Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing.
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Base Sequence↗
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Results for “data reproducibility”
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Improving read lengths by recomputing the matrices of Model 377 DNA Sequencers.
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Algorithms↗
Applications of single-molecule detection to the analysis of pathogenic DNA.
We have devised a new technique based on single fluorescent molecule detection for the analysis of specific sequences of DNA. The method consists of synthesizing a fluorescent reporter molecule using a polymerase extension reaction and labeled nucleotides. The fluorescent reporter products are analyzed in a laser-based single-molecule detection system. We have applied this method to the detection of pUC19 and Bacillus anthracis DNA targets. We expect that this method will have applications in rapid detection and identification of DNA from pathogens as well as other sources, and that it will be used for processing of large number of samples in a short period of time.
Bacillus anthracis↗
Simian virus 40 detection in human mesothelioma: reliability and significance of the available molecular evidence.
Simian virus 40 was discovered as a contaminant of early poliovirus vaccines that were inadvertently administered to millions of people in Europe and the United States between 1955 and 1963. Although SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro, its role in human cancer could not be proven epidemiologically. The matter was forgotten until 1993 when SV40 was accidentally found to cause mesotheliomas in hamsters injected intra-cardially. Subsequently, DNA sequences associated with its powerful oncogenic principal, the large T antigen, were found with high frequency in human pleural mesothelioma using the polymerase chain reaction (PCR). Since then many laboratories have confirmed the human findings. However, a few laboratories have failed to reproduce these data and the authors of the studies have claimed that the detection of SV40 DNA may simply represent PCR contamination artefacts. The controversy raised by this viewpoint is reviewed in this article together with a critical appraisal of the reliability of the molecular techniques used to detect SV40 DNA, in order to evaluate the potential aetiopathogenic role of SV40 in human mesothelioma.
Animals↗
Assessment of immunobiological effects induced by chemicals, drugs or food additives. I. Tier testing and screening approach.
The indications of immunologic dysfunction following low level exposure of rodents or even man to certain chemicals and drugs have raised concern regarding methodology and approaches for routine assessment of immunobiological effects. The immunogiological effect observed may either indicate impaired immunopotentiation or hypersensitization. The assays selected for testing should be relevant to the human experience and adaptable to certain practical considerations such as cost, reproducibility of data, ease of performance and application to routine toxicology studies. Using these considerations, a tier approach was proposed consisting of assays for screening for immunologic effects (Tier I) and assays to help define the mechanisms responsible for the immunobiological effects observed (Tier II). A tier of assays was also proposed for measuring the sensitization potential of certain compounds. Certain assays from the screening tier were assembled into a hypothetical and practical test battery to screen for immunological effects of a chemical with potential immunosuppressive properties. Information provided by this test battery should provide a reasonable and sensitive data base from which a standard of evaluation could be made regarding the safety of the test (1) compound.
Animals↗
The use of implanted closed-tip catheters for the introduction of thermometry probes during local hyperthermia treatment series.
In the past two and a half years custom-made closed-tip catheters have been used to allow extensive temperature monitoring with multi-junction thermocouple probes during local hyperthermia. Data from animal experiments indicate that the use of thermometry probes within such a catheter provides reliable temperature measurements. In order to reduce stress to the patient the catheters were fixed so that they could stay in place during the total treatment series. The catheters, outer diameter 1.22 mm, were introduced through hollow needles under sterile conditions before the first hyperthermia session, and fixed using Histo-acryl (tissue adhesive) and Tegaderm (transparent adhesive). Recently, the data concerning the first 180 catheters placed in 74 treatment areas were evaluated. In one patient, an abscess developed 10 days after removal of the (3) catheters, which required surgical intervention, but it was not clear whether these problems had developed as a result of catheter placement. There were no problems with 139 of the remaining 177 catheters (79 per cent) and in 50 treatment fields (68 per cent), respectively, for a duration of 9-55 (mean 23) days. Ten catheters (6 per cent) were lost during the treatment series after 1-22 (mean 10) days. With 28 catheters (16 per cent) infection was observed, 14-27 (mean 19) days after insertion, which necessitated catheter removal before the last treatment session in 11 cases (6 per cent). The complication rate decreased with increasing experience; from 29 per cent (25/85) in the early period to 14 per cent (13/92) in the later period. We conclude that the use of closed-tip catheters under proper control generally causes few, if any, problems. This results in the acquisition of a substantial amount of reliable temperature data at reproducible sites, and it is well tolerated by the patient.
Adenocarcinoma↗
Technical note: an improved McCutchen miniviscometer for connective tissue fluids.
McCutchen's miniviscometer is a Couette-type instrument that requires only 0.2 ml of synovial or other test fluid. The modification described here adds to the capabilities of the original: control of temperature good to +/- 0.2 degrees C; increase of the maximum measureable viscosity at low shear rates (20 sec-1) to 3000 centipoise; and expansion of the usable range of shear rates to (20--1500 sec-1. The device is rugged and data are reproducible to +/- 1.2% (S.D.) of the mean over the full range of shear rates.
Body Fluids↗
Objective evaluation of two markers of HIV-1 infection (p24 antigen concentration and CD4+ cell counts) by a self organizing neural network.
The aim of the present work is to obtain groups of patients with similar profiles of p24 antigen concentration and of CD4+ cell counts. These two markers were chosen because their evaluation represents a significant step in the clinical follow up of HIV-1 infected subjects. The detection methods for p24 antigen concentration and for CD4+ cell counts are well assessed and guarantee easy reproducibility of data obtained in different laboratories. A set of observations with the same time intervals were derived from a continuous function obtained for each patient by a back-propagation neural net trained on the raw data from the patient. The classifications were obtained by a Kohonen neural net trained in three ways: with p24 antigen profiles only, with CD4+ cell count profiles only and with both sets of profiles. The results show that the clustering fashion of the two parameters closely resembles the clustering fashion of CD4+ only, rather than that of p24Ag, both with reference to cluster formation and with reference to distances between clusters.
Acquired Immunodeficiency Syndrome↗
Regulatory T cells overexpress a subset of Th2 gene transcripts.
There is now compelling evidence for subpopulations of CD4+ T cells whose role is to prevent immune pathology in both autoimmunity and transplantation. We have cloned CD4+ T cells against a male transplantation Ag that, unlike Th1 or Th2 clones, suppresses the rejection of male skin grafts and are therefore considered examples of regulatory T cells. We have identified, using serial analysis of gene expression, transcripts that are overexpressed in regulatory T cells compared with Th1 and Th2 clones. Some of these transcripts are increased in tolerated rather than rejecting skin grafts and in addition are expressed by the natural regulatory CD4+CD25+ subpopulation of naive mice. These genes include prepro-enkephalin, GM2 ganglioside activator protein, glucocorticoid-induced TNFR superfamily member 18, and integrin alpha(E)beta(7). They seem to represent a subset of transcripts shared with Th2 cells, suggesting that transplantation tolerance and normal immunoregulation may represent a unique form of Th2-like differentiation.
Adoptive Transfer↗
Bacterial artificial chromosome libraries for mouse sequencing and functional analysis.
Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.
Animals↗
Database search based on Bayesian alignment.
The size of protein sequence database is getting larger each day. One common challenge is to predict protein structures or functions of the sequences in databases. It is easy when a sequence shares direct similarity to a well-characterized protein. If there is no direct similarity, we have to rely on a third sequence or a model as intermediate to link two proteins together. We developed a new model based method, called Bayesian search, as a means to connect two distantly related proteins. We compared this Bayesian search model with pairwise and multiple sequence comparison methods on structural databases using structural similarity as the criteria for relationship. The results show that the Bayesian search can link more distantly related sequence pairs than other methods, collectively and consistently over large protein families. If each query made one error on average against SCOP database PDB40D-B, Bayesian search found 36.5% of related pairs, PSI-Blast found 32.6%, and Smith-Waterman method found 25%. Examples are presented to show that the alignments predicted by the Bayesian search agree well with structural alignments. Also false positives found by Bayesian search at low cutoff values are analyzed.
Algorithms↗
[Tridimensional skin models recording percutaneous absorption]
Today there is a lack of generally accepted alternatives to animal experiments for the evaluation of percutaneous absorption or skin penetration. To evaluate the potential of commercially available reconstructed epidermis we compared the barrier function of the three dimensional human skin equivalents Episkintrade mark, EpiDermtrade mark, and Skinethictrade mark to those of excised human skin. Moreover the batch-to-batch variation of skin equivalents was determined. Methods for evaluation included the determination of transepidermal water loss (TEWL) and of the prednisolone (PD) penetration. Whereas TEWL values of Skinethictrade mark and EpiDermtrade mark were very close (15 g·m-2·h-1), PD-penetration showed a clearly superior barrier function with the Skinethictrade mark model. Drug penetration was only 50% of the EpiDermtrade mark model and PD concentrations in the acceptor medium amounted to 150 ng/ml as compared to 300 ng/ml. The highest values were obtained with the Episkintrade mark model (20 g·m-2·h-1, 500 ng/ml). Therefore this model has the poorest barrier function. The TEWL value for human skin was 9 g·m-2·h-1 and PD concentrations in the acceptor medium were below the limit of detection (10.1 ng/ml). Because of the superior barrier function of the Skinethictrade mark the following evaluation was only performed using this model. Batch-to-batch variation was less than with human skin (interindividual variation of 2 h TEWL 12% and > 19%). Despite of the slightly less well-developed barrier, this skin model clearly appears superior with respect to the reproducibility of data. Therefore future investigations on the potential of human skin equivalents for the determination of percutaneous absorption appear very promising.
Journal Article↗
Variable CYP2A6-mediated nicotine metabolism alters smoking behavior and risk.
Nicotine is the psychoactive substance responsible for tobacco dependence; smokers adjust their cigarette consumption to maintain brain nicotine levels. In humans, 70 to 80% of nicotine is metabolized to the inactive metabolite cotinine by the enzyme CYP2A6. CYP2A6 can also activate tobacco smoke procarcinogens [e.g., NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. In initial studies we found that there was an under-representation of individuals carrying defective CYP2A6 alleles in a tobacco-dependent population, and that among smokers, those with deficient nicotine metabolism smoked fewer cigarettes. We have since reproduced this data in a prospective smoking study (400 male and female, heavy and light smokers) examining the role of the CYP2A6 genotype on carbon monoxide levels, plasma and urine nicotine and cotinine levels, and cigarette counts. We have also recently identified deletion and duplication variants in the CYP2A6 gene locus and have examined their impact on smoking. These data provide the impetus to examine how inhibition of CYP2A6 activity might be useful in a therapeutic context. Both kinetic and behavioral experiments in human smokers demonstrated that inhibiting CYP2A6 in vivo decreased nicotine metabolism and smoking behavior. This article summarizes the preliminary results from our studies.
Aryl Hydrocarbon Hydroxylases↗
[Assessment of car driving skills in brain injured individuals].
Independent driving is considered as very important for a full independence and social integration of disabled people. In these patient with disabilities or handicaps, the driving skills must be carefully assessed, especially in presence of a moderate dysexecutive syndrome. A precise neurological and neuropsychological examination is usually necessary to evaluate aptitudes for driving. Standard neuropsychological tests are often limited to value the brain injury patients. At the moment, assessment on driving simulators really improves the opinion of medical experts, as they give qualitative and quantitative data on reproducible conditions. In fact, in more than 80% of cases, the tests on driving simulators allow to determine if the patient is able to drive or no, with or without a specially adapted vehicle. However, in a small percentage of cases, the evaluation of open-road driving by a driving instructor is necessary, proving that driving simulators will never take over from reality.
Automobile Driver Examination↗
[Sentinel lymph node for breast cancer: remove less to know more].
BACKGROUND: Our aim was to study the value of sentinel lymph node (SLN) biopsy in patients with breast cancer seen at a community hospital. METHODS: Consecutive cases receiving primary treatment for unicentric breast cancer less than 3 cm in diameter were prospectively studied from January 1999 to July 2000. All patients signed a detailed informed consent. The majority of patients (89%) underwent a combined technique of intradermal injection of 0.3-1.2 mCi of (99)Tc and 1-3 cc of Patent Blue at the biopsy site. Intraoperative localization was performed with a hand-held gamma probe. The first 15 patients underwent routine back-up lymphadenectomy. Thereafter, only patients with positive SLN, suspicious findings, or personal preference underwent formal axillary dissection. RESULTS: One hundred eight cases were included in the study with a median age of 61 years and a median diameter of the breast tumor of 1.5 cm. Success rate for identification of SLN was 94% (101/108 cases). A total of 917 additional lymph nodes were removed after SLN biopsy (median 6.5 lymph nodes/patient). Correlation between SLN and the final axillary status was 98%. In 20/36 patients (61%) with positive axillary status the sentinel lymph node was the only positive one. Ten patients had only microscopic foci of cancer found in the SLN. Sixty-seven patients (62%) could have avoided axillary dissection becouse the SLN was found, it was negative, and there were no other intraoperative suspicious findings. CONCLUSIONS: SLN biopsy is accurate and easily reproduced. Our data confirms that the majority of breast cancer patients may no longer need routine axillary lymphadenectomy.
Adult↗
[HPLC determination of six flavonoid constituents in Ginkgo biloba leaves].
Six flavonoid constituents (quercetin, isorhamnetin, kaempferol, bilobetin, ginkgetin and sciadopitysin) were isolated from Ginkgo biloba leaves and determined by reversed phase HPLC using salvianolic acid B as internal standard. The column employed was Zorbax ODS (150 mm x 4 mm ID, 5 microns). The mobile phase consisted of solvent A (methanol) and solvent B [tetrahydrofuran--water--formic acid (34:65:1)] for gradient elution. The flow rate was 1 ml.min-1 and detection was effected at 350 nm. This method is accurate, rapid and reproducible. Analytical data for various samples were given.
Biflavonoids↗
Contrast-enhanced three-dimensional transcranial color-coded sonography of intracranial stenoses.
BACKGROUND AND PURPOSE: Intracranial stenoses are associated with a considerable number of strokes each year. The clinical value of a workstation-based three-dimensional (3D) reconstruction system for transcranial color-coded sonography was evaluated in patients with intracranial stenosis or occlusion. METHODS: Twenty-six patients (13 men, 13 women; mean age, 57 years +/- 12 [SD]) with 36 intracranial stenoses or occlusions, as detected at two-dimensional (2D) color Doppler imaging (CDI) and digital subtraction angiography (DSA), underwent Levovist-enhanced power Doppler imaging (PDI), with subsequent 3D reconstruction. A workstation connected to a magnetic sensor capable of spatial localization of the probe was used to reconstruct 3D images of the circle of Willis from serial PDI images. RESULTS: At DSA, seven (19%) stenoses were estimated to less than 50%, 24 (67%) were 50% or more, and five (14%) were occluded. DSA and 3D-PDI estimates of the degree of stenosis agreed in 33 cases (92%), with a weighted kappa value of 0.86. Disagreement occurred with two subtotal basilar artery stenoses and one subtotal middle cerebral artery stenosis, which were evaluated as being complete occlusions at 3D-PDI. Interobserver agreement between two experienced 3D investigators in estimating the percentage of stenosis was high (correlation coefficient,.98). CONCLUSION: 3D-PDI enables the investigator to reconstruct virtually any arbitrary viewing angle. Compared with conventional CDI, 3D-PDI offers easier spatial assessment of intracranial stenoses, and its findings are sufficiently correlated with angiographic findings. Because different investigators can postprocess the same 3D data, improving reproducibility and reducing investigator dependency in transcranial color-coded sonography may be possible.
Aged↗
[In vivo measurements of shoulder movements by a goniometer with six degrees of freedom: preliminary study].
PURPOSE OF THE STUDY: The purpose of this work was to develop a goniometer with six degrees of freedom (6DOF) to achieve global measurement of the main movements of the shoulder and to examine the reproducibility of the measurements obtained. MATERIAL AND METHODS: Movement amplitudes, inter- and intra-observer reproducibility of the methodology and the device were tested in a population of ten young subjects with asymptomatic shoulders. RESULTS: Maximal amplitudes obtained for each movement were: abduction 111.8 +/- 11 degrees; flexion 127.9 +/- 12.7; external rotation 86.3 +/- 14 degrees; free elevation 129.3 +/- 9 degrees. The curves traced in the plane of rotation and rotation were plotted against elevation. Comparison between amplitudes on the right and left were not significantly different. DISCUSSION AND CONCLUSION: This preliminary study demonstrated the potential contribution of a 6DOF-goniometer for a 3D assessment of shoulder movement. Measurements made when positioning the goniometer demonstrated good reproducibility. Our data are similar to others reported in the literature. The plane of the scapula and the position in physiological internal rotation, as described in the literature, were also observed. Studying shoulder movements, particularly their association, should provide a new means of assessing function in patients with complex disease of the shoulder joint. The device should be helpful in obtaining an objective measurement of the shoulder kinetics that would be useful for patient follow-up and assessment of corporal damage.
Adult↗