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Prospective outcome analysis of a protocol for the surgical and rehabilitative management of burns to the hands.

Treatment protocols for the management of burned hands are essential for integrating team efforts and achieving optimal functional results. Standard protocols are especially useful during mass casualty incidents, when the admission of multiple patients with large burns and/or associated injuries may reduce the priority usually accorded the hands. We prospectively evaluated a surgical and rehabilitative treatment protocol for burned hands during a mass casualty incident, after which 43 burn patients with 82 burned hands were admitted to one burn center. Soft-tissue management was individualized to achieve, if possible, wound closure within 14 days, and included the use of topical antimicrobials, cutaneous debridement and/or tangential excision, biologic dressings, and split-thickness autografts. Range of motion therapy was based on daily measurement of active motion of the metacarpophalangealjoints. Static splinting alternating with continuous passive motion every 4 hours was utilized for sedated patients. Continuous passive motion alternating with active ranging and night splinting was utilized for metacarpophalangeal flexion <70 degrees. Active ranging and progressive resistance was utilized for metacarpophalangeal flexion > or =70 degrees. Sixty-four hands required excision and grafting, with 89 percent having at least one autografting procedure completed by postburn day 16. Total active motion of the hands treated averaged 220.6 degrees on discharge and 229.9 degrees at 3 months after injury. Mean hand grip strength was 60.8 pounds at discharge and 66.0 pounds at 3 months after injury. Adherence to a standard hand burn protocol resulted in timely wound coverage and recovery of hand function for a large group of patients treated at a single burn facility after a mass casualty incident.

Accidents, Aviation↗

Dynamic ligand design and combinatorial optimization: designing inhibitors to endothiapepsin.

The dynamic ligand design (DLD) algorithm, an automated method for the creation of novel ligands, links up small functional groups that have been placed in energetically favorable positions in the binding site of a target molecule. The positions and orientations of the small functional groups can be determined using the multi-copy simultaneous search approach (MCSS) or experimental data. In this work the original DLD methodology is extended by using a modified version of the pseudo-potential energy function. A novel simulated annealing protocol is presented for optimizing the pseudo-potential energy of ligands in the binding site; the protocol is expected to be applicable to other optimization problems. The utility of the method is illustrated by designing an inhibitor for endothiapepsin. The binding affinity of the inhibitor is assessed using a thermodynamic cycle that decomposes the binding free energy into a sum of translational, rotational, configurational, hydrophobic, and electrostatic contributions. The calculations suggest that the designed molecule will bind endothiapepsin with high affinity.

Algorithms↗

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Buffers↗

Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity.

Different protocols of protease (pepsin) treatment were compared in the immunostaining for intermediate filament (IF) proteins in formaldehyde-fixed and paraffin-embedded tissues. Without protease treatment, the immunoreactivity for all IF proteins was poor in such material. Appropriate pepsin pretreatment improved the immunoreactivity of formaldehyde-fixed tissues for all types of IF proteins, except for the 68 K neurofilament protein, which could not be immunostained with the antibody used. The optimal time for the pepsin treatment was varying in different tissues, and too long treatment caused progressive loss of immunostaining. Thus, for instance when demonstrating cytokeratins, renal adenocarcinomas were more sensitive to protease and needed a shorter treatment than other carcinomas. Therefore, a nonoptimal protease treatment protocol may cause false negative results and false cell type selective IF immunostaining. Prolonged fixation made it necessary to prolong the protease treatment. In tissues fixed up to four years in formalin, cytokeratin immunoreactivity could still be restored by a long pepsin treatment (up to 2-3 hours). For most tissues fixed for 24 hours in formaldehyde, an optimal protocol was the following: 0.05% pepsin (2 U/ml) in HCl, pH 1.8, at +37 degrees C for 20-30 minutes. The protease treatment did not produce false positive results. Alcohol-fixed material was good for IF immunostaining without any protease treatment, but such tissue blocks mostly lost the immunoreactivity during long term storage.

Endopeptidases↗

Optimization of foot-and-mouth disease vaccination protocols by surveillance of neutralization antibodies.

An appropriate immunization program for pigs in a foot-and-mouth disease (FMD) endemic area was proposed based on data analysis obtained from serological surveillance in Taiwan, after an intensive vaccination program. To provide an adequate passive immunity for piglets, gilts that have completed two basic vaccinations must be boosted once before breeding. To achieve an efficient response to the FMD vaccine for piglets born to well vaccinated sows, vaccination need to be delayed until 10-12 weeks of ages for the first immunization, followed by a boost 4 weeks later.

Animals↗

Optimization and validation of a docking-scoring protocol; application to virtual screening for COX-2 inhibitors.

To exploit available structural information about the cyclooxygenase enzyme for the virtual screening of large chemical libraries, a docking-scoring protocol was tuned and validated. The screening accuracy was assessed using a series of known inhibitors and a set of diverse a priori inactive compounds that was seeded with known active ligands. The major parameters of the DOCK algorithm were investigated. A large improvement of the results was obtained on tweaking some of them. The generated complexes were rescored using six scoring functions. In this way, the striking importance of this step was demonstrated, as well as the good performances of DOCK energy and SCORE for this target. The results were further improved via a consensus approach. As a first application, a subset of a large compound library was screened using this protocol. Among the compounds that were selected for biological testing, a third of them turned out to have a significant enzyme inhibition.

Crystallography, X-Ray↗

Ultra-rapid freezing of very low numbers of sperm using cryoloops.

BACKGROUND: With the availability of ICSI, men with severe oligozoospermia (<5x10(6)/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (approximately 100) was feasible using cryoloops. METHODS: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. RESULTS: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. CONCLUSION: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.

Cell Survival↗

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants.

BACKGROUND: Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and cryoprotectants in cryopreservation of testicular tissue. METHODS: Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants. RESULTS: Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.

Adult↗

Emergency physicians can reliably assess emergency department patient cardiac output using the USCOM continuous wave Doppler cardiac output monitor.

OBJECTIVES: 1 To develop a training package for ultrasonic cardiac output monitor (USCOM) cardiac output assessments and determine the number of proctored studies necessary for skill acquisition. 2 To develop criteria for acceptance of cardiac output results obtained with the USCOM. 3 To evaluate the reliability of USCOM cardiac output assessments in the ED. METHODS: The authors developed an audiovisual training package. Four emergency physicians and one geriatrician subsequently underwent hands-on training, and skill acquisition was assessed at the fifth, 10th, 15th and 20th examinations. Six image-scoring criteria were developed to assess acoustic image quality. Upon completion of training a protocol was developed to optimize interassessor reliability. Two trained emergency physicians then performed blinded examinations on ED patients using the protocol and interassessor reliability was evaluated. RESULTS: During training average image score improved between the fifth and 20th assessed patient from 4.6 (95% CI 4.0-5.3) to 5.5 (95% CI 5.0-6.0, Pt-test=0.02) out of 6 and average intra-assessor cardiac output difference improved from 17% (95% CI 4-25) to 5% (95% CI 0-11, Pt-test=0.02). Analysis of 52 cardiac output assessments in 21 ED patients demonstrated excellent interassessor correlation (r=0.96, 95% CI 0.90-0.98, P<0.001). The average interassessor difference in cardiac output and index was 0.2 L/min (4%, 95% CI 3-6) and 0.1 L/min/m2 (4%, 95% CI 2-6), respectively. CONCLUSION: Emergency physicians with no prior ultrasonographic experience can be trained to obtain reliable cardiac output estimations upon conscious ED patients with the USCOM over the course of 20 patient assessments.

Cardiac Output↗

Isolation of FRET-positive cells using single 408-nm laser flow cytometry.

BACKGROUND: Flow cytometry may be used to isolate large amounts of living, fluorescently labeled cells. Certain fluorescent labels, like enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), allow the assessment of direct protein-protein interaction in situ, by fluorescence resonance energy transfer (FRET). However, current flow cytometric methods either require elaborate technical adaptations or, using a single laser protocol, are hampered by background signal. We optimized a single 408-nm laser protocol to detect FRET between ECFP/EYFP-tagged proteins. METHODS: Cell lines stably expressing ECFP and/or EYFP or an EYFP-ECFP fusion protein were used to design the settings for the flow cytometer to detect FRET-positive cells using a single 408-nm laser. Using these settings, interactions between the subunits of the transcription factor NF-Y were studied. RESULTS: Flow cytometric analysis of the cells expressing an EYFP-ECFP fusion protein yielded a discrete FRET-positive population. Using the same settings, in cells expressing NF-YB-CFP and NF-YC-YFP fusion proteins, FRET could also be detected. These cells were sorted and FRET was confirmed by confocal microscopy. CONCLUSION: FRET-positive cells, expressing ECFP- and EYFP-tagged proteins, can be detected using single 408-nm laser excitation, with low background signal. This allows high-throughput analysis and isolation of viable FRET-positive and -negative cells for subsequent biological experiments.

Bacterial Proteins↗

Which experimental design for phase I clinical trials? Proposal for an investigational approach.

The choice of a protocol for the first administration to man of a new drug remains an unresolved problem for the researcher. There are few studies concerning dose-ranging experiments in phase I to determine the threshold and plateau doses of a new drug in a population. A simulation approach may help in evaluating different protocols, by considering how they could be optimized, and by selecting the best protocol for a given case.

Drug Evaluation↗

A semi-empirical treatment planning model for optimization of multiprobe cryosurgery.

A model is presented for treatment planning of multiprobe cryosurgery. In this model a thermal simulation algorithm is used to generate temperature distribution from cryoprobes, visualize isotherms in the anatomical region of interest (ROI) and provide tools to assist estimation of the amount of freezing damage to the target and surrounding normal structures. Calculations may be performed for any given freezing time for the selected set of operation parameters. The thermal simulation is based on solving the transient heat conduction equation using finite element methods for a multiprobe geometry. As an example, a semi-empirical optimization of 2D placement of six cryoprobes and their thermal protocol for the first freeze cycle is presented. The effectiveness of the optimized treatment protocol was estimated by generating temperature-volume histograms and calculating the objective function for the anatomy of interest. Two phantom experiments were performed to verify isotherm locations predicted by calculations. A comparison of the predicted 0 degrees C isotherm with the actual iceball boundary imaged by x-ray CT demonstrated a spatial agreement within +/-2 mm.

Algorithms↗

Application of an optimized electroporation procedure for replacement of the polyhydroxyalkanoate synthase I gene in Nocardia corallina.

To develop a system for gene replacement in Nocardia corallina, a protocol for electroporation was optimized by systematic alterations of growth conditions, field strength, time constant and the electroporation buffer. Transformation efficiencies of 0.5 x 10(6) - 3 x 10(6) transformants/microgram plasmid DNA were obtained routinely. The gene encoding the polyhydroxyalkanoate (PHA) synthase I of N. corallina was cloned and interrupted by insertion of a kanamycin-resistance gene. The resulting plasmid was introduced into N. corallina by electroporation to inactivate the wild-type gene by homologous recombination. Kanamycin-resistant clones were screened by Southern hybridization for the absence of the wild-type gene and analyzed for PHA accumulation.

Acyltransferases↗

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils.

AIM: To establish a rapid, improved soil environmental DNA extraction and purification protocol. METHODS AND RESULTS: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. CONCLUSIONS: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.

Bacillus↗

Iterative optimal design of PET experiments for estimating beta-adrenergic receptor concentration.

To estimate in vivo myocardial beta-adrenergic receptor concentration with sufficient precision and to reduce the experimental complexities in positron emission tomography (PET), an iterative optimal design method is applied. An initial three-injection protocol, utilising [F-18]-labelled (R)- and (S)-fluorocarazolol and unlabelled (S)-fluorocarazolol, is optimised for ligand dosages and administration times to maximise the precision of all model parameters using the D-optimal criterion. Using this experimental protocol, PET data are collected in porcine studies, and model parameters are estimated. All model parameters are identified with satisfactory precision. The in vivo myocardial beta-receptor concentration is 7.5+/-0.6 pmol x ml(-1), which corresponds to the in vitro result of 10.1+/-1.3 pmol x ml(-1). With more accurate parameter values, a simplified two-injection protocol is optimally designed, utilising only radiolabelled and unlabelled (S)-fluorocarazolol, based on a new criterion to maximise the precision of the beta-receptor concentration. This revised optimum design predicts that the in vivo beta-receptor concentration can be estimated with good precision but reduced experiment complexity.

Adrenergic beta-Antagonists↗

Protein extraction from woody plants.

In this chapter we present a protocol for total protein extraction optimized for wood-forming tissue (differentiating secondary xylem). The protocol is then used for a series of other organs (root, leaf, pollen, bud, flower, cambium, and phloem) in broadleaf (oak and poplar) and conifer (pine) species. Proteins are first extracted from tissue powdered in liquid nitrogen using the TCA-acetone method and then solubilized in an optimized buffer. The resulting 2D gels can be viewed at http://cbi.labri.fr/outils/protic/index.php.

Chemical Precipitation↗

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Biomarkers↗

Improved method for the detection of cytokeratin 19-positive cells in the peripheral blood of breast cancer patients.

To determine the correlation of the levels of circulating tumor cells (CTC) in breast cancer patients with clinical parameters like disease-free survival and response to therapy, we employed a previously reported quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR) assay for cytokeratin-19 (Ck19; Ann Oncol 2001; 12: 39-46). Our preliminary results based on this assay (Protocol A) prompted us to optimize the experimental conditions. Here we report an improved assay (Protocol B), which more sensitively and specifically detects CTC in breast cancer patients.

Base Sequence↗