized conformation analysis protocols.

In order to develop a selective mutation screening strategy for BRCA1, one of the gene responsible for hereditary predisposition to breast cancer, we analysed by single-strand conformation polymorphism (SSCP) and conformation-sensitive gel electrophoresis (CSGE) a cohort of 20 Bulgarian breast cancer patients, prescreened for nonsense mutations by the protein truncation test. By assaying the complete coding sequence of the gene applying both methods, we were able to detect 12 sequence alterations: 11 nucleotide substitutions and one deletion. Two of the alterations are intronic polymorphisms, the rest are exon sequence variants. Of the 12 polymorphisms identified, 11 are described and one is new. All sequence changes were detected by CSGE and eight of them were also shown by SSCP analysis. There was no sequence alterations which could be detected by SSCP analysis only. We propose that because of the specificity of most sequence variants detected (nucleotide substitutions) and the comparatively high percentage of AT content of the BRCA1 gene (58.4%), CSGE turned out to be the more sensitive technique in our assay. This observation is in agreement with other accepted analysis strategies for BRCA1 and it may prove useful for mutation screening of AT-rich, multi-exon genes.

Adult↗

PubMedDate not supplied

A nomogram to select the optimal treadmill ramp protocol in subjects with high exercise capacity: validation and comparison with the Bruce protocol.

PURPOSE: Guidelines suggest that individualized ramp protocols with treadmill times targeted between 8 and 12 minutes are most suitable to estimate exercise capacity. However, previous methods to determine individualized ramp rates and comparisons between ramp and standardized protocols have been limited to clinically referred populations. METHODS: Forty-three healthy volunteers [median (interquartile range), age 36 (30-41) years; 10 women] performed an individualized ramp and a Bruce treadmill exercise protocol in random order. The Veterans Specific Activity Questionnaire [VSAQ, resulting in metabolic equivalents (METs)VSAQ] combined with a modified variant of the VSAQ nomogram (resulting in METs(NOMOGRAM)) was used to individualize the ramp protocol. Exercise capacity estimated from speed and grade of the treadmill (METs(ESTIMATED)) and that derived from directly measured peak oxygen uptake (VO2) [METs(MEASURED)] were compared with the pretest estimates of exercise capacity. RESULTS: Median values for METs(VSAQ), METs(NOMOGRAM), METs(ESTIMATED), and METs(MEASURED) for the ramp protocol were 12.0 (10-12), 15.0 (14-16.5), 16.7 (15.9-17.8), and 15.2 (13.5-16.7), respectively. For the ramp protocol, all 43 participants achieved a treadmill time between 8 and 12 minutes, whereas with the Bruce protocol only 6 (14%) participants fell within this range (P < .0001). Peak VO2 [ramp: 53.1 (47.4-58.3) versus Bruce: 53.5 (48.7-58.3) mL/kg/min; P = .008] was slightly lower using the ramp protocol. CONCLUSIONS: The modified variant of the VSAQ nomogram is a useful tool to estimate an individual's exercise capacity and to select a treadmill ramp protocol to yield the recommended exercise duration for moderately to highly fit, healthy individuals. The individualized ramp and the Bruce protocols are similar with regard to directly measured peak VO2 achieved.

Adult↗

PubMed2005

Cervical spine ROM measurements: optimizing the testing protocol by using a 3D ultrasound-based motion analysis system.

The aim of this study was to evaluate the intra- and inter-examiner reliability and validity of neck range of motion (ROM) measurements. Thirty-five healthy subjects were assessed in all neck movements from two initial positions, sitting and standing, actively (open and closed eyes) and passively by using a 3D ultrasound-based motion analysis device (Zebris). Three tests were employed to assess intra-examiner reliability and two examiners used for the inter-examiner reliability. X-rays in neck flexion and extension were used to validate the Zebris system. The standing position yielded higher intraclass correlation coefficient (ICC) values (>0.86) with less error [smallest detectable difference (SDD)<13.8%] than sitting (ICC>0.79, SDD<14%). Passive assessment of neck ROM presented better reproducibility than active assessment with open or closed eyes in both positions. The inter-examiner reliability was moderate (ICC=0.43-0.68). The correlation between the Zebris system and X-rays was high in both flexion and extension movements. The results showed that the most reliable protocol for assessment of neck ROM is a passive measurement in the standing position. The measurements were well validiated against X-rays and the experience of the investigators must be considered before any comparison among studies is employed.

Adult↗

PubMed2006

Quantitative analysis of levels of serum immunoglobulin G against botulinum neurotoxin type D and association with protection in natural outbreaks of cattle botulism.

The recent outbreaks of cattle botulism in vaccinated Israeli dairy cattle prompted us to determine vaccine efficacy and reasons for vaccine failure. Analysis of clinical signs, feeding practice, vaccination history, and epidemic curves enabled us to define a study population in two outbreaks, where high doses of Clostridium botulinum neurotoxin type D (BoNT/D) were evenly consumed by the affected animal groups. Attack rates among unvaccinated 6- to 24-month-old heifers were 96% (55/57) and 85% (53/62). The attack rates in vaccinated parity 1, 2, and >or=3 cows were 40.4% (21/52), 14.3% (4/28), and 5.6% (3/54), respectively. Vaccine efficacies for these cow groups were 52.5%, 83.2%, and 93.4%, respectively. In younger, unvaccinated 2- to 6-month-old calves, presumably protected by maternal antibodies, the attack rate was 24% (17/71). These differences correlated with significant differences in levels of specific anti-BoNT/D antibody in serum by an enzyme-linked immunosorbent assay (ELISA). The ELISA performance for predicting protection was analyzed by receiver operating characteristic analysis and was found to be highly significant, with an area under the curve of 0.941 (standard error, 0.034; 95% confidence interval, 0.875 to 1.008; P < 0.000). No animals with serum ELISA unit levels above 0.33 were affected in these exposed groups. At this cutoff level, the specificity of the ELISA was 100%, sensitivity was 67%, and accuracy was 92%. We concluded that botulinum toxoids can confer adequate protection against natural exposure to lethal doses of BoNT/D; however, the vaccination protocols should be optimized. Our in-house ELISA system will enable us to optimize vaccination protocols in the animal population.

Animals↗

PubMed2005

Object-specific attenuation correction at SPECT/CT in thorax: optimization of respiratory protocol for image registration.

Institutional review board approval was obtained for multiple imaging examinations in healthy volunteers and patients and for the analysis of images. The purpose of the study, and the risks associated with radiation exposure with regard to stochastic effects that might result in cancer and/or genetic mutations, were explained to all subjects, and all questions from subjects were answered. Each subject provided written informed consent. The purpose of the study was to prospectively determine the respiratory protocol at computed tomography (CT) that results in the best registration of CT images with images acquired at single photon emission computed tomography (SPECT) in the thorax. Errors of registration between myocardial SPECT images and CT images obtained with different respiratory protocols (postinhalation breath hold, postexhalation breath hold, and free breathing) in 13 healthy subjects were compared. CT scans obtained with free breathing and postexhalation breath hold better matched SPECT images than did those obtained with postinhalation breath hold (one-way analysis of variance, P < .01). Fewer SPECT/CT images showed artifacts with registration performed by using internal landmarks (four, two, and one of 13 images with postinhalation breath-hold, postexhalation breath-hold, and free-breathing protocols, respectively) than with registration performed by using external markers (nine, four, and two of 13 images). CT data acquisition with a free-breathing or postexhalation breath-hold protocol and image registration by using internal landmarks are recommended for attenuation correction.

Adult↗