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Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction.

We have developed a simple digestion-polymerase chain reaction (PCR) assay for a simultaneous transgene detection and sexing of pronucleus-injected bovine preimplantation embryos. Bovine embryos were microinjected with dam-methylated gene construct and cultured in vitro for 6-7 days after the injections. The developed blastocysts and compact morulae were bisected and the embryonic biopsies representing mainly trophoblasts were subjected to the digestion-PCR, while the biopsied embryos remained in culture. Embryonic DNA was released with proteinase K and the samples were digested with a Dpnl-Bal31 mixture before the PCR amplification of the transgene, bovine alpha S1-casein, and bovine Y-chromosome fragments in the same reaction. The whole assay from biopsy to electrophoresis took less than 6 hr. The digestion removed up to 50 fg of dam-methylated transgene copies (unintegrated or contaminants) and also a few hundred copies of contaminating PCR products from the embryonic samples. The digestion-PCR assay eliminated all transgene contaminations from noninjected blastocysts, which were exposed to the microinjection DNA during the stay in injection chambers, and reduced the amount of transgene-positive embryos among pronucleus-injected blastocysts as compared with unmodified PCR. Analysis of 486 microinjected bovine embryo biopsies in 13 separate experiments revealed that we were able to sex 398 (82%) of the biopsies and 77 (19%) of the biopsies were scored as transgene positive and 57 (14%) as transgene questionable. Upon reanalysis of 41 of the biopsied embryos, 38 (93%) of the embryos were observed to be transgene negative and 2 questionable in both assays and uneven distribution of transgene copies was observed in one embryo. The results from sexing were in accordance with biopsies and remaining embryos in 38 (93%) of the embryos.

Animals↗

Use of an automated chromium reduction system for hydrogen isotope ratio analysis of physiological fluids applied to doubly labeled water analysis.

The doubly labeled water method is commonly used to measure total energy expenditure in free-living subjects. The method, however, requires accurate and precise deuterium abundance determinations, which can be laborious. The aim of this study was to evaluate a fully automated, high-throughput, chromium reduction technique for the measurement of deuterium abundances in physiological fluids. The chromium technique was compared with an off-line zinc bomb reduction technique and also subjected to test-retest analysis. Analysis of international water standards demonstrated that the chromium technique was accurate and had a within-day precision of <1 per thousand. Addition of organic matter to water samples demonstrated that the technique was sensitive to interference at levels between 2 and 5 g l(-1). Physiological samples could be analyzed without this interference, plasma by 10000 Da exclusion filtration, saliva by sedimentation and urine by decolorizing with carbon black. Chromium reduction of urine specimens from doubly labeled water studies indicated no bias relative to zinc reduction with a mean difference in calculated energy expenditure of -0.2 +/- 3.9%. Blinded reanalysis of urine specimens from a second doubly labeled water study demonstrated a test-retest coefficient of variation of 4%. The chromium reduction method was found to be a rapid, accurate and precise method for the analysis of urine specimens from doubly labeled water.

Chromium↗

The value of frequent treatment verification films in reducing localization error in the irradiation of complex fields.

Using treatment verification film to detect geometric miss or localization error (LE), a follow-up analysis of LE for patients treated with extended mantle fields was made for the period 1973-1974. There were 451 treatment verification films for 19 patients reviewed and 67 errors were detected for an error rate of 15%. This rate represented a continued decrease compared to the previous rate of 35% (330 errors for 902 films) during 1969-73. There was a continued reciprocal increase in number of films per patient per course of treatment which was thought, in part, to account for the improvement in technical precision. Reanalysis of localization error by site within the field showed a decrease in rate of error for the axillae from 1969-73 to 1973-1974 and a relative increase in rate of error for the upper abdominal nodes. The decrease in error rate for the axillae was ascribed to increased attention to leaving adequate margin between pulmonary shield and axilla and the increase in error rate for upper abdominal nodes was ascribed to inadequate margin between shield and spine. The relative increase in errors for the upper abdominal nodes correlated with the physicians who made the original decision about margin between spine and shield. Propagation of LE by site has been noted and can be prevented by monitoring treatment fields with verification film and correcting errors as they are noted.

Hodgkin Disease↗

Early deaths in newly diagnosed cases of pediatric acute leukemia: a Southwest Oncology Group Study.

A reanalysis of all ten clinical trials conducted by the Southwest Oncology Group from 1958 thorugh 1976 in newly diagnosed pediatric acute leukemia was carried out to quantify the "early death" rate (i.e., rate of deaths during the first four weeks of remission induction) and to determine factors related to these deaths. Out of 1,964 patients registered, there were 126 (6.4%) early deaths. For the 1,375 acute lymphocytic leukemia (ALL) patients only, there were 75 (5.5%) early deaths. During the last eleven years on this study (1965 through 1976), the death rate for ALL patients appears to have remained stable at five deaths per 100 patients, despite the general improvement in overall survival. The most important prognostic factor was the initial platelet count: patients with a platelet count below 25,000 had a death rate over three times higher than that of patients with a platelet count over 75,000. Infants under one year of age also had a very high early death rate (6 of 41, 15%).

Age Factors↗

Chromosome abnormalities and karyotypic evolution in 83 patients with myelodysplastic syndrome and predictive value for prognosis.

In a chromosome study of 83 patients with myelodysplastic syndrome (MDS), 50 showed a clonally abnormal karyotype. The most frequent abnormalities were the whole or a partial loss of the long arm of chromosome 7 (-7 or 7q-) (14 patients) and a partial loss of the long arm of chromosome 5 (5q-) (11 patients). Twenty patients with 5q- and/or -7 or 7q- had a shorter survival (median, 5 months) than those with other abnormal karyotypes (22 months) or those with a normal karyotype (28 months). In this series 30 patients were examined cytogenetically on two or more occasions during the course of their illness. Ten patients showed a further karyotypic alteration from the initial findings, and, concomitantly, their disease progressed in severity including overt leukemia. These patients had a shorter survival (median, 2 months) after the chromosome reanalysis than the other 20 patients who did not have further karyotypic changes (21 months). Thus, the prognosis of patients with MDS can be predicted more accurately by reanalyzing the chromosomes after the initial analysis.

Adult↗

Sex differences in WAIS item performance.

Although the WAIS IQ scores of males and females differ only negligibly, significant sex differences do exist on many WAIS subtests. The purpose of the present investigation was to identify any WAIS items within these subtests that showed reliable sex differences. Within a cross-validation methodology that employed 521 adults (264 males; 257 females), 21 items were found to show reliable sex differences. A reanalysis by age groups provided similar results. The nature of the items was discussed, and an index of stereotyped sex-role experiences was proposed.

Adult↗

Survival analysis of response to group psychotherapy in bulimia nervosa.

A reanalysis of treatment response and relapse was performed using survival analysis in a 12-week clinical trial of cognitive behavioral group psychotherapy for the treatment of bulimia nervosa. One hundred forty-three (143) bulimic women with high incidence of binge eating, self-induced vomiting, and/or laxative abuse were randomly assigned to one of four possible treatment conditions that consisted of a combination of two factors: (1) emphasis on abstinence (high and low), and (2) treatment intensity (high and low). "Initial" and "maintained" response to treatment based on "total" and "near" abstinence criteria were determined using self-reported binge eating, vomiting, and laxative use data. Results suggest that an emphasis on abstinence appears important in achieving initial abstinence, whereas intensity of treatment may be important in maintaining abstinence.

Adolescent↗

Mass media interventions for promoting HIV testing.

BACKGROUND: Use of the mass media is one of the important strategies in communicating behavioral change in relation to HIV/AIDS prevention. Mass media are used to promote voluntary HIV counseling and testing and to sustain test-seeking behavior. OBJECTIVES: To assess the effect of mass media interventions and the most effective form of mass media intervention at a general population level or in specific target populations, in relation to changes in HIV testing, compared with a control group or with pre-intervention levels. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (The Cochrane Library Issue 2, 2004), MEDLINE (1966 to April 2004), EMBASE (1980 to April 2004), NLM Gateway, CINAHL (1982 to April 2004), AIDSearch (1980 to April 2004), and PsycINFO (1974 to April 2004), Sociological abstracts (1982 to April 2004), and Communication studies (1982 to April 2003). The reference lists of related reviews were searched and experts in the field were contacted to identify ongoing research. Relevant web sites of international agencies (UNAIDS, WHO, UNFPA, World Bank, and Centers for Disease Control and Prevention) were also searched. All these searches were done without language restriction. All databases were searched up to April 2004. SELECTION CRITERIA: Randomized controlled trials, including cluster-randomized trials and controlled clinical trials, that compared either multimedia interventions or one type of media strategy with a control in relation to promotion of HIV testing were included. Interrupted time series analyses that assessed the effect of mass media against no media or an alternative intervention to promote HIV testing were also included. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed trial quality and extracted data. Study authors were contacted for additional information. Types of mass media interventions, participants, and outcomes were extracted in every possible instance. MAIN RESULTS: Of the 35 references that were identified, two randomized controlled trials, three non-randomized controlled studies, and nine interrupted time series were included in the final analysis. All individual studies concluded that mass media were effective, and this was confirmed by reanalysis of the interrupted time series studies which all had initial impact. Mass media interventions for promotion of HIV testing showed significant immediate (Random effect: Estimated mean = 5.487, 95%CI = 2.370 to 8.605) and overall (Random effect: Estimated mean = 6.095, 95%CI = 1.812 to 10.378) effect. No long-term effects were seen on mass media interventions for promotion of HIV testing (Random effect: Estimated mean = 4.447, 95%CI = -0.188 to 9.082). AUTHORS' CONCLUSIONS: Mass media interventions have immediate and overall effects in promotion of HIV testing. No long-term effects were seen. There was no significant impact of detecting seropositive status after mass media intervention for promoting HIV testing, and this finding was limited to a small number of studies. Further research is required to identify possible effects on seropositivity status after mass media intervention for promotion of HIV testing among high-risk groups in epidemic countries. Additional research is needed to identify the effectiveness of different types of mass media interventions, the cost effectiveness of the interventions, and characteristics of messages.

Controlled Clinical Trials as Topic↗

Plastic microchip electrophoresis for genetic screening: the analysis of polymerase chain reactions products of fragile X (CGG)n alleles.

Clinical screening of abnormal chromosomes associated with fragile X syndrome (FXS) demands a high-throughput method including DNA sizing and detection of the amplified products. This study is to explore the use of polymer microchip electrophoresis for the analysis of polymerase chain reaction (PCR) products of fragile X (CGG)n alleles to facilitate a fast exclusion test of FXS. The sequences flanking the CGG-repeat of FMR1 gene was amplified by betaine-PCR and the amplified products were desalted and then analyzed by microchips which were fabricated on poly(methyl methacrylate) (PMMA) substrate. The PCR bands with more than six CGG-repeats in difference could be clearly distinguished in less than 3 min by microchip electrophoresis with a separation length of 6 cm. It was found that the signal was greatly enhanced with the use of both covalent (Cy5) and intercalating dye (TORRO-3), which has never been demonstrated before. We tested the method by reanalysis of twelve samples from males and six samples from females. For female samples with less than six repeat differences, Southern blotting method was performed to confirm or exclude the findings from microchips. It was found that the test results from all male and female samples show a 100% correlation between the microchip electrophoresis and the existing methods.

Electrophoresis, Capillary↗

Comparison of sulfasalazine and placebo for the treatment of axial and peripheral articular manifestations of the seronegative spondylarthropathies: a Department of Veterans Affairs cooperative study.

OBJECTIVE: To determine if the peripheral articular manifestations of the seronegative spondylarthropathies (SNSA) respond differently than the axial manifestations to treatment with sulfasalazine (SSZ). METHODS: This is a reanalysis of a previously reported series of randomized, double-blind, placebo-controlled, multicenter trials comparing the effects of SSZ, 2,000 mg/day, and placebo on the axial and peripheral articular manifestations of ankylosing spondylitis (AS), psoriatic arthritis (PsA), and reactive arthritis (ReA; Reiter's syndrome). Patients were classified as treatment responders on the basis of meeting predefined improvement criteria in 4 outcome measures: namely, patient and physician global assessments in all patients, morning stiffness and back pain in patients with axial manifestations, and joint pain/tenderness scores and joint swelling scores in patients with peripheral articular manifestations. RESULTS: Six hundred nineteen SNSA patients (264 AS, 221 PsA, and 134 ReA) were studied. One hundred eighty-seven of these patients had only axial manifestations of their disease, while 432 patients had peripheral articular manifestations. Of the patients with axial disease, 40.2% of the SSZ group and 43.3% of the placebo group met the predefined response criteria (P = 0.67). Of the peripheral articular group, 59.0% of the SSZ-treated patients and 42.7% of the placebo-treated patients showed a response (P = 0.0007). CONCLUSION: In a large group of affected individuals, the response of SNSA patients to SSZ appears to be related to the articular manifestations of their disease. These data demonstrate that the axial and peripheral articular manifestations of SNSA respond differently to treatment with SSZ. In SNSA patients with persistently active peripheral arthritis, SSZ is safe, well tolerated, and effective.

Adult↗

Self-reported symptoms of neurotoxicity and agricultural injuries among Ohio cash-grain farmers.

BACKGROUND: In 1993, the Ohio Farm Family Health and Hazard Surveillance Program (OFFHHSP) was initiated to assess the health status of cash-grain farmers and their families. The objective of this study was to examine the postulated relationship between symptoms of neurotoxicity and risk of agriculture-related injury among Ohio cash-grain farmers. METHODS: A reanalysis of the data from a previously published case-control study of cash-grain farmers [Crawford et al. (1998) Am J Ind Med 34:588-599] was performed. The Principal Operator (PO) of each farm was asked to complete a self-administered questionnaire which included items about injuries experienced during the past year. A section of the questionnaire contained a 24-item instrument ("Q24") that queried symptoms of neurotoxicity experienced during the previous 12 months. Design-based multiple logistic regression analyses were conducted to evaluate associations between neuropsychological subsets ("domains") of the 24-item instrument and injury risk among the white male POs (who accounted for >99% of all POs). RESULTS: Significantly elevated odds ratios (ORs) were found in 8 of 11 domains. POs answering in the affirmative to the single item "Have you been bothered by lack of coordination or loss of balance?" were 3.12 times more likely to have reported an injury than POs responding negatively (95% CI 1.68-5.81). CONCLUSIONS: Farmers with higher scores on several Q24 domains, and for particular questionnaire items, appear to be at increased risk of agricultural injury.

Accidents, Occupational↗

Genetic and environmental determinants of periodontal disease.

Reanalysis of data on periodontal disease in 241 families [Chung et al, 1977b], based on an extended and more satisfactory path model [Rao et al, 1979], failed to detect significant heritability, and concluded in favor of cultural inheritance only, without maternal effects or intergenerational differences. The most parsimonious hypothesis yields a relative variance component of 0.338 +/- 0.024 due to indexed environment for both children and adults, the remainder (1.0--0.338 = 0.662) being due to residual nongenetic factors.

Culture↗

Pseudomosaicism in prenatal diagnosis: two simultaneous, independent reciprocal translocations.

Cytogenetic evaluation of cultured amniotic fluid cells showed mosaicism of three karyotypes: 46,XY; 46,XY [t(13;17)(q13;q25]; and 46, XY [t(11;12)(p11;q13)]. Reanalysis of cells from the four original culture flasks harvested individually revealed that both translocations derived from the same flask. Repeat amniocentesis, as well as peripheral blood obtained postnatally from a phenotypically normal male infant, demonstrated only normal chromosomes (46,XY). This observation represents a case of pseudomosaicism containing two different reciprocal translocations.

Adult↗

A genetic study of hyper-alpha-lipoproteinemia.

Because of its association with longevity and reduced incidence of coronary heart disease, it becomes important to find out how elevated HDL-cholesterol levels are determined. Analyses of family data from Cincinnati initially suggested environmental factors common to sibs; however, some form of dominant inheritance could not be ruled out. Reanalysis of the Cincinnati data by Iselius and Lalouel concluded against a major locus, but did identify three families as possibly segregating for a major locus. Analysis of an additional 26 kindreds from the same population in Cincinnati by Siervogel and associates concluded that a major gene could be causing familial aggregation of high density lipoprotein in white kindreds. In this analysis, we pooled all the white Cincinnati kindreds (n = 31), and investigated the familial transmission using complex segregation analysis. We failed to obtain clear evidence for major locus determination. Under the parsimonious hypothesis of no major locus, the polygenic heritability and common sibling environmental correlation were estimated as 0.531 and 0.263, respectively, consistent with other evidence.

Adult↗

Pedigree discriminant analysis: a method to identify monogenic segregation.

We describe a method for obtaining a linear discriminant function to identify monogenic segregation in multivariate pedigree data. It differs from Fisher's linear discriminant function in that it does not assume that the genotype of each individual in the pedigree already known. The method consists of finding that linear function of the variables that maximizes the likelihood of a set of pedigree data, under the hypothesis of single gene segregation, subject to the constraint that the total sample variance of the function remains constant. To simplify the computation the variables are first transformed to their standardized principal components. Reanalysis of a set of pedigree data suggests that age and powers of age should be considered as extra variables from which the principal components are obtained, and virtually all of the variance should be accounted for by the principal components used to obtain the discriminant function.

Age Factors↗

Monoamine oxidases and alcoholism. II. Studies in alcoholic families.

Thirty-five alcoholic families have been studied to investigate the relationship between DNA markers at the monoamine oxidase (MAO) loci and 1) platelet activity levels and 2) alcoholism. A quantitative linkage analysis failed to reveal any evidence that the variation in activity levels cosegregates with the DNA markers. A sib-pair analysis did not reveal a significant excess of MAO haplotype sharing among alcoholic sibs, although the deviation from random sharing was in the direction consistent with an X-linked component. A reanalysis of platelet MAO activity levels in a subset of these families revealed that the lower levels previously found in alcoholics is more likely due to the differences between males and females. Only among males and only when a "broad" definition of alcoholism is used (and MAO activity levels are transformed to normality) does it appear that alcoholics have depressed activities compared to nonalcoholics. Finally, when the confounding due to gender difference is removed, no differences between type I and type II alcoholics are found in these families.

Alcoholism↗

Genetic structure of Mediterranean populations revealed by Y-chromosome haplotype analysis.

The allelic variability at six Y-chromosome-specific polymorphisms (YAP, DYS19, DYS389-I, DYS390, DYS391, and DYS392) was used to generate male-specific haplotypes in 333 males representing 12 population samples from the region around the Mediterranean sea. Extreme interindividual variation was observed, as more than 160 distinct Y-chromosome variants could be defined as six-locus haplotypes. Concomitant with this high variability, low levels of population genetic structure were observed. In particular, a "core" of populations directly facing the north and the east of the Mediterranean basin, from the Middle East to the Italian Peninsula, was found to be genetically undifferentiated. This observation, supported by a reanalysis of Y-specific binary polymorphisms in the same populations, suggests that at least part of the male-specific gene pools of these populations has either a very recent common origin (that could be related with the Neolithic demic diffusion hypothesis), and/or that gene flow has played a significant role in shaping the patterns of genetic variability in this region. In agreement with both hypotheses, we found that the spatial distribution of DYS392 alleles revealed a marked differentiation between the East and the West of the Mediterranean area. Through the analysis of microsatellite variation, the time to the most recent common ancestor (TMRCA) of the YAP(+) sublineage 4 has been estimated. The estimations, based on two different data sets, turn out to be quite recent (7,000-11,000 YBP), suggesting that this lineage may have been first introduced into Southern Europe through Neolithic migrations from the Middle East.

Chromosomes, Human, Y↗