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Selected AbstractsQuantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassaysJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2008T. E. WARKENTIN Summary.,Background:,Many laboratories test for heparin-induced thrombocytopenia (HIT) using a PF4-dependent enzyme-immunoassay (EIA). An advantage of the EIA is its simplicity; a disadvantage is that it only indirectly detects heparin-dependent, platelet-activating antibodies (,HIT antibodies'). Objectives:,To determine whether the magnitude of a positive EIA result, expressed in optical density (OD) units, predicts risk of HIT antibodies, defined as a strong-positive platelet serotonin-release assay (SRA) result (,50% serotonin release). Patients/methods:,We determined the risk of a strong-positive SRA result for five categories of OD reactivity (<0.40, 0.40,<1.00, 1.00,<1.40, 1.40,<2.00, and ,2.00 OD units) using two EIAs (commercial anti-PF4/polyanion IgG/A/M and in-house anti-PF4/heparin,IgG). Results:,For patient sera investigated for HIT antibodies, a weak-positive result (0.40,<1.00 OD units) in either EIA indicated a low probability (,5%) of a strong-positive SRA; the risk increased to ,90% with an OD , 2.00 units. Quantifying the EIA,SRA relationship for 1553 referred patient sera, we found that for every increase of 0.50 OD units in the EIA,IgG, the risk of a strong-positive SRA result increased by OR = 6.39 [95% confidence interval (CI), 5.13, 7.95; P < 0.0001]. For every increase of 1.00 OD units in the EIA,IgG, the risk increased by OR = 40.81 (95% CI, 26.35, 63.20; P < 0.0001). Conclusions:,The probability of HIT antibodies (strong-positive SRA result) inferred by a positive PF4-dependent EIA varies considerably in relation to the magnitude of the EIA result, expressed as OD values. In our laboratory, the probability of HIT antibodies being present reached ,50% only when the OD level was ,1.40 units. [source] Mechanisms of morphogen movementDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2005Maura Strigini Abstract Morphogens are defined as signaling molecules that are produced locally, yet act directly at a distance to pattern the surrounding field of cells in a concentration-dependent manner. In recent years many laboratories have devoted their attention to how morphogens actually reach distant cells. Several models have been proposed, including diffusion in the extracellular space and planar transcytosis. A combination of genetic, developmental, and cell-biological approaches have been taken to tackle this issue. I will present the models and discuss the types of experiments that have been designed to test them. It stands out that most of the work has been carried out in Drosophila. Morphogens contribute to patterning of the vertebrate nervous system, and the same signaling molecules have recently been shown to play important, possibly instructive, roles in axon guidance. Little, if anything, is known about the movement of morphogens in the context of nervous system development. The long-standing tradition of biophysical studies on diffusion in the brain extracellular space, along with the sophisticated in vitro culture systems developed in neurobiology laboratories, may provide new tools and ideas to test these models in a new context. © 2005 Wiley Periodicals, Inc. J Neurobiol 64: 324,333, 2005 [source] The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature,,§DIAGNOSTIC CYTOPATHOLOGY, Issue 6 2007Patricia A. Fetsch M.T. (ASCP) Abstract The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2,3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250. Diagn. Cytopathol. 2007;35:319,328. © 2007 Wiley-Liss, Inc. [source] Diagnostic effects of prolonged storage on fresh effusion samples,,DIAGNOSTIC CYTOPATHOLOGY, Issue 1 2007Frances Manosca M.D. Abstract The effects on morphology and diagnostic interpretation of delayed processing of refrigerated effusion samples have not been well documented. The potential for cellular degeneration has led many laboratories to reflexively fix samples rather than submit fresh/refrigerated samples for cytologic examination. We sought to determine if effusion specimens are suitable for morphologic, immunocytochemical, and DNA-based molecular studies after prolonged periods of refrigerated storage time. Ten fresh effusion specimens were refrigerated at 4°C; aliquots were processed at specific points in time (days 0, 3, 5, 7, 10, 14). Specimens evaluated included four pleural (3 benign, 1 breast adenocarcinoma) and six peritoneal (2 ovarian adenocarcinomas, 1 malignant melanoma, 2 mesotheliomas, 1 atypical mesothelial) effusions. The morphology of the cytologic preparations from the 10 effusions was preserved and interpretable after 14 days of storage at 4°C. The immunocytochemical profile of the samples (AE1/AE3, EMA, calretinin, and LCA) was consistent from day 0 to day 14. Amplifiable DNA was present in all samples tested on day 14. We conclude that cytopathologic interpretation of effusion samples remains reliable with refrigeration at 4°C even if processing is delayed. Diagn. Cytopathol. 2007;35:6,11. © 2006 Wiley-Liss, Inc. [source] Time-based analysis of silver-stained proteins in acrylamide gelsELECTROPHORESIS, Issue 10 2006Bertram Becher Dr. Abstract Silver staining of proteins after PAGE often remains the method of choice in many laboratories. Nevertheless, it is known that quantification of protein levels is keenly restricted to a small range of protein concentrations leading to an over- or underestimation of protein amounts. To overcome this, a time-based analysis method was developed to avoid the saturation effect of the silver-staining reaction, thus resulting in an improved dynamic range of the gel image produced and therefore better quantification of proteins. Instead of the well-known end-point image analysis, gray intensities of time series images of a developing gel are determined and times until a threshold gray value is reached are calculated. These times are used to calculate a new grayscale image which can be analyzed using commercial image processing software. [source] Test method for concrete spalling using small electric furnaceFIRE AND MATERIALS, Issue 4 2010Ren Zhao Abstract Concrete spalling can cause severe damage to concrete structure when exposed to fire. The spalling mechanisms are not very well understood. For the testing of spalling, full-scale structural members should be used, as spalling tests are sensitive to size effects. Full-scale testing in large furnace is costly and is not suitable for testing large number of concrete mixture trials. The standard and hydrocarbon fire time,temperature curves have rapid temperature rise during the initial phase. This temperature rise requires a gas furnace with high heating capacity and cannot be generated by electric muffle furnace commonly available in many laboratories. This paper presents a method to carry out spalling test in small-scale specimens with exposure to rapid temperature rise using a commonly available electric furnace in the laboratories. The tests are based on 150,mm diameter cylinders that are laterally confined to simulate full-scale structural members. The cylinder surface is exposed to rapid temperature rise by exposing through vertical and/or horizontal holes in pre-heated small electric furnace. Some unconfined 100,mm diameter cylinders were also exposed horizontally to test the performance of confinement. The paper shows that the hydrocarbon fire and standard fire exposure can be simulated by manipulating the exposure location of the surface of the concrete cylinder. Ordinary Portland cement concrete cylinders with different strengths were tested and different spalling patterns were observed. The spalling patterns matched the test results from a gas furnace fire test simulating the fire curves. The tests demonstrated that the method is an effective and convenient technique to predict the spalling risk of a concrete. Copyright © 2009 John Wiley & Sons, Ltd. [source] Role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeuticsIMMUNOLOGICAL REVIEWS, Issue 1 2008Marc Feldmann Summary: Advances in cDNA and monoclonal antibody technology in the 1980s fuelled the discovery and characterization of the properties of cytokines. It became apparent that because cytokines were expressed in tissues derived from autoimmune diseases, they were likely to be of fundamental importance in disease pathogenesis and developing a new class of biological therapeutics. In this review, we describe the history of bench to bedside translation of work that led to the identification of tumor necrosis factor (TNF) as a key regulator of the loss of homeostatic immune-inflammatory responses in rheumatoid arthritis (RA) and a good therapeutic target. First in human clinical trials in collaboration with a biotechnology company, the safety and efficacy of TNF blockade with a chimeric monoclonal antibody was substantiated in patients refractory to standard anti-rheumatoid drugs. Abnormal immune-inflammatory responses after therapy showed improvement and remain a focus of ongoing research in many laboratories. Longer term multi-center studies that followed with several anti-TNF biologicals have demonstrated the augmented efficacy, including inducing clinical remission, of low dose methotrexate and anti-TNF therapy co-therapy, but serious infections and lymphomas in a low frequency have been observed. In the course of the past decades, three ,blockbuster' anti-TNF biologicals are in the clinic. Over a million patients with RA and other immune-mediated diseases have been successfully treated, and a better perspective on the risk of harm and its management has become part of good clinical practice. This success has encouraged a burgeoning industry of biologicals for chronic diseases. [source] A simple and low-cost solution for the automation of X-ray powder diffractometers with chart recorder outputJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2006M. Jayaprakasan X-ray powder diffraction is an established method for the qualitative identification of crystalline materials and their quantitative analysis. The new generation of X-ray diffraction systems are based on expensive digital/embedded control technology and computer interfaces. Yet many laboratories use conventional manual-controlled systems with XY strip-chart recorders. Since the output spectrum is a strip chart (hard copy), raw data, essential for structural and qualitative analysis, are not readily available for further analysis. Upgrading to modern computerized diffractometers is very expensive. The proposed automation design described here is intended to enable the conventional diffractometer user to collect, store and analyze data quickly. The design also improves the resolution by five times compared with the conventional setup. For the automation, a PC add-on card has been designed to control and collect the timing and intensity counts from the conventional X-ray diffractometer, and suitable software has been developed to collect, process and present the X-ray diffraction data for both qualitative and quantitative analysis. Moreover, a major advantage of this design is that it does not warrant any physical modification of the hardware of the conventional setup; it is simply an extension to enhance the performance of collecting raw data with a higher resolution at desired intervals/timings. [source] Survey of the year 2005: literature on applications of isothermal titration calorimetryJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2007Adessamad Ababou Abstract Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naïve, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding. Copyright © 2006 John Wiley & Sons, Ltd. [source] Endpoint quantitative PCR assays for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitansJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2003J. D. Rudney Background:, Conventional polymerase chain reaction (PCR) assays for periodontal pathogens are so sensitive that they detect infections of no clinical significance. Quantitative PCR (qPCR) may provide a solution to this problem. However, most qPCR systems require expensive real-time thermal cyclers. Objective:, Our goal was to develop qPCR assays which would allow endpoint quantification. Materials and methods:, 16S rRNA primers for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans were adapted to the AmplifluorÔ qPCR system, which incorporates fluorescein into the PCR product so that endpoint fluorescence is proportional to the original amount of template. DNA dilutions representing known numbers of cells were used as standard curves. Pooled subgingival plaques from the four deepest pockets of 21 severe adult periodontitis patients were assayed. Buccal molar supragingival plaque from 35 dental students provided healthy controls. Endpoint fluorescence was measured with a microplate reader. Results:, Optimized standard curves were linear in log,log or semilog fits over a range of 100,106 cells. Countable B. forsythus was present in all patients, with counts (as logs) from 2.4 to 7.3 (mean = 5.0), and 11 controls with counts from 2.1 to 4.5 (mean = 3.0). P. gingivalis was present in 11 patients and no controls, with counts from 2.2 to 4.7 (mean = 3.2). A. actinomycetemcomitans was present in two patients, with counts of 1.5 and 3.5. Conclusions:, AmplifluorÔ qPCR assays discriminated between plaque samples differing by one log or more, allowing major infections to be distinguished from minor ones. This approach allows high-throughput qPCR of plaque samples, using equipment available to many laboratories. [source] An Integrin and Rho GTPase-Dependent Pinocytic Vacuole Mechanism Controls Capillary Lumen Formation in Collagen and Fibrin MatricesMICROCIRCULATION, Issue 1 2003GEORGE E. DAVIS ABSTRACT A major question that remains unanswered concerning endothelial cell (EC) morphogenesis is how lumens are formed in three-dimensional extracellular matrices (ECMs). Studies from many laboratories have revealed a critical role for an ECM-integrin-cytoskeletal signaling axis during EC morphogenesis. We have discovered a mechanism involving intracellular vacuole formation and coalescence that is required for lumen formation in several in vitro models of morphogenesis. In addition, a series of studies have observed vacuoles in vivo during angiogenic events. These vacuoles form through an integrin-dependent pinocytic mechanism in either collagen or fibrin matrices. In addition, we have shown that the Cdc42 and Rac1 guanosine triphosphatases (GTPases), which control actin and microtubule cytoskeletal networks, are required for vacuole and lumen formation. These GTPases are also known to regulate integrin signaling and are activated after integrin-matrix interactions. Furthermore, the expression of green fluorescent protein-Rac1 or -Cdc42 chimeric proteins in ECs results in the targeting of these fusion proteins to intracellular vacuole membranes during lumen formation. Thus, a matrix-integrin-cytoskeletal signaling axis involving both the Cdc42 and Rac1 GTPases regulates the process of EC lumen formation in three-dimensional collagen or fibrin matrices. [source] The role of cell-specific circadian clocks in metabolism and diseaseOBESITY REVIEWS, Issue 2009M. S. Bray Summary Biological rhythms are an integral component of essentially all aspects of life. These rhythms are controlled in part by circadian clocks, transcriptionally based mechanisms that synchronize the organism to its changing environment. The central circadian clock is located within the suprachiasmatic nucleus of the brain, while peripheral clocks are located within virtually all cells outside of the suprachiasmatic nucleus. Although our understanding of central clock structure and function is well advanced, the role of peripheral clocks in whole body energy metabolism is just beginning to be elucidated. Both central and peripheral circadian clocks likely regulate many physiological functions, including insulin sensitivity, endocrine regulation, energy homeostasis, satiety signalling, cellular proliferation and cardiovascular function. Widely varying phenotypes have been reported following global genetic disruption of the clock mechanism in mice, with phenotype dependent on both the clock component targeted and genetic background. The inconsistency in phenotypes associated with clock disruption may be due, in part, to cell-specific effects of the circadian clocks. To address this question, many laboratories have begun generating animal models of cell type-specific clock disruption. In this review, we summarize the existing literature on tissue-specific models of circadian clock disruption and provide a focus for future research in this area. [source] ,Proteomic Basics , Sample Preparation and Separation': The 1st European Summer School in Kloster Neustift 12,18 August, 2007 Brixen/Bressanone, South Tyrol, ItalyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008Katrin Marcus Dr. Abstract Proteomics is rapidly developing into a routine approach for protein analysis in many laboratories. The series of European-wide Summer Schools ,Proteomics Basics' (http://www.proteomic-basics.eu/) aims at teaching of comprehensive knowledge in proteomics research and applied technologies for master and graduate students and postdocs currently moving into the field of proteomic research. In the next 3,years the series will cover the theoretical basis of the fundamental topics in the various areas of proteomic analysis, i.e. sample preparation and handling, mass spectrometry, post-translational modifications and quantitation given by leading experts in the field. This summer school series embodies a unique advantage in comparison with conventional scientific meetings and university curricula: internationally renowned experts will give a detailed perspective view of the fundamentals of their particular proteome research area, something which is usually not encountered at conferences and congresses. Here, we give a report on the first European Summer School ,Sample Preparation and Handling' within the series ,Proteomic Basics' that was held at the monastery in Neustift close to Bressanone/Brixen, Italy from August 12 to 18, 2007. [source] A semi-automated solid-phase extraction liquid chromatography/tandem mass spectrometry method for the analysis of tetrahydrocannabinol and metabolites in whole blood,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2009Eshwar Jagerdeo Marijuana is one of the most commonly abused illicit substances in the USA, making cannabinoids important to detect in clinical and forensic toxicology laboratories. Historically, cannabinoids in biological fluids have been derivatized and analyzed by gas chromatography/mass spectrometry (GC/MS). There has been a gradual shift in many laboratories towards liquid chromatography/mass spectrometry (LC/MS) for this analysis due to its improved sensitivity and reduced sample preparation compared with GC/MS procedures. This paper reports a validated method for the analysis of ,9 -tetrahydrocannabinol (THC) and its two main metabolites, 11-nor-9-carboxy-,9 -tetrahydrocannabinol (THC-COOH) and 11-hydroxy-,9 -tetrahydrocannabinol (THC-OH), in whole blood samples. The method has also been validated for cannabinol (CBD) and cannabidiol (CDN), two cannabinoids that were shown not to interfere with the method. This method has been successfully applied to samples both from living people and from deceased individuals obtained during autopsy. This method utilizes online solid-phase extraction (SPE) with LC/MS. Pretreatment of samples involves protein precipitation, sample concentration, ultracentrifugation, and reconstitution. The online SPE procedure was developed using Hysphere C8-EC sorbent. A chromatographic gradient with an Xterra MS C18 column was used for the separation. Four multiple-reaction monitoring (MRM) transitions were monitored for each analyte and internal standard. Linearity generally fell between 2 and 200,ng/mL. The limits of detection (LODs) ranged from 0.5 to 3,ng/mL and the limits of quantitation (LOQs) ranged from 2 to 8,ng/mL. The bias and imprecision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and imprecision as <9%, for all components at each quantity control level. Published in 2009 by John Wiley & Sons, Ltd. [source] Putting the colours into chromogenic in situ hybridization (CISH)THE JOURNAL OF PATHOLOGY, Issue 1 2006J Shipley Abstract Recurrent genomic alterations are the hallmarks of particular cancers. Application of molecular cytogenetic technologies to tumour material in order to detect these alterations has become important for molecular diagnostics and research. A dual-colour chromogenic in situ hybridization (dc-CISH) method described recently in the Journal of Pathology has the advantage of visualizing two probes simultaneously with the ability to discern morphological features. In addition, the bright field microscopy required is readily accessible to many laboratories. The approach has been validated by comparison of results with standard analyses for HER-2 amplification status in formalin-fixed, paraffin-embedded breast cancers and is applicable to the analysis of other clinically relevant genomic aberrations as well as of use in research investigations. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Proceedings of the Australian Physiological and Pharmacological Society Symposium: New Frontiers in Muscle Research Gene transfer: manipulating and monitoring function in cells and tissuesCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2001Rekha G Panchal SUMMARY 1. The ectopic expression of genes has proven to be an extremely valuable tool for biologists. The most widely used systems involve electrically or chemically mediated transfer of genes to immortalized cell lines and, at the other end of the spectrum, transgenic animal models. As would be expected, there are compromises to be made when using either of these broad approaches. Immortalized cell lines have limited ,physiological relevance' and transgenic approaches are costly and out of the reach of many laboratories. There is also significant time required for the de novo generation of a transgenic animal. 2. As a viable alternative to these approaches, we describe the use of recombinant adenovirus and Sindbis virus to deliver genes to cells and tissues. 3. We exemplify this approach with studies from our laboratories: (i) an investigation of Ca2+ handling deficits in cardiac myocytes of hypertrophied hearts using infection with recombinant adenovirus encoding either green fluorescent protein (GFP) or the sarcoplasmic/endoplasmic reticulum calcium-ATPase (Serca2a); (ii) a study of the mechanism of macrophage/microglial migration by infection of embryonic phagocytes with a GFP-encoding virus and coculture with brain slices to then track the movement of labelled cells; and (iii) we are also exploiting the natural tropism of the Sindbis virus to label neurons in hippocampal brain slices in culture to resolve high-resolution structure and to map neuronal connectivity. 4. Further development of these approaches should open new avenues of investigation for the study of physiology in a range of cells and tissues. [source] Life before birth: effects of cortisol on future cardiovascular and metabolic function,ACTA PAEDIATRICA, Issue 7 2003PW Nathanielsz The concept of fetal programming is an area that is now under rigorous investigation in many laboratories throughout the world. We need to engender a fascination in all segments of society, not just pregnant women, about life in the womb. Conclusion: Everyone needs to understand that improving the condition of the fetus will have personal, social and economic benefits. The time has come to realize that, in a sense, it is not just women who are pregnant but it is the family and the whole of society. [source] Physiological effects of dominance hierarchies: laboratory artefacts or natural phenomena?JOURNAL OF FISH BIOLOGY, Issue 1 2002K. A. Sloman Studies of fish behaviour have demonstrated the existence of social interactions that result in dominance hierarchies. In environments in which resources, such as food, shelter and mates, are limited, social competition results in some fish becoming dominant and occupying the most profitable positions. This behaviour has been observed in natural environments and also in many laboratory-based experiments. When two fish have been confined in a small tank, one of them usually has exhibited behaviour that suggests it is dominant over the other submissive animal. Physiological consequences of social interaction can be seen in both dominants and subordinates but are more extreme in the subordinate. However, this scenario is without doubt an artificial situation. Fewer experiments have been conducted using laboratory experiments that are more socially and physically complex than those experienced by dyads in tanks. In simple fluvial tanks, through which water is recirculated, the physiological responses of fish to social competition have generally been qualitatively similar to those recorded among dyads. However, when environmental disturbances, complex resource distributions, increase in water flushing, presence of predators and competing species of fish have been included in experimen-tal designs, there have been fewer, diminished or no physiological dierences between dominant and subordinate fish. There have been very few studies of physiology in relation to dominance in natural habitats, and those that have been conducted suggest that under some circumstances hierarchies may cause less intense physiological responses than have been suggested based on results of laboratory studies in simple environments. Possible reasons for these variations are discussed. The need is identified for a well structured experimental approach to the investi-gation of the causes and consequences of hierarchies if the ecology of wild fish is to be modelled eectively based on physiological processes. It is also suggested that the further development and application of techniques for monitoring physiologies of fish in the wild is important. [source] | ||||||||||||||||