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At least 775 records · Page 43Linked to original sources
PubMed2002

Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media.

PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.

Acute Disease↗
PubMed2006

A real-time quantitative PCR detection method specific to widestrike transgenic cotton (event 281-24-236/3006-210-23).

In compliance with global regulations on transgenic crops, a real-time quantitative PCR method specific to Widestrike transgenic cotton (event 281-24-236/3006-210-23, OECD Unique Identifier DAS-24236-5/DAS-21023-5) was established on the basis of the DNA sequences in the junction between the transgene insert and cotton genome. The optimized method consists of a DNA extraction method for cotton seeds and three PCR systems corresponding to a cotton-specific endogenous reference DNA sequence SAH7 (Sinapis Arabidopsis Homolog 7) and specific detection of event 281-24-236 and event 3006-210-23. The method performance including specificity, sensitivity, accuracy, and precision was determined at a dynamic range of Widestrike DNA levels from 0.04% to 5.0%. The limits of detection (LOD) and quantification (LOQ) were < or =0.04% and < or =0.09%, respectively, at 100 ng of DNA sample per reaction. The quantification results using either the event 281-24-236 or 3006-210-23 system were consistent, and the relative deviation from the expected (true) value was in the range of +/-25%. The robustness of the method was demonstrated by a series of tests with deviations from the optimized assay parameters such as annealing temperature, extension time, PCR instrument, interlaboratory transferability, etc. All the measurements from these tests met the criteria set by EU JRC-CRL (European Commission Joint Research Centre-Community Reference Lab). This real-time quantitative PCR method is accurate and robust, and is recommended as a global benchmark method for the detection and quantification of Widestrike cotton. The method including description, protocol, and performance results is available on the JRC-CRL website (http://gmo-crl.jrc.it/statusofdoss.htm).

DNA, Plant↗
PubMed2002

Viral binding proteins as antibody surrogates in immunoassays of cytokines.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative approach explored here is the use of pathogen-encoded cytokine-binding proteins, which have Kd in the nanomolar range. It is anticipated that pathogens have evolved binding proteins, antagonists, and/or specific neutralizing phenotypes directed against key signaling and effector molecules involved in the multifaceted host defense system. Thus, by screening the genomes of a wide range of microbial agents, we would expect to find coding sequences for binding proteins for the most important cytokines. Consistent with this view is the identification of poxvirus genes encoding binding activities for TNF type I and type II interferons, interleukin (IL)-1beta, IL-18, and beta-chemokines. These high-affinity receptors have the potential to act as surrogate antibodies in a number of applications in cytokine quantification and purification and could be potentially useful reagents to complement the existing panel of anti-cytokine, monoclonal, polyclonal, or engineered antibodies that are currently available.

Antibodies↗
PubMed2026

Telomere length in patients with non-functional adrenal incidentalomas.

Telomeres maintain genomic integrity during cell replication by preventing chromosomal fusions. Beside genetic influences, telomere length is affected by environmental factors such as oxidative stress and inflammation. These mechanisms also contribute to metabolic syndrome components linked to cellular aging. We aim to evaluate whether telomere length is shortened in patients with non-functional adrenal incidentaloma (NFAI) compared to the control group. This study was designed as a prospective, single-center study. The total of 88 participants included were 44 patients aged between 40 and 60 years with NFAI in our endocrinology clinic and 44 control subjects. An Absolute Human Telomere Lengths Quantification qPCR Assay kit (Nucleotestbio, Budapest, Hungary) was used for analyses. There was no significant difference between the NFAI and control groups regarding age and sex distribution. Telomere length was significantly shorter in the NFAI group (NFAI group: 3.680 &#xb1; 1.970 kb; control group: 4.469 &#xb1; 1.672 kb; p = 0.046). While no significant difference was found in telomere lengths in subgroup analyses, patients with basal adrenocorticotropic hormone (ACTH) levels <15 pg/mL had significantly shorter telomeres than those with basal ACTH levels &#x2265;15 pg/mL (p = 0.034). A strong positive correlation was observed only between telomere length and ACTH level (p = 0.001). This study demonstrated that telomere length is significantly shortened in NFAI patients. Here, we propose that the underlying cause of telomere length shortening in the NFAI group may be related to increased cardiovascular risk and an elevated inflammatory state, even in the presence of cortisol levels within the normal range.

Humans↗
PubMed1997

Methylation of cytosines in nonconventional methylation acceptor sites can contribute to reduced gene expression.

Epigenetic silencing of gene expression is often correlated with extensive DNA methylation at cytosine residues in the promoter and the coding region of silenced genes. Increasing evidence indicates that, in such cases, DNA methylation can also occur in sequence contexts other than CG and CNG, resulting in genomic regions with almost complete modification of cytosines. Whether this nonconventional methylation at CNN sites also contributes to gene repression is not known. We constructed genes with a promoter and a coding region devoid of the conventional methylation acceptor sites CG and CNG in addition to constructs with the corresponding wild-type sequences containing these sites. We generated unmethylated and completely methylated DNA by the polymerase chain reaction and performed expression assays in plant protoplasts. Quantification of transcript levels by RNase protection assay demonstrated that DNA methylation at positions other than CG or CNG sites contributes to the reduction in gene expression.

Amino Acid Sequence↗
PubMed2025

vcfgl: a flexible genotype likelihood simulator for VCF/BCF files.

MOTIVATION: Accurate quantification of genotype uncertainty is pivotal in ensuring the reliability of genetic inferences drawn from NGS data. Genotype uncertainty is typically modeled using Genotype Likelihoods (GLs), which can help propagate measures of statistical uncertainty in base calls to downstream analyses. However, the effects of errors and biases in the estimation of GLs, introduced by biases in the original base call quality scores or the discretization of quality scores, as well as the choice of the GL model, remain under-explored. RESULTS: We present vcfgl, a versatile tool for simulating genotype likelihoods associated with simulated read data. It offers a framework for researchers to simulate and investigate the uncertainties and biases associated with the quantification of uncertainty, thereby facilitating a deeper understanding of their impacts on downstream analytical methods. Through simulations, we demonstrate the utility of vcfgl in benchmarking GL-based methods. The program can calculate GLs using various widely used genotype likelihood models and can simulate the errors in quality scores using a Beta distribution. It is compatible with modern simulators such as msprime and SLiM, and can output data in pileup, Variant Call Format (VCF)/BCF, and genomic VCF file formats, supporting a wide range of applications. The vcfgl program is freely available as an efficient and user-friendly software written in C/C++. AVAILABILITY AND IMPLEMENTATION: vcfgl is freely available at https://github.com/isinaltinkaya/vcfgl.

Software↗
PubMedDate not supplied

Non-viral vector-mediated uptake, distribution, and stability of chimeraplasts in human airway epithelial cells.

BACKGROUND: Chimeraplasty is a novel methodology that uses chimeric RNA/DNA oligonucleotides (chimeraplasts) to stimulate genomic DNA repair. Efficient uptake and nuclear localization of intact chimeraplasts are key parameters to achieve optimal correction of mutation defects into specific cell types. METHODS: A 5'-end FITC-labeled 68-mer RNA/DNA oligonucleotide was complexed with the polycation polyethylenimine (PEI) and the cationic lipids Cytofectin and GenePorter. Flow cytometry was employed to evaluate chimeraplast uptake under different conditions. Intracellular chimeraplast distribution and co-localization with endocytosis markers were assessed by confocal microscopy. Relative quantification of chimeraplast metabolism was performed by denaturing PAGE and GeneScan(trade mark) analysis. RESULTS: In airway epithelial cells, optimized chimeraplast uptake reached near 100% efficiency with the carriers tested. However, chimeraplast nuclear localization could only be achieved using PEI or Cytofectin. Chimeraplast/GenePorter lipoplexes were retained in the cytoplasm. PEI polyplexes and Cytofectin lipoplexes displayed different uptake rates and internalization mechanisms. Chimeraplast/PEI polyplexes were internalized at least partially by fluid-phase endocytosis. In contrast, phagocytosis may have contributed to the internalization process of large-sized chimeraplast/Cytofectin lipoplexes. Moreover, significant chimeraplast degradation was detected 24 h after transfection with both PEI polyplexes and Cytofectin lipoplexes, although the latter seemed to confer a higher degree of protection against nuclease degradation. CONCLUSION: Both Cytofectin and PEI are efficient for chimeraplast nuclear uptake into airway epithelial cells. However, despite the distinct structures and trafficking pathways of the corresponding complexes, none of them could prevent nuclease-mediated metabolism of the chimeric oligonucleotides. These findings should be taken into account for future investigations of chimeraplast-mediated gene repair in airway epithelial cells.

Base Sequence↗
PubMed1994

Detection and quantification of hepatitis C virus RNA replication in the liver.

To investigate the correlation between the replication of hepatitis C virus in liver and the clinical and histopathological features, we detected and quantified plus and minus strands of HCV-RNA in plasma and in livers of patients with chronic hepatitis C by a quantitative polymerase chain reaction. RNA was extracted from the plasma and liver tissue of ten patients with biopsy-proven chronic hepatitis C. The plus and minus strands of HCV-RNA were detected by a strand-specific reverse transcription with either sense or anti-sense oligonucleotide primers deduced from the hepatitis C virus genome, and a standard HCV-RNA with an enzyme restriction site was used to quantify the amount of HCV-RNA. Both plus and minus strands of HCV-RNA were detected from the liver tissue of all patients included. The amount of plus-stranded HCV-RNA in the liver was 10 times higher than that of minus-stranded HCV-RNA. Plus-stranded HCV-RNA was detected in the plasma in all patients, while the minus strand was not detected in any patient. There was a weak correlation between the amount of both strands of HCV-RNA in the liver and that of the plus strand in plasma. There was no significant correlation between the amount of liver HCV-RNA and serum alanine transaminase and aspartate transaminase levels, or histopathological findings in the liver. The present method of detecting and quantifying liver HCV-RNA is simple and sensitive; it may be used to detect residual hepatitis C virus replication after the disappearance of plasma HCV-RNA in acute hepatitis or in chronic hepatitis after interferon treatment.

Adult↗
PubMed1998

Strong conservation of the constitutive activity of the IE1/2 transcriptional control region in wild-type strains of human cytomegalovirus.

The IE1/2 transcriptional control region of human cytomegalovirus (HCMV) drives the expression of the HCMV major immediate-early genes (UL123-122), which encode proteins crucial for initiation of the virus replicative cycle. Nucleotide sequence polymorphism in this region of the viral genome could account for variations in the replication of HCMV wild-type strains. In order to test this hypothesis, the constitutive transcription-enhancing activity of the IE1/2 transcriptional control region derived from 12 clinical isolates of HCMV was compared. This was done by PCR amplification of the respective elements followed by cloning up-stream of a beta-globin reporter gene. After transient expression in various cell types, including human teratocarcinoma cell lines, and quantification of RNA levels, the activating function of this complex cis-element was shown to be strongly conserved. This was mirrored by high nucleotide sequence conservation, even within the so-called modulator region. This strong evolutionary conservation of sequence and of transcription-enhancing function strengthens the assumption that the IE1/2 transcriptional control region plays an essential role in initiation of the HCMV replicative cycle.

Base Sequence↗
PubMed2026

Discovery of acridone analogs as novel entry inhibitors targeting e protein of dengue virus.

The envelope (E) protein of the Dengue virus (DENV) is critical for virion attachment and membrane fusion with the host cell, as well as the release of the viral RNA genome into the cytoplasm. In this study, we describe the design, synthesis, and biological evaluation of novel viral entry inhibitors containing an acridone core. Notably, compound 13e demonstrated potent cellular antiviral activity (IC50 = 8.6 &#x3bc;M and selectivity index = 21.4). Compound 13e was evaluated using several methods, including time-of-addition and virus entry/binding assays, which revealed that it selectively blocked DENV2 infection by inhibiting virion attachment. Furthermore, compound 13e exhibited potent antiviral efficacy, as evidenced by viremia quantification and histopathological analysis results, without causing significant body weight loss or other toxicities. Furthermore, target engagement assay supported the role of compound 13e as an E protein binder, consistent with its function as an entry inhibitor.

Dengue Virus↗
PubMed2004

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro.

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.

Active Transport, Cell Nucleus↗
PubMed1999

A standard deviation based quantification differentiates coding from non-coding DNA sequences and gives insight to their evolutionary history.

A method quantifying the randomness of nucleotide sequences is developed, based on the introduction of a standard deviation type of quantity involving locally computed means and a length scale around which is assessed the clustering of nucleotides. It is pointed out that the value taken by this modified standard deviation may distinguish between coding rich and non-coding rich sequences. Moreover, the approach described herein allows the determination of some minimal characteristics of an evolutionary scenario which can account for the origin of the clustering in the nucleotide distribution of the different parts of the genome.

Animals↗
PubMed2001

Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay.

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log(10). Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10(6) PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10(6) PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5' end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4(+) cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.

Anti-HIV Agents↗
PubMedDate not supplied

Quantifying viral propagation in vitro: toward a method for characterization of complex phenotypes.

For a eukaryotic virus to successfully infect and propagate in cultured cells several events must occur: the virion must identify and bind to its cellular receptor, become internalized, uncoat, synthesize viral proteins, replicate its genome, assemble progeny virions, and exit the host cell. While these events are taking place, intrinsic host defenses activate in order to defeat the virus, e.g., activation of the interferon system, induction of apoptosis, and attempted elicitation of immune responses via chemokine and cytokine production. As a first step in developing an imaging methodology to facilitate direct observation of such complex host/virus dynamics, we have designed an immunofluorescence-based system that extends the traditional plaque assay, permitting simultaneous quantification of the rate of viral spread, as indicated by the presence of a labeled viral protein, and cell death in vitro, as indicated by cell loss. We propose that our propagation and cell death profiles serve as phenotypic read-outs, complementing genetic analysis of viral strains. As our virus/host system we used vesicular stomatitis virus (VSV) propagating in hamster kidney epithelial (BHK-21) and murine astrocytoma (DBT) cell lines. Viral propagation and death profiles were strikingly different in these two cell lines, displaying both very different initial titer and cell age effects. The rate of viral spread and cell death tracked reliably in both cell lines. In BHK-21 cells, the rate of viral propagation, as well as maximal spread, was relatively insensitive to initial titer and was roughly linear over several days. In contrast, viral plaque expansion in DBT cells was contained early in the infections with high titers, while low titer infections spread in a manner similar to the BHK-21 cells. The effect of cell age on infection spread was negligible in BHK-21 cells but not in DBTs. Neither of these effects was clearly observed by plaque assay.

Animals↗
PubMed2005

Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.

Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His(6)-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% +/- 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.

Arabidopsis↗
PubMed1999

Quantification of JC virus DNA in the cerebrospinal fluid of patients with human immunodeficiency virus-associated progressive multifocal leukoencephalopathy--a longitudinal study.

In progressive multifocal leukoencephalopathy (PML) the JC virus (JCV) load in the cerebrospinal fluid (CSF) is discussed as a parameter for disease progression. To investigate the evolution of viral shedding into the CSF, the JCV DNA concentration was quantified by competitive polymerase chain reaction (PCR) in multiple CSF samples from prior to and during an unsuccessful intrathecal salvage therapy in 2 human immunodeficiency virus-infected patients with biopsy-proven PML. With continuous clinical progression the virus load varied considerably intra- and interindividually, ranging from nondetectable to 1.2x108 genome equivalents/10 microliter CSF. Whereas an overall increase during progressive disease was confirmed, the virus burden was either constant or fluctuated irregularly during the intermediate stage of disease. This shows a variability of viral shedding during active disease that must be taken into account when the JCV load is measured by quantitative PCR for both the diagnosis of PML and monitoring under investigational treatment.

AIDS-Related Opportunistic Infections↗
PubMed2005

High-throughput double quantitative competitive polymerase chain reaction for determination of genetically modified organisms.

Quantitative competitive polymerase chain reaction (PCR), especially the double competitive PCR methods (DC-PCR), have evolved as reliable approaches to quantification of genetically modified organisms (GMO) in food. However, DC-PCR is a low-throughput method because it requires titration of each sample with various amounts of a competitive internal standard, a protocol that involves several PCRs per sample followed by electrophoresis and densitometry. To address this drawback, we have developed a new method for GMO quantification, namely, a high-throughput double quantitative competitive PCR (HT-DCPCR). In HT-DCPCR, electrophoresis and densitometry are replaced by a rapid, microtiter well-based bioluminometric hybridization assay and there is no need for titration of each sample. The determination of GM soya was chosen as a model. We have constructed internal standards (DNA competitors) both for the 35S promoter sequence and for a plant-specific reference gene (lectin). The competitors have identical size and share the same primer binding sites with the target sequences but differ in a 24-bp internal segment. Each target sequence (35S and lectin) is coamplified with a constant amount (1000 copies) of the respective competitor. The four amplified fragments are hybridized with specific probes and captured on a universal solid phase to achieve simplicity and high throughput. The hybrids are determined by using streptavidin conjugated to the photoprotein aequorin. The ratio of the luminescence values obtained for the target and the competitor is linearly related to the starting amount of target DNA. The limit of quantification for the 35S promoter is 24 copies. The proposed method was evaluated by determining the GMO content of soybean powder certified reference materials. Also HT-DCPCR was compared to real-time PCR in a variety of real samples.

Calibration↗
PubMed2006

Within-tree transcriptome profiling in wood-forming tissues of a fast-growing Eucalyptus tree.

Despite the availability of high-throughput transcript profiling technology, little is known about tissue-specific gene expression patterns in the wood-forming tissues of Eucalyptus plantation tree species. We used cDNA-amplified fragment length polymorphism (AFLP) analysis in combination with infrared fragment detection and semi-automated band quantification to profile gene expression in a 6-year-old, fast- growing Eucalyptus tree. The expression profiles of 6385 transcript-derived fragments (TDFs) were analyzed across four major woody tissues (mature xylem, immature xylem, phloem and cork) collected from two stem positions, to provide a global view of transcript abundance and variability in the Eucalyptus stem. About 21% of the TDFs were differentially expressed and could be grouped into clusters representing co- expressed genes. A total of 71 TDFs representing different gene clusters were isolated and characterized. These included genes implicated in cell fate, signal transduction and cell wall biosynthesis, processes closely associated with xylogenesis. Analysis of the expression levels of selected TDFs by quantitative RT-PCR corroborated the TDF quantification and confirmed that cDNA-AFLP analysis is a highly efficient and accurate tool for transcript profiling and gene discovery in wood-forming tissues of tree species.

Cluster Analysis↗