PCR mediated analysis of RNA sequences.
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Base Sequence↗
SEARCH · PubMed Health
Results for “mediation analysis”
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Partial sequence and polymerase chain reaction-mediated analysis of expression of the human CYP2C18 gene.
We describe the isolation of the human CYP2C18 gene, a new member in the complex CYP2C subfamily, associated with the genetically-determined polymorphism for (S)-mephenytoin hydroxylase. The 5' end of CYP2C18 gene was isolated from a human genomic library using a probe derived from the CYP2C10 cDNA and the 5' flanking region, exons 1 to 4 and intron-exon junctions were sequenced. With respect to intron-exon boundaries, the partial gene structure was identical to that of rat and rabbit CYP2C genes. Consensus sequences for putative 'glucocorticoid responsive elements' were observed in the 5' flanking region and in intron 1, an interesting feature in a so-called constitutively-expressed gene subfamily. The knowledge of CYP2C gene sequences is a prerequisite to genomic analysis by PCR techniques. Using oligonucleotides derived from the gene sequence, we were able to specifically detect CYP2C18 mRNAs in human liver.
Animals↗
PCR-mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families.
We compare the kinetics of appearance of supernatant enzyme activities (lignin peroxidase, manganese peroxidase, and cellulase) and gene expression (LIG, mnp, and cbhI gene families and the unique cbhII gene) in Phanerochaete chrysosporium ME446 when grown on four different carbon sources: ball-milled straw, representing the natural substrate lignocellulose; Avicel as a crystalline cellulose; and high and low concentrations of glucose, in all cases with limiting nitrogen. PCR-based technology utilizing pairs of primers specific for particular genes showed that there is differential expression between and within the families. There were a number of instances of mRNA species being present only on a single day, implying tight regulation of lignocellulose degradation at the mRNA level. The patterns of extracellular enzyme activities and mnp and cbh gene expression are similar whereas LIG gene expression can be detected when no corresponding enzyme activity is observed in the extracellular supernatant. The enzyme produced under these conditions is presumably sequestered by the mycelium and is likely to be functionally significant. Another striking result is that cellulose, in the form of Avicel, elicits the expression of three LIG gene for which there is no expression under the same conditions with the other carbon sources.
Base Sequence↗
Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids.
For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.
Acetylation↗
PCR-mediated analysis of human rotaviruses.
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Cloning, Molecular↗
Genotypic characterization of an MHC class II locus in lake trout ( Salvelinus namaycush) from Lake Superior by single-stranded conformational polymorphism analysis and reference strand-mediated conformational analysis.
This study compares the genotypic information provided by reference strand-mediated conformational analysis and single-stranded confirmational polymorphism (SSCP) analysis for the major histocompatibility complex (MHC) II locus in lake trout. For this study 80 wild-caught animals from the Apostle Islands of Lake Superior were genotyped using both RSCA and SSCP analysis. Their genotypes were recorded using both methods and compared. The genotypic information provided by the 2 methods was essentially the same although some inconsistencies were observed. Both methods detected approximately 65 genotypes, and both were able to distinguish heterozygous and homozygous animals. The analyses determined that only approximately 20% of alleles were shared between 2 morphologically different populations within the sample set, and identified the dominant alleles. SSCP analysis was quicker, simple, and more robust than RSCA. SSCP analysis using fluorescence technologies could be the method of choice for future genotypic analysis of the MHC II locus in salmonids.
Animals↗
Differential DNA methylation in blood as potential mediator of the association between ambient PM2.5 and cerebrospinal fluid biomarkers of Alzheimer's disease among a cognitively normal population-based cohort.
Fine particulate matter (PM2.5) is a known risk factor for Alzheimer's disease (AD), with emerging evidence showing its effects detectable in the pre-clinical stage through cerebrospinal fluid (CSF) biomarkers of AD. While studies have linked PM2.5 exposure and AD to DNA methylation (DNAm) alterations, the role of DNAm as potential mediator in the association between PM2.5 and AD biomarkers in cognitively normal individuals remains largely unexplored, and formal mediation analyses addressing this question are scarce. Genome-wide DNAm profiles (Illumina EPIC BeadChips) in whole blood and CSF Aβ42 concentrations were assessed in 536 cognitively normal individuals from the Emory Healthy Brain Study (EHBS). Residential PM2.5 exposure for the year preceding participants' blood collection was estimated. A multi-stage analytical pipeline, incorporating single-mediator analysis, high-dimensional mediation analysis, and causal mediation analysis, was applied. Nine CpG sites were identified as noteworthy mediators of the relationship between PM2.5 and decreased CSF Aβ42 concentrations. Causal mediation analysis confirmed significant natural indirect effects (NIE) for eight CpGs, with effect estimates ranging from -0.015--0.029 per 1 ug/m3 increase in PM2.5 exposure. The proportion mediated ranging from 14-43%. Six CpGs are annotated to genes implicated in neuroinflammatory pathways. These findings suggest that differential DNAm, particularly in genes related to neuroinflammation, mediates the association between PM2.5 exposure and CSF Aβ42 concentrations, highlighting the utility of blood DNAm in detecting and studying biological pathways underlying PM2.5 toxicity in the pre-clinical stages of AD.
Humans↗
HLA typing by Reference Strand Mediated Conformation Analysis (RSCA).
Reference Strand Mediated Conformation Analysis (RSCA) is a novel conformational method that offers high resolution and high sample throughput for typing the HLA class I and class II genes. This technique differs from conventional sequence-based typing methodologies in that the HLA type is assigned on the basis of accurate measurement of conformation-dependent DNA mobility in polyacrylamide gel electrophoresis (AGE). RSCA utilises a fluorescent-labelled locus-specific reference DNA to selectively modify the molecular conformation of the tested DNA. The use of laser-based instrumentation and computer software, in addition to internal DNA markers for correction of gel variability, allows the discrimination of HLA alleles which differ by one nucleotide in a DNA fragment nearly as large as a kilobase in length. RSCA has been successfully applied in blind studies of HLA typing, demonstrating that is reproducible, able to identify new alleles and able to resolve ambiguous heterozygous combinations.
DNA↗
A novel approach to analysis of phage clones by reference strand mediated conformation analysis.
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Amino Acid Sequence↗
Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction.
Using phenyl-azide-mediated photocrosslinking, we show that the alpha carbon of amino acid 2 of the helix-turn-helix motif of bacteriophage lambda Cro is within 12 A of the bottom-strand nucleotides at positions 2 and 3 of the DNA half site in the Cro-DNA complex in solution. This result is in excellent agreement with the crystallographic structure of the Cro-DNA complex. The results of phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction, together with the previously reported results of phenyl-azide-mediated photocrosslinking analysis of CAP-DNA interaction, establish that phenyl-azide-mediated photocrosslinking is generalizable and provide information regarding the structural requirements for phenyl-azide-mediated photocrosslinking. Comparison of the results of phenyl-azide-mediated photocrosslinking to the results of EDTA: iron-mediated affinity cleaving indicates that phenyl-azide-mediated photocrosslinking yields superior resolution.
Azides↗
Resolution of complex feline leukocyte antigen DRB loci by reference strand-mediated conformational analysis (RSCA).
The DRB genes of the domestic cat are highly polymorphic. Studies based on clonal sequence analysis have suggested the existence of two distinct loci within individual animals and good evidence for 24 distinct FLA-DRB alleles. This variability, the complexity of clonal sequence analysis and its susceptibility to PCR-induced artefacts has represented a bottleneck to further progress. In this study we have applied reference strand-mediated conformational analysis (RSCA) to FLA-DRB. This protocol has been shown to be highly reproducible. Using five reference strands including two derived from non-domestic felines, we could distinguish 23 FLA-DRB alleles. We used RSCA to explore genetic polymorphism of FLA-DRB in 71 cats including 31 for which clonal sequence analysis was also available. On average, RSCA identified 0.9 more alleles within cats than clonal sequence analysis. Reference strand-mediated conformational analysis was also able to identify animals containing new alleles that could be targeted for sequence analysis. Analysis of allele patterns showed clear evidence for different allele distributions between breeds of cats, and suggested the Burmese breed may have highly restricted FLA-DRB polymorphism. Results from two families provided clear evidence for variation in the number of DRB genes on different haplotypes, with some haplotypes carrying two genes and some containing three. This study highlights the utility of RSCA for the resolution of complex amplicons containing up to six distinct alleles. A simple, rapid method for characterizing FLA-DRB makes possible studies on vaccine response and susceptibility/resistance to viral infections, which are a significant clinical problem in cats.
Alleles↗
Received social support, self-efficacy, and finding benefits in disease as predictors of physical functioning and adherence to antiretroviral therapy.
OBJECTIVE: The study investigated whether received social support, self-efficacy, and finding benefits in disease are related to physical functioning and adherence to antiretroviral medication among men and women infected with HIV. METHODS: Data were collected among 104 patients of three HIV clinics in India. The measures included general self-efficacy scale, Berlin social support scales, questionnaire on taking antiretroviral medication, and SF-20 (physical functioning). RESULTS: The results of path analysis and mediation analysis revealed that finding benefits and self-efficacy were directly related to both adherence and physical functioning. Additionally, finding benefits mediated the relation between patients' self-efficacy and adherence as well as physical functioning. Although received support was unrelated to adherence directly, effects of received support on adherence were mediated by self-efficacy. CONCLUSIONS: Besides personal and social resources, benefit finding was related to better adherence to antiretroviral medication. PRACTICE IMPLICATIONS: Identifying patients receiving low social support, with weak general self-efficacy and finding no benefits in being diagnosed with HIV may help to elicit those people who are at risk for poorer adherence and physical functioning.
Activities of Daily Living↗
Identification of medically important pathogenic fungi by reference strand-mediated conformational analysis (RSCA).
This report describes the application of reference strand-mediated conformational analysis (RSCA), a novel DNA typing technique, for the identification of clinically significant fungal pathogens. RSCA is a heteroduplex-based conformational method which relies on detecting differences in the DNA conformation of heteroduplexes generated in this study by the annealing of different fungal 18S rRNA amplicons to a common fluorescent-labelled reference (FLR). These heteroduplexes are then observed with laser-based instrumentation and computer software to detect differences in the DNA conformation reproducibly. This technique was shown to generate unique and reproducible profiles for the 18S rRNA gene sequences of a number of medically important fungi, distinguishing different Candida species (C. albicans, C. kefyr, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. tropicalis, C. krusei, C. glabrata, C. sake and C. parapsilosis), and in some cases detecting single nucleotide differences between 18S rRNA sequences. The RSCA technique was further evaluated with 50 human clinical isolates of Candida spp., previously identified by culture techniques, and was shown to identify the isolates correctly. This technique displays enormous potential as an alternative to DNA sequence determination and has the potential to become an automated technique that can be implemented in the routine setting.
Candida↗
Brain Health Loss Mediates the Effect of Infarct Volume on Functional Outcome in Ischemic Stroke.
IMPORTANCE: Brain health may facilitate resilience to detrimental consequences from neurological diseases. Infarct volume is associated with poor functional outcome after acute ischemic stroke (AIS), but potential mediating effects through stroke-related brain health loss have not been investigated. OBJECTIVE: To determine whether stroke-related brain health loss, quantified by change in MRI derived effective Reserve (eR), mediates the effect of acute infarct volume on functional outcome after AIS. DESIGN: Observational multicenter cohort study. SETTING: We analyzed data from the GASROS (n=488) and MRI-GENIE (n=560) cohorts, collected 2003-2011. PARTICIPANTS: Adult patients consecutively diagnosed with AIS, with available admission MRI. EXPOSURE: At admission, white matter hyperintensity (WMH) and normal-appearing brain volumes were assessed on T2-FLAIR, and acute infarct volume on diffusion weighted imaging. WMH was normalized by brain volume, creating WMH load. We quantified brain health using eR, a latent variable incorporating age, WMH load, and normal-appearing brain volume. ΔeR reflected the change in eR when acute infarct volume was included, representing stroke-related brain health decline. Mediation analysis was used to determine if ΔeR mediates the effect of infarct volume on functional outcome (modified Rankin Scale [mRS] at 90 days). MAIN OUTCOME MEASURE: Proportion of mediating effect. RESULTS: We included 1,048 patients (median age 67y, 38% females). At baseline, median NIHSS score was 3 (IQR 1-7), median infarct volume 3.1mL (IQR 0.9-15.5). At 90 days, median mRS score was 1 (IQR 1-3) and 51 (5%) patients had died. In mediation analysis, ΔeR significantly mediated 36% (95% CI 16-56%) of the total effect of infarct volume on functional outcome (direct effect (ß=0.15 [95% CI 0.09-0.22], p<0.001; indirect effect mediated through ΔeR: ß=0.09 [95% CI 0.04 to 0.14], p=0.001). In subgroup-analyses, the mediative effect was apparent among female but not male, and among patients aged >67y but not ≤67y. CONCLUSIONS AND RELEVANCE: Stroke-related structural brain health loss mediates about one third of the effect of acute infarct volume on functional outcome after ischemic stroke, with important sex and age differences. Brain health significantly influences outcome and recovery potential, and may be considered a key biomarker when modeling outcome after AIS.
acute ischemic stroke↗
[Clinical application for reference strand mediated conformation analysis system in HLA-A genotyping].
OBJECTIVE: To establish and stabilize a new HLA typing strategy, reference strand mediated conformation analysis (RSCA), and to conform its advantages by using HLA-A as target gene. METHODS: 20 standard DNA samples from international RSCA cooperative team were used to establish and stabilize RSCA. 84 DNA samples of related hematopoietic stem cell transplant donor-recipients were extracted from peripheral blood cells. HLA-A loci were typed both by RSCA and PCR-SSP. RESULTS: RSCA results of 20 standard samples were identical to those of international RSCA cooperative team, and the accuracy and replication rate were both 100%. Among the 84 samples, 82/84 (97.6%) cases could be designated definitely, and 33/84 (39%) cases could be typed to allelic level. In addition, 2/84 (2.4%) cases could not be detected by RSCA for their poor PCR results. 20 samples were randomly selected to identify the replication rate of RSCA, and the results demonstrated that the replication rate was 100%. Among the PCR-SSP typing results, there were about 10% samples needed to be typed for 1 - 3 times to confirm their types. CONCLUSION: RSCA has some advantages compared with the PCR-SSP typing method, which are high resolution, high sensitivity, high accuracy, high replication, capability to find new alleles, high throughput, low cost and suitability for unrelated hematopoietic stem cell transplant donor-recipient HLA typing. But there are still some defects in this new strategy, which are time-consuming for only one sample a time and higher DNA quality requirement. In addition, RSCA database needs to be further improved.
Adolescent↗
HLA-A typing by reference strand-mediated conformation analysis (RSCA) using a capillary-based semi-automated genetic analyser.
HLA typing of class I loci by reference strand-mediated conformation analysis (RSCA) using a slab gel genetic analyser has been described. This study adapted the method for use in the capillary based ABI PRISM 310. Control DNA samples were used to create a database of mobility values for 37 HLA-A alleles. The technique was validated by comparing RSCA and sequence-specific oligonucleotide probe (SSOP)/sequence-specific primer amplification (SSP) HLA-A locus typing results from 214 cord blood samples. Of the samples tested, 6.5% required confirmatory typing by SSP, compared with a repeat rate of 10-40% for SSOP. In 200 samples where no SSP was necessary, there was 100% concordance between RSCA and previous results. The ABI PRISM 310 RSCA method defines HLA-A types at medium resolution and is quick and easy to implement.
Electrophoresis, Capillary↗
Reference strand mediated conformation analysis resolves HLA-DRB1 typing ambiguities when matching for unrelated bone marrow transplantation.
We show here the use of reference strand mediated conformation analysis (RSCA) to unambiguously resolve the HLA-DRB1 typing of two individuals which were selected as potential unrelated donors for bone marrow transplantation (BMT). In the first case, both sequence-specific primer (SSP) amplification and sequence-specific oligonucleotide probing (SSO), routinely used in different tissue typing laboratories gave, for the two unrelated donors, the same ambiguous typing of HLA-DRB1*04011+*0403 or DRB1*0407+*0413. In this case sequence-based typing (SBT) was not the method of choice to resolve the situation, due to the sequence ambiguities of these two given combinations. RSCA of both samples, using homozygous typing cells (HTCs) for DRB1*04011, *0403 and *0407 as internal controls, gave the unambiguous result that both donors were HLA-DRB1*04011+*0403. In the second case, a donor was typed as DRB1*1102+1103 by SSP, while SSO excluded the DRB1*1102 allele. The patient was unambiguously typed as DRB1*1101+1103 by both techniques. RSCA, using DNA from reference cell lines as internal controls, gave the unambiguous typing that the donor was DRB1*1103 homozygous.
Alleles↗
Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.
We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.
Amino Acid Sequence↗