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PubMedDate not supplied

Adrenocortical carcinoma: experience in 45 patients.

Forty-five patients with adrenocortical carcinoma (13 nonfunctioning and 32 functioning carcinomas) were retrospectively studied. Five-year survival rate was 29% overall; for patients at stage I-II (n = 15) it was 70%, and for patients at stage III-IV (n = 30) it was 12%. In patients given mitotane + chemotherapy survival rate was similar to that observed in patients given chemotherapy alone, and significantly longer than in patients given mitotane alone (p < 0.05). There were no differences in disease-free interval and survival between adjuvant mitotane and no treatment. Optimization of therapeutic protocols in addition to early recognition may improve prognostic aspects of this type of malignancy for which treatment outcome is still unsatisfactory.

Adolescent↗
PubMed1997

Aspects of assessment and management of velopharyngeal dysfunction in developing countries.

Proper assessment of patients with velopharyngeal valve incompetence is a mandatory prerequisite for optimal management. The protocol of assessment of Ain Shams University, Phoniatric Department, uses three levels of assessment of velopharyngeal valve incompetence according to the complexity of the armamentarium used. This allows application of those parts of the protocol that suit the needs of the different socioeconomic levels and geographical locations. Firstly, the elementary diagnostic procedures, which are rather simple, noninvasive, but essentially subjective. Despite the clinical feasibility of these procedures, documentation of the data is made utilizing the tools at the second level of assessment in that protocol (clinical diagnostic aids). This level comprises video-nasofiberscopy and high fidelity voice recording. An attempt to extract quasi-quantitative measures from the hitherto qualitative video-nasofiberscopy is made. The third level of assessment, namely additional instrumental measures, comprises CT scanning of the velopharyngeal port, aerodynamics, and acoustic analysis. The results of the three levels of the protocol are presented. Their significance and clinical efficacy are discussed. Some community-related problems that have faced the cleft palate team are outlined. Their sociocultural significance in a developing country is discussed.

Adult↗
PubMed1995

Myocardial 18F-FDG-PET. Experiences with the euglycemic hyperinsulinemic clamp technique.

Myocardial positron emission tomography (PET) with 18F-Fluordeoxyglucose (FDG) is increasingly used for the detection of viable tissue in the infarcted myocardium. Previous studies show that the variable metabolic conditions determine the regional distribution of this tracer and that the inhomogeneities of uptake often observed even in the normal myocardium may relate to substrate availability. The authors tried to stimulate the myocardial FDG uptake by either the technically easier method of glucose loading or by the euglycemic hyperinsulinemic clamp (EHC) technique. In their hands both methods could be considered as equally practicable but differing in some important details in regard to both the study protocol (tracer dose, optimal scanning time) and the reproducibility of results. The EHC allows a quick stabilization of the metabolic environment and resulted in an earlier and markedly increased FDG uptake. However, the important standardization of the method was performed by a computer-controlled system only for the glucose and insulin infusions. Their experiences show that the EHC provides a useful framework for assessing altered cardiac metabolism and possibly describes changes after therapeutic interventions more precisely than the commonly used glucose-loading technique.

Deoxyglucose↗
PubMed1993

A review of nutrition support for transplant patients.

Renal, hepatic, and cardiac transplantation are now recognized as acceptable methods for treating end-stage organ failure. Obtaining optimum results in such patients requires not only skillful surgical technique and postoperative immunosuppression but also stabilization of the patient preoperatively. This stabilization includes a number of physiologic parameters. One of the most important of these is the correction of preexisting nutritional deficits. Each type of end-stage organ disease creates unique nutritional problems. This article reviews these deficiencies and makes recommendations as to the appropriate nutrition protocols that can optimize results in the patient who undergoes organ transplantation.

Humans↗
PubMed1994

Pregnancy in systemic lupus erythematosus: a prospective study.

The objective of this work was to analyse the course of maternal disease and fetal outcome in pregnant patients with systemic lupus erythematosus (SLE) counselled and followed according to a protocol intended to optimize maternal and fetal outcome. The prospective study included all pregnancies between 1987 and 1993 in SLE patients known at least 6 months before pregnancy at the Lupus Clinic of our hospital. In 25 patients there were 35 pregnancies. Thirty-four (97%) started at sustained remission of disease; 11 (31%) were in women with antiphospholipid antibodies (aPL); 14 (40%) in women with a history of biopsy-proven lupus nephritis; one (3% in a woman with a serum creatinine above 125 mumol/l. In 29 pregnancies (82%) maternal disease remained inactive during gestation. In three pregnancies (9%) active disease was treated with prednisone. There were no serious post-partum flares of disease. Pregnancy resulted in 25 (71%) live births, 8 (23%) first trimester abortions, and one intrauterine fetal death. One pregnancy was terminated because of hydrocephalus. Nine of 25 (36%) live births were delivered by caesarean section. For 6 of 9 (67%) caesarean sections the indication was fetal distress and pre-eclampsia. In the majority of patients with SLE who conceive at remission, the disease does not flare in pregnancy. With optimal obstetric care, close follow-up and treatment with low-dose aspirin if aPL are present, a high success rate (71%) can be achieved.

Adult↗
PubMedDate not supplied

Water and cryoprotectant permeability characteristics of isolated human and canine pancreatic islets.

Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22 degrees C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (Lp), (ii) the permeability coefficient of the CPA (Ps), and (iii) the associated reflection coefficient (sigma). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest Ps values for canine islets were observed following exposure to 2 M EG, and the highest Ps values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 microm/min/atm, 2.47 microm/s, and 0.90 (for Lp, Ps, and phi, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 microm/min/atm, 3.48 microm/s, and 0.85 (for Lp, Ps, and sigma, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.

Animals↗
PubMed2005

Development and application of an automated solution stability assay for drug discovery.

Screening of solution stability provides an early alert on potential liabilities of drug candidates so that strategies can be developed to overcome the challenges. A fully automated solution stability assay has been developed to accelerate traditional manual operation. The assay uses the advanced capabilities of a high-performance liquid chromatography instrument that is present in many pharmaceutical research laboratories. The samples are prepared automatically by a temperature-controlled autosampler. The samples are delivered to the stability matrices, mixed, incubated, and injected at selected time points during the reaction time course. This automated process occurs without operator intervention, thus allowing 96 experiments to be run with 0.5 h of a scientist's time compared to 8 h for the same study when performed manually. Automation not only eliminates the manual operation but also improves accuracy and throughput. The assay protocol has been optimized to achieve homogenous mixing and eliminate carryover. The assay is robust, flexible, and high throughput. It can be used to study stability for a large number of samples under multiple incubation conditions and has a wide range of applications in drug discovery and development, such as screening compound stability in biological assay media, obtaining a stability-pH profile, surveying compound stability in physiological fluids, and performing development forced degradation and excipient compatibility.

Automation↗
PubMed2004

Surveillance of duodenal adenomas in familial adenomatous polyposis reveals high cumulative risk of advanced disease.

PURPOSE: The development of high-grade dysplasia (HGD) on duodenal or jejunal adenomas and of late-stage (stage IV) duodenal polyposis are major clinical events for familial adenomatous polyposis (FAP) patients. Our aim was to determine their respective frequency, risk factors, and cumulative risk. PATIENTS AND METHODS: A prospective, optimized, endoscopic surveillance protocol was applied to 58 FAP patients in a university hospital. The number, size, and histology of duodenojejunal polyps were assessed, and the Spigelman's score was calculated at each endoscopy. Cox regression and linear regression analysis were used to determine risk factors for HGD development and the cumulative risk of stage IV duodenal polyposis, respectively. RESULTS: During a median (+/- standard deviation) follow-up of 47.9 +/- 15.6 months, 35 patients with at least two consecutive examinations had 107 duodenojejunal examinations. The Spigelman's score increased in 21 patients (60.0%), and HGD developed in 12 patients (34.2%). High initial Spigelman's score (> 7 points), but not age or APC mutation site, was a risk factor for HGD development. Estimated cumulative risk of developing stage IV duodenal polyposis was of 42.9% at age 60 (95% CI, 35.7% to 50.0%) and 50.0% at age 70 (95% CI, 42.9% to 57.1%). CONCLUSION: This prospective series shows a higher duodenal polyposis progression rate and cumulative risk of late-stage (stage IV) duodenal polyposis in FAP patients compared with previous series. These results suggest that current modalities for surveillance and management of these patients need revision.

Adenoma↗
PubMed2006

Rat embryo quality and production efficiency are dependent on gonadotrophin dose in superovulatory treatments.

The present study was performed to optimize a superovulation protocol in rats in order to produce a large number of good-quality embryos suitable to develop rat embryonic stem (rES) cells. We first evaluated the ovulation kinetics of three rat strains: Wistar, Fisher and ACI/N. Animals (n=30 per strain) were treated with 50 IU of pregnant mare serum gonadotrophin (PMSG), and ovulation was induced with 50 IU of human chorionic gonadotrophin (hCG) 50 h apart. Next, we evaluated the dose-response curves of PMSG and hCG in Wistar rats in order to obtain the highest number of embryos. The parameters evaluated for superovulation efficiency were: percentage of mated females, percentage of pregnant females and the average number of embryos collected per female. The results of these experiments suggested that the best dose combination was 50 IU for each hormone. Subsequent experiments, again with Wistar rats, were designed to test which of four hormonal combination treatments (30/30, 30/50, 50/30, and 50/50 IU of PMSG/hCG) will produce the largest numbers of good-quality embryos. Embryo quality was evaluated by embryo development uniformity, embryo morphology, embryo survival in an in vitro culture and embryo ability to generate rES-like cells. Results from these experiments showed that 30/50 IU of PMSG/hCG was the treatment that induced the best embryo quality. In conclusion, our results indicated that, in Wistar rats, the most appropriate hormonal combination dose for superovulation protocols with high number of good-quality embryos was 30 IU of PMSG and 50 IU of hCG given 50 h apart. We are performing further studies with rES-like cells produced with the present methodology to evaluate if they are able to participate in the production of germ-line chimeras.

Animal Husbandry↗
PubMed2004

TCR zeta-chain abnormalities in human systemic lupus erythematosus.

A growing number of studies have revealed that the expression of many genes is abnormal in T lymphocytes of patients with systemic lupus erythematosus (SLE). Although aberrant expression of signaling molecules may arise intrinsically or in response to the environment, these abnormalities play a significant role in the pathogenesis of this autoimmune disease. Modern research on lymphocyte signaling abnormalities in SLE has been directed toward identifying defective expression of various signaling molecules, understanding the molecular basis of the deficiency, and dissecting the T-cell signaling abnormalities that result from abnormal gene expression. The developments suggest that interplay of abnormal transcriptional factor, aberrant messenger RNA processing/editing, ubiquitination, proteolysis, oxidative stress, and changes in chromatin structure invariably contribute to the abnormal expression of numerous signaling molecules in SLE T cells. The contribution of each of these mechanisms in the abnormal expression of signaling molecules in SLE T cells is not known. In addition to abnormalities in gene expression, multiple factors, including altered cellular distribution of the protein, rewiring of the receptor, modulation of membrane clustering, and lipid raft distribution of signaling molecules and defective signal-silencing mechanisms play a key role in delivering the anomalous T-cell receptor/CD3-mediated intracellular calcium response in SLE T cells. The optimized methods and protocols described here pertaining to TCR zeta-chain expression and related T-cell signaling abnormalities can be very well applied to other molecules aberrantly expressed in SLE T cells.

Base Sequence↗
PubMed2006

Development and adaptation of the PRINS technology: an overview.

The advent of molecular genetic techniques has brought forth new procedures for in situ chromosomal analysis. One of these techniques is the primed in situ labeling (PRINS) procedure, which constitutes a fast and efficient alternative to conventional fluorescence in situ hybridization for nucleic acid detection. Based on the use of chromosome-specific primers, the PRINS method combines the high sensitivity of the PCR reaction with the cytological localization of DNA sequences. Since its introduction, the PRINS protocol has been optimized, and numerous applications have been developed. The technique has thus proved to be a useful tool for in situ chromosomal screening, and has become a simple and efficient complement to conventional and molecular cytogenetic methods.

Animals↗
PubMed2007

X-ray data collection from macromolecular crystals.

Instruments, methods, and software for modern macromolecular crystallography is becoming so effective that molecular biologists often can solve structures from their crystals by working "without a license." In this chapter, the authors attempt to demystify some of the apparatus and techniques by providing a roadmap. Current methods for collecting X-ray diffraction data from macromolecular crystals are described. The principles of operation of the required X-ray sources, optics, goniometers, and detectors are outlined, and a typical data collection protocol is presented. Optimization of data quality is a pivotal stage in the whole crystallographic process, so much attention is given to the detailed setting up of the experiment. This is followed by a summary of the basic ideas behind the diffraction image-processing packages and their application to data reduction. Despite the increasingly "black box" nature of these computer programs, understanding how they extract the intensities, errors, and indices from the data can make subsequent structure solution and refinement much easier.

Crystallization↗
PubMed1999

Combining periodic and continuous sodium hypochlorite treatment to control biofilms in dental unit water systems.

BACKGROUND: This study evaluated the efficacy of combined intermittent and continuous treatment with diluted sodium hypochlorite, or NaClO, to improve dental unit water quality in a clinical setting. METHODS: In this prospective study, 10 dental units were fitted with separate water reservoir systems. Dental units were maintained with weekly rinses with 1:10 NaClO. Treatment water consisted of 750 milliliters of tap water and one drop of undiluted commercial bleach. Bacterial contamination in the effluent coolant water was assayed via microbiologic culture on a weekly basis. At the end of the study, scanning electron microscopy of the inner surfaces of the dental unit waterlines corroborated the results. RESULTS: All 10 dental units consistently delivered water with less than 10 colony-forming units per milliliter, or CFU/mL, with a mean bacterial contamination of less than 1 CFU/mL. Baseline scanning electron microscopy demonstrated biofilm formation. Scanning electron microscopy at the end of the study demonstrated the lack of features consistent with biofilm formation. Although tri-halomethanes were detected in output water, all samples were below Environmental Protection Agency limits for drinking water. CONCLUSIONS: Weekly treatment with 5.25 percent NaClO diluted 1:10, and concomitant use of chlorinated treatment water (3 parts per million chlorine) consistently attained the proposed American Dental Association goal of fewer than 200 CFU/mL in the unfiltered output. The effects of continuous treatment on dentin and enamel bond strength may require further evaluation. CLINICAL IMPLICATIONS: The success of this protocol suggests that optimal attainment of dental water quality goals may require a combination of approaches.

Biofilms↗
PubMed2001

Safety and effectiveness of ibutilide in a community hospital.

To determine the safety and effectiveness of ibutilide, we conducted a retrospective study of patients in a community hospital from December 1997-January 2000, reviewing hospital and pharmacy records for 12 inpatients (aged 68+/-13 yrs) receiving ibutilide for atrial fibrillation or flutter. The cumulative conversion rate with ibutilide 0.012+/-0.004 mg/kg was 58%. Serious cardiovascular adverse events were documented in six patients; of those patients, five had baseline QTc intervals above 440 msec, two experienced ventricular fibrillation, and four experienced symptomatic bradycardia (range 49-60 beats/minute) requiring medical intervention. The effectiveness of ibutilide was similar to that found in previous studies. However, more complications were seen in this setting where ibutilide was used infrequently and without stringent patient selection criteria or standardized administration protocols. To ensure optimal patient care, institutions should consider implementing guidelines or education strategies to reduce the potential for serious adverse effects associated with improper screening, dosing, and monitoring of patients receiving ibutilide.

Aged↗
PubMed2001

Induction and rejoining of DNA double-strand breaks in normal human skin fibroblasts after exposure to radiation of different linear energy transfer: possible roles of track structure and chromatin organization.

DNA double-strand breaks are nonrandomly induced by high-LET radiation. Differences in the induction and rejoining of DSBs after irradiation with ions having different LET were detected by fragment analysis. The data obtained indicate that the track structure of the traversing particle and its interaction with the different chromatin structures of the cellular DNA influence the yield as well as the distribution of the induced damage. The induction and rejoining of clustered DSBs induced by the same nitrogen ion fluence at LETs of 80-225 keV/microm were investigated by a detailed analysis of the DNA fragmentation patterns in normal human fibroblasts. The DSBs in the cells were allowed to rejoin during incubations for 0-20 h. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of fragments in the size ranges 1-6 Mbp and 5 kbp-1.5 Mbp. A strong influence of LET on the level of DSB induction was evident. The DSB yield increased from 4.5 +/- 0.2 to 10.0 +/- 0.3 DSBs per particle traversal through the cell nucleus when LET increased from 80 to 225 keV/microm. Further, the size distribution of the DNA fragments showed a significant dependence on radiation quality, with an excess of fragments at 50-200 kbp and around 1 Mbp. Differences in repair kinetics were also evident, with slower rejoining for increasing LET, and the initial nonrandom fragment distributions were still present after 1 h of repair.

Chromatin↗
PubMed2006

96-well electroporation method for transfection of mammalian central neurons.

Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.

Animals↗
PubMed2000

A concise guide to cDNA microarray analysis.

Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and have developed strategies for data normalization and analysis.

DNA, Complementary↗
PubMed2001

Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridization probes.

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in LightCycler assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.

DNA-Directed DNA Polymerase↗