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A double scanning microphotometer for image analysis: hardware, software and biomedical applications.

A new image processing system designed for densitometry and pattern analysis of microscopic specimens is described with special regard to the hardware, the software and the biologic applications. The data acquisition procedure involves the combination between the scanning of the preparation by means of a motorized stage and the scanning of successive fields by a mechanical device. The signal provided by the photomultiplier is converted into digital values which are directed to an on-line computer. The data processing is based on a one-pass computation involving automata theory and therefore it avoids the storage of the image in the computer memory. In so doing, an entire and continuous image of the whole preparation can be processed at the highest magnification of the microscope whatever the size of the analyzed specimen may be. A biologic application of the system is reported and concerns the automatic identification and counting of cells in the various phases of the mitotic cycle.

Animals↗

High-sensitivity cytotoxicity assays for nonadherent cells.

High-sensitivity cytotoxicity assays refer to assays that can detect high levels of cell kill, to many powers of 10, and that can detect, ideally, a single remaining viable cell. Two such assays are described here, which have been used with Raji B-lymphoma cells, and are applicable to other nonadherent target cells. The first is a cell-counting assay, performed over a 3-wk period, which provides a simple, reliable, and sensitive assay of cytotoxicity. By determining the time required for 16-fold multiplication, the apparent fraction surviving can be calculated. This assay does not correct for treatment-induced delays in cell division and is dependent on maintaining the cells in exponential growth. The second assay measures colony-forming units using a limiting dilution method. Feeder cells are required to obtain a high cloning efficiency. Each dilution is plated in 48 wells of a 96-well plate, and positive wells are scored rapidly, by eye, after two wk.

Antibodies↗

Use of monoclonal antibodies to refine flow cytometric differential cell counting of canine bone marrow cells.

OBJECTIVES: To evaluate use of monoclonal antibodies to increase accuracy of flow cytometric differential cell counting of canine bone marrow cells. SAMPLE POPULATION: Bone marrow specimens from 15 dogs. PROCEDURES: Specimens were labeled with monoclonal antibodies that detected CD18, major histocompatability antigen class-II (MHC class-II), CD14, and Thy-1. Location of fluorescent and nonfluorescent cells within gates of a template developed for canine bone marrow differential cell counting was determined, the template was revised, and 10 specimens were analyzed by use of the old and revised templates and by labeling cells with anti-MHC class-II and anti-CD14. RESULTS: Data confirmed the presumptive location of marrow subpopulations in scatter plots, permitted detection of lymphocytes and monocytemacrophages, and was used to revise the analysis template used for differential cell counting. When differential cells counts determined by the original and revised templates were compared with results of manual differential cell counts, the revised template had higher correlation coefficients and more similar mean values. Labeling cells with anti-MHC class-II and anti-CD14 permitted identification of lymphoid and monocyte-macrophages cells in bone marrow specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the revised flow cytometric analysis template combined with anti-CD14 and anti-MHC class-II antibody labeling provides reliable differential cell counts for clinical bone marrow specimens in dogs. These techniques have potential applications to clinical bone marrow examination and preclinical toxicity studies.

Animals↗

After the Indian Ocean tsunami: Singapore's contribution to the international disaster victim identification effort in Thailand.

This paper describes the international disaster victim identification (DVI) response mounted in Thailand, with particular reference to Singapore's contribution to this process, in the wake of the Asian tsunami of 26 December, 2004, which devastated parts of more than 10 countries in and around the Indian Ocean and claimed more than 200,000 lives. Although Singapore was unscathed by this natural calamity, over 30 Singaporean visitors were counted amongst the thousands of deceased victims, mostly in Thailand. The systematic application of forensic pathology, forensic dentistry, DNA profiling, and fingerprinting to human identification, especially of the bodies of various nationalities that were in advanced states of putrefaction, was crucial to the entire DVI process. The authors perceive that the resource implications arising from such a disaster, which is unprecedented in both its scale and reach in the international history of DVI, are immense. Forward planning, adequate funding and international cooperation are essential to mounting an effective response to any major mass disaster of the future.

Bayes Theorem↗

[Treatment of stage IV neuroblastoma with high-dose melphalan and autologous bone marrow transplantation following in vitro preliminary treatment of the bone marrow with the active cyclophosphamide derivative Asta Z-7654].

The case of a 4 year 8 months old boy with neuroblastoma of unknown primary, metastatic to the bone and to the bone marrow is presented. After achieving a partial remission with six cycles of conventional chemotherapy, the patient was given supraconventional chemotherapy (melphalan 220 mg/m2 bolus i.v.) in an effort to eliminate residual disease. Prior to the administration of the drug, 560 cc of autologous bone marrow, morphologically free of tumor was harvested (total 110 X 10(8) nucleated cells) and concentrated to a mononuclear cell fraction with a total of 10 X 10(8) cells. After in vitro purging with the stable metabolite of 4-hydroperoxycyclophosphamide ASTA Z 7654 (40 micrograms/2 X 10(7) mononuclear cells/ml), the mononuclear cell suspension was retransfused 10 hours following the application of high dose melphalan. Hemopoietic reconstitution was delayed with a platelet count reaching 70,000/microliter only after seven months. At the time of this writing (20 months after diagnosis and 16 months after autologous bone marrow transplantation) there is no evidence for active disease according to the bone scan and multiple bone marrow biopsies. In view of the dismal prognosis of patients with neuroblastoma, stage IV it is recommended that further patients should be treated with a slightly modified protocol of the cooperative austrian neuroblastoma study.

Antineoplastic Combined Chemotherapy Protocols↗

Assessment of cellular mass and lean body mass by noninvasive nuclear techniques.

With the nuclear techniques employed in this study, it has been possible to measure total body potassium (TBK), lean body mass (LBM), and body cell mass (BCM). The latter two are derived independently of TBK. LBM is derived from the sum of total body water (TBW), total body protein, and bone mineral ash measurements. The BCM value is, in turn, based on the difference between the LBM (as measured above) and the sum of the extracellular water and extracellular solids. It was demonstrated in 123 normal individuals that, although the TBK/LBM ratio decreases with age, the TBK/BCM ratio does not change significantly with age. It can be deduced that the ratio of TBK to intracellular water should be relatively constant with age. This relative constancy with age was demonstrated independently with the absolute measurements of TBK and intracellular water in normal individuals. Because the BCM is physiologically and chemically more homogeneous than LBM and because it reflects the actively metabolizing cellular compartment more accurately than LBM, it is the preferred parameter to be used for reference or normalization in body composition studies. For most applications, BCM is readily derived from TBK measurement by either whole body counting or isotope dilution techniques.

Adult↗

Evaluation of the disinfective efficacy of povidone-iodine with the use of the transparent film dressing OpSite Wound.

To determine the duration of efficacy of disinfection with povidone-iodine (PVP-I) conducted before application of OpSite Wound (Smith and Nephew, UK), bacteriological evaluation was carried out. In 9 healthy adults, 3 sites on the forearms or other parts were chosen for application of OpSite Wound after adequate disinfection with PVP-I. As compared to the bacterial count score of 1.67-2.00 before disinfection, a significant reduction of the score was observed on removal of OpSite Wound on days 1, 2 and 3. The score of 0.5-0.67 immediately after disinfection did not change or deteriorate until removal of OpSite Wound. This finding supports the fact that unnecessary dressing changes could be avoided postoperatively for at least 3 days by disinfection with PVP-I and application of OpSite Wound particularly for operations categorized as 'clean surgery'.

Adult↗

Comparisons of four methods of estimating physical activity in adult women.

PURPOSE: To compare four different methods of measuring physical activity (PA) in adult women under free-living conditions. METHODS: Twelve women participated in a 7-d period during which PA was assessed via self-report, accelerometry and step-counting. Subjects wore at the waist a Tritrac-R3D accelerometer, a Computer Science Application Inc. activity monitor (CSA), both of which measure bodily accelerations in various planes, and a Yamax Digi-Walker-500 that records steps. After the 7-d period subjects responded to a 7-d Physical Activity Recall interview (PAR). RESULTS: Physical activity-related energy expenditure (PAEE) determined from PAR was significantly higher than PAEE estimated from either the Tritrac, CSA, or the Yamax data. Time spent in light, moderate, and hard physical activity was not significantly different between PAR, CSA, and Tritrac. Log-transformed activity counts from CSA were highly correlated with log-transformed Tritrac results (r > 0.90). Number of steps recorded by the Yamax was significantly correlated with untransformed Tritrac and CSA results. CONCLUSIONS: Based upon comparisons with PAR, Tritrac, CSA, and Yamax underestimate the amount of PAEE by 25, 46, and 48%, respectively. The correspondence between the Tritrac and CSA results suggests that both devices produce similar estimates of bodily movement in free-living women. The relationships among results for Tritrac, CSA, and Yamax indicate that the number of steps recorded by the Yamax is representative of the amount of physical activity performed during the day as estimated by Tritrac and CSA. These results should be useful when selecting a method to measure PA in individuals under free-living conditions.

Activities of Daily Living↗

Amount of peripheral reticulocytes as biologic dosimetry of ionizing radiation. Experiments in the rabbit.

The amount of reticulocytes in peripheral blood generally reflect the bone-marrow status of erythropoiesis and may therefore be useful in evaluating radiation injury. The blood reticulocytes in rabbits exposed to various doses of ionizing radiation were examined using an apparatus capable of microfluorometric quantification of reticulocytes as well as the conventional technique of reticulocyte count. A dose-dependent decrease of reticulocytes was observed. The possibilities of further improvements of the technique using flow cytometry and its application for screening human population are discussed.

Accidents↗

Therapeutic and protective efficacy of doramectin injectable against gastrointestinal nematodes in cattle in New Zealand: a comparison with moxidectin and ivermectin pour-on formulations.

Two similar studies were conducted in New Zealand to compare the therapeutic and persistent activity of doramectin injectable, moxidectin pour-on, ivermectin pour-on and oxfendazole oral drench when administered to nematode-infected cattle which were then grazed on common pastures. On day 0 (treatment day), 40 cattle were weighed, faecal sampled and allocated on the basis of day--3 faecal egg counts (FEC) to four treatment groups. Cattle were then treated with either doramectin by subcutaneous (s.c.) injection, moxidectin and ivermectin by topical application, or oxfendazole orally using label-recommended dosages. Oxfendazole treatment served primarily as a control to monitor reinfection without persistent activity. Faecal samples for nematode egg counts and coprocultures for larval differentiation were collected six times between day 0 and day 56 and all cattle were reweighed on day 56. Doramectin reduced pretreatment FEC by 99.1% in the first study and by 100% in the second study when assessed at 14 days posttreatment. Corresponding reductions for moxidectin were 80.8% and 85.2%, for ivermectin 86.0% and 80% and oxfendazole 78.3% and 100%, respectively. Posttreatment rise in FEC indicated that reinfection of moxidectin-treated animals occurred at the same time as oxfendazole controls in both trials. Posttreatment rise in FEC with ivermectin pour-on was similar to moxidectin and oxfendazole in one study, but in the other study ivermectin pour-on delayed the rise by 14-21 days. The rise in FEC for doramectin was delayed for 14-21 days in one study and at least 21 days in the other. The better parasite control provided by doramectin resulted in greater weight gains for cattle over the 56-day period as compared to moxidectin pour-on, ivermectin pour-on and oxfendazole in both trials. Gains of doramectin treated cattle were significantly (p < 0.05) greater than those of ivermectin and moxidectin groups in one trial and the oxfendazole group only in the other.

Administration, Oral↗

Descriptors you can count on? Normalized and filtered pharmacophore descriptors for virtual screening.

The three-dimensional (3D) binary pharmacophore fingerprints find wide application as descriptors in applications ranging from virtual screening through library design. While the 3D content they capture is an intuitively attractive feature of such measures, maximizing their signal to noise ratio has proven to be a tricky balancing act. This issue surfaces primarily due to the potential of such fingerprints to create an explosion of pharmacophores as molecular complexity and flexibility increases. In this article, we describe a modification to the fingerprint generation process that normalizes pharmacophore occurrence frequency by the conformational ensemble size used to derive the descriptor. By including pharmacophore frequency and conformational count, the importance of a given pharmacophore is weighted by the probability of its existence within a given conformational ensemble, rather than treating each pharmacophore equally. In addition, a number of filters have been added to permit the removal of unwanted pharmacophores from the descriptor set. These filters are based on pharmacophore composition (e.g. permutations made up primarily of lipophilic and/or aromatic centers), and size (pharmacophore perimeter length relative to the largest perimeter length found in the molecule). The highly uneven nature of pharmacophore distributions across the conformational ensemble used to generate them is highlighted, as are enrichment comparisons with their binary fingerprint peers. In addition, the limitations in descriptor comparison validation are high-lighted as an illustration of the need for more extensive validation experiments.

Drug Design↗