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Selected AbstractsMulticenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2007Stuart H Ralston MD Abstract Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated. Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR,based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB. Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis. Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV. Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR,based techniques. [source] Curiosity and cure: Translational research strategies for neural repair-mediated rehabilitationDEVELOPMENTAL NEUROBIOLOGY, Issue 9 2007Bruce H. Dobkin Abstract Clinicians who seek interventions for neural repair in patients with paralysis and other impairments may extrapolate the results of cell culture and rodent experiments into the framework of a preclinical study. These experiments, however, must be interpreted within the context of the model and the highly constrained hypothesis and manipulation being tested. Rodent models of repair for stroke and spinal cord injury offer examples of potential pitfalls in the interpretation of results from developmental gene activation, transgenic mice, endogeneous neurogenesis, cellular transplantation, axon regeneration and remyelination, dendritic proliferation, activity-dependent adaptations, skills learning, and behavioral testing. Preclinical experiments that inform the design of human trials ideally include a lesion of etiology, volume and location that reflects the human disease; examine changes induced by injury and by repair procedures both near and remote from the lesion; distinguish between reactive molecular and histologic changes versus changes critical to repair cascades; employ explicit training paradigms for the reacquisition of testable skills; correlate morphologic and physiologic measures of repair with behavioral measures of task reacquisition; reproduce key results in more than one laboratory, in different strains or species of rodent, and in a larger mammal; and generalize the results across several disease models, such as axonal regeneration in a stroke and spinal cord injury platform. Collaborations between basic and clinical scientists in the development of translational animal models of injury and repair can propel experiments for ethical bench-to-bedside therapies to augment the rehabilitation of disabled patients. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Multicenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2007Stuart H Ralston MD Abstract Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated. Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR,based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB. Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis. Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV. Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR,based techniques. [source] Effects of assay conditions in life history experiments with Drosophila melanogasterJOURNAL OF EVOLUTIONARY BIOLOGY, Issue 2 2001M. Ackermann Selection experiments with Drosophila have revealed constraints on the simultaneous evolution of life history traits. However, the responses to selection reported by different research groups have not been consistent. Two possible reasons for these inconsistencies are (i) that different groups used different environments for their experiments and (ii) that the selection environments were not identical to the assay environments in which the life history traits were measured. We tested for the effect of the assay environment in life history experiments by measuring a set of Drosophila selection lines in laboratories working on life history evolution with Drosophila in Basel, Groningen, Irvine and London. The lines measured came from selection experiments from each of these laboratories. In each assay environment, we measured fecundity, longevity, development time and body size. The results show that fecundity measurements were particularly sensitive to the assay environment. Differences between assay and selection environment in the same laboratory or differences between assay environments between laboratories could have contributed to the differences in the published results. The other traits measured were less sensitive to the assay environment. However, for all traits there were cases where the measurements in one laboratory suggested that selection had an effect on the trait, whereas in other laboratories no such conclusion would have been drawn. Moreover, we provide good evidence for local adaptation in early fecundity for lines from two laboratories. [source] Testing high SPF sunscreens: a demonstration of the accuracy and reproducibility of the results of testing high SPF formulations by two methods and at different testing sitesPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 4 2002Patricia Poh Agin Background/Purpose: The goals of this study were (i) to demonstrate that existing and widely used sun protection factor (SPF) test methodologies can produce accurate and reproducible results for high SPF formulations and (ii) to provide data on the number of test-subjects needed, the variability of the data, and the appropriate exposure increments needed for testing high SPF formulations. Methods: Three high SPF formulations were tested, according to the Food and Drug Administration's (FDA) 1993 tentative final monograph (TFM) ,very water resistant' test method and/or the 1978 proposed monograph ,waterproof' test method, within one laboratory. A fourth high SPF formulation was tested at four independent SPF testing laboratories, using the 1978 waterproof SPF test method. All laboratories utilized xenon arc solar simulators. Results: The data illustrate that the testing conducted within one laboratory, following either the 1978 proposed or the 1993 TFM SPF test method, was able to reproducibly determine the SPFs of the formulations tested, using either the statistical analysis method in the proposed monograph or the statistical method described in the TFM. When one formulation was tested at four different laboratories, the anticipated variation in the data owing to the equipment and other operational differences was minimized through the use of the statistical method described in the 1993 monograph. Conclusions: The data illustrate that either the 1978 proposed monograph SPF test method or the 1993 TFM SPF test method can provide accurate and reproducible results for high SPF formulations. Further, these results can be achieved with panels of 20,25 subjects with an acceptable level of variability. Utilization of the statistical controls from the 1993 sunscreen monograph can help to minimize lab-to-lab variability for well-formulated products. [source] 019 The COLIPA Standard for solar simulators failed to standardize sunscreen SPFSPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 2 2002R.M. Sayre The COLIPA Standard for solar simulators was developed to insure that SPF tested in different laboratories was not different because of the solar simulator used. Indeed for products with lower SPFs 2-10, the solar simulator standard reasonably assures similar SPFs when tested in different laboratories. For products with SPFs greater than 15, the SPF for the same product could be tested at 15 in one laboratory but as an SPF 100 in another. Differences in SPF due to solar simulator filtration will occur only for sunscreen products that exhibit absorption like cut-off filters. Products which generally absorb all UV wavelengths equally will not exhibit differences in SPF due to solar simulator filtration. In addition because of different amounts of UVA allowed within the COLIPA standard, the actual response for a given exposure may in one laboratory produce persistent pigment darkening and in another a simple sunburn. Ways to correct this flaw will be examined. [source] Process-related risk of beryllium sensitization and disease in a copper,beryllium alloy facility,AMERICAN JOURNAL OF INDUSTRIAL MEDICINE, Issue 3 2005Christine R. Schuler PhD Abstract Background Chronic beryllium disease (CBD), which primarily affects the lungs, occurs in sensitized beryllium-exposed individuals. At a copper,beryllium alloy strip and wire finishing facility we performed a cross-sectional survey to examine prevalences of beryllium sensitization and CBD, and relationships between sensitization and CBD and work areas/processes. Methods Current employees (185) were offered beryllium lymphocyte proliferation testing (BeLPT) for sensitization, clinical evaluation for CBD (if sensitized), and questionnaires. We obtained historical airborne beryllium measurements. Results Participation was 83%. Prevalences of sensitization and CBD were 7% (10/153) and 4% (6/153), respectively; this included employees with abnormal BeLPTs from two laboratories, four diagnosed with CBD during the survey, and one each diagnosed preceding and following the survey. Potential BeLPT laboratory problems were noted; one laboratory was twice as likely to have reported an abnormal result (P,<,0.05, all tests), and five times as likely to have reported a borderline or uninterpretable result (P,<,0.05, first blood draw and all tests). CBD risk was highest in rod and wire production (P,<,0.05), where air levels were highest. Conclusions Sensitization and CBD were associated with an area in which beryllium air levels exceeded 0.2 ,g/m3, and not with areas where this level was rarely exceeded. Employees at this copper,beryllium alloy facility had similar prevalences of sensitization and CBD as workers at facilities with higher beryllium air levels. Am. J. Ind. Med. 47:195,205, 2005. Published 2005 Wiley-Liss, Inc. [source] The human plasma proteome: Analysis of Chinese serum using shotgun strategyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Ping He Abstract We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20,healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1),removal of six high-abundant proteins; (2),tryptic digestion of low- and high-abundant proteins of serum; (3),separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944,nonredundant proteins were identified under a stringent filter condition (Xcorr,,,1.9, ,2.2, and ,3.75, ,Cn,,,0.1, and Rsp,,,4.0) in both pooled male and female samples, in which 594 and 622,entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206,proteins were identified with at least two distinct peptides per protein and 185,proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement,C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery. [source] Inter-laboratory comparison of radiometric culture for Mycobacterium avium subsp. paratuberculosis using raw milk from known infected herds and individual dairy cattle in VictoriaAUSTRALIAN VETERINARY JOURNAL, Issue 7 2010SE Ridge Objective To compare the results of radiometric culture conducted in three Australian laboratories for Mycobacterium avium subsp. paratuberculosis (Mptb) using bulk vat and individual animal milk samples. Procedure Milk samples were collected from 15 cows exhibiting clinical signs of Johne's disease, and subsequently confirmed as infected with Mptb, and from the bulk milk vats on 91 farms running herds known to be infected with Mptb. Each milk sample was divided into three equivalent samples and one of each of the replicates was forwarded to the three participating laboratories. The identity and nature of the samples was protected from the study collaborators. The laboratories processed the samples and undertook radiometric culture for Mptb using their standard method. Results of testing were provided to the principal investigator for collation and analysis. Results In total, 2 (2.2%) of 91 vat-milk samples and 8 (53.3%) of 15 individual cows' milk samples returned positive radiometric milk culture results. Only one sample, from a clinical case of Johne's disease, was identified as positive by more than one laboratory. There were differences in the absolute frequency with which Mptb was identified in the milk samples by the collaborating laboratories. Conclusions Mptb was cultured from a very small percentage of Australian raw bulk milk samples sourced from known infected herds. By contrast, Mptb was successfully cultured from half of the milk samples collected from clinically affected cows. There was no statistical difference between laboratories in the proportion of vat samples or individual animal milk samples in which Mptb was detected. 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