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At least 865 records · Page 48Linked to original sources
PubMed2002

RNA expression profiling as prognostic tool in renal patients: toward nephrogenomics.

Damage to the kidney generally elicits tissue repair mechanisms, but these processes themselves conversely may result in the progression of chronic renal disease. In a majority of patients chronic renal insufficiency progresses to a common histological end point, marked by the presence of a vast amount of scar tissue, that is, glomerulosclerosis and interstitial fibrosis. These lesions are the result of an excessive production of extracellular matrix (ECM) components. Studies on RNA expression in experimental kidney disease have shown that renal mRNA levels for ECM components and cytokines can function as prognostic tools. This suggests that mRNA levels potentially predict outcome and reaction to therapy in patients with renal diseases. Timely detection of molecular alterations could allow early therapeutic intervention that slows down or even prevents the development of sclerotic and fibrotic lesions. This review first provides a short introduction on mechanisms of initiation and progression of renal disease. Molecular techniques are available to identify renal RNA sequences potentially involved in disease progression. We discuss several molecular techniques that are being used in kidney research for quantitation and detection of mRNA. This is followed by a brief overview of investigation in experimental renal diseases, which reveal that alterations in tissue ECM mRNA levels precede histological damage and can function as predictors of clinical outcome. In particular, studies in human kidney biopsies that evaluate the prognostic value of mRNA levels with respect to renal function are examined, paying special attention to the pitfalls that potentially are encountered when interpreting the results of such studies. Then, we elaborate on ways of optimal exploitation of mRNA quantification as a prognostic tool. The potential and limitations of microarray technology in the search for genes specifically involved in progression of renal disease are reviewed, including RNA expression profiling and large-scale DNA mutation screening. Finally, the future utilities of microarray in nephrology and renal pathology are discussed.

Gene Expression Profiling↗
PubMed1995

Induction of paternal genome loss by the paternal-sex-ratio chromosome and cytoplasmic incompatibility bacteria (Wolbachia): a comparative study of early embryonic events.

Paternal genome loss (PGL) during early embryogenesis is caused by two different genetic elements in the parasitoid wasp, Nasonia vitripennis. Paternal sex ratio (PSR) is a paternally inherited supernumerary chromosome that disrupts condensation of the paternal chromosomes by the first mitotic division of fertilized eggs. Bacteria belonging to the genus Wolbachia are present in Nasonia eggs and also disrupt paternal chromosome condensation in crosses between cytoplasmically incompatible strains. Cytoplasmic incompatibility Wolbachia are widespread in insects, whereas PSR is specific to this wasp. PGL results in production of male progeny in Nasonia due to haplodiploid sex determination. The cytological events associated with PGL induced by the PSR chromosome and by Wolbachia were compared by fluorescent light microscopy using the fluorochrome Hoescht 33258. Cytological examination of eggs fertilized with PSR-bearing sperm revealed that a dense paternal chromatin mass forms prior to the first metaphase. Quantification of chromatin by epifluorescence indicates that this mass does undergo replication along with the maternal chromatin prior to the first mitotic division but does not replicate during later mitotic cycles. Contrary to previous reports using other staining methods, the paternal chromatin mass remains condensed during interphase and persists over subsequent mitotic cycles, at least until formation of the syncytial blastoderm and cellularization, at which time it remains near the center of the egg with the yolk nuclei. Wolbachia-induced PGL shows several marked differences. Most notable is that the paternal chromatin mass is more diffuse and tends to be fragmented during the first mitotic division, with portions becoming associated with the daughter nuclei. Nuclei containing portions of the paternal chromatin mass appear to be delayed in subsequent mitotic divisions relative to nuclei free of paternal chromatin. Crosses combining incompatibility with PSR were cytologically similar to Wolbachia-induced PGL, although shearing of the paternal chromatin mass was reduced. Wolbachia may, therefore, block an earlier stage of paternal chromatin processing in the fertilized eggs than does PSR.

Animals↗
PubMed2024

On the Hunt for the Histone Code.

Our genome is not made of naked DNA but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site-specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don's clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab's effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don's pioneering work in this area, many labs have been able to jump into the epigenetics field and "Hunt" down their own histone targets. A walkthrough of those early histone years in the Hunt Lab is described by three of us who were fortunate enough to be at the right place, at the right time.

Animals↗
PubMed2003

Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel.

We developed a completely homogeneous and isothermal method of detecting RNA sequences and demonstrated ultrarapid and direct quantification of pathogenic gene expression with high sensitivity. The assay is based on performing isothermal RNA sequence amplification in the presence of our novel DNA probe, an intercalation activating fluorescence DNA probe, and measuring the fluorescence intensity of the reaction mixture. When detecting mecA gene expression of methicillin-resistant Staphylococcus aureus, we quantified starting copies ranging from 10 to 10(7) copies within 10min. The primer sequences were designed to bind to secondary structure-free sites of the target RNA, which enabled a totally isothermal protocol to quantify mRNA specifically in a sample of existing genomic DNA. When we applied this to quantifying the expression of marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG strain, the results correlated well with the viability of each bacterium. We also demonstrated monitoring Pab gene expression of M. bovis BCG during cultivation with antibiotics. The present method can potentially realize rapid antimicrobial susceptibility testing of slowly growing organisms, such as tuberculosis.

Base Sequence↗
PubMed2025

Quantitative Real-Time PCR for Circular RNA Detection and Analysis.

In eukaryotes, nearly 2% of the genome represented by the coding proteins. However, emerging evidence suggest more than 75% of the human genome referred to as noncoding part also plays a crucial role in governing major regulatory pathways. Noncoding RNAs can be categorized into several groups, such as microRNAs (miRNAs), small nuclear RNA (snRNAs), small nucleolar RNA (snoRNAs), transfer RNA (tRNA), and circular RNA (circRNAs), which contribute to this regulatory landscape. Circular RNAs (circRNAs) are identified as a new class of regulatory noncoding RNAs with gene regulatory roles by acting as miRNA or RNA binding protein sponges or interacting with proteins. Researchers employ quantitative real-time PCR methods to examine circular RNA expression utilizing divergent primers for identification and quantification.

RNA, Circular↗
PubMed2002

Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid.

A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method based on TaqMan technology was developed to determine the presence and amount of enterovirus RNA. In order to prevent false-negative results, a one-step multiplex RT-PCR was optimized. It contains two dual-labelled fluorogenic probes to quantify the 5' noncoding region of enterovirus and detect an internal positive control. In the present study, 104 cerebrospinal fluid samples collected during an outbreak of enteroviral meningitis were analyzed using this method. Amplification of the internal positive control was effective in all but two specimens, confirming the absence of PCR inhibitors and allowing the results of amplification to be validated. The sensitivity of the RT-PCR was 96.8%, while that of cell culture was 34.9%. Genomic viral loads found ranged between 3.3 and 5.9 log(10) copies per milliliter of cerebrospinal fluid (mean, 4.8 log(10) copies/ml). This fluorogenic enterovirus RT-PCR allows large numbers of samples to be screened rapidly. Moreover, its sensitivity and reproducibility make it highly reliable. With these characteristics, the enterovirus RT-PCR can be a useful tool that may offer considerable benefit in the clinical management of patients with enteroviral infections.

Adolescent↗
PubMed2002

Depuration dynamics of viruses in shellfish.

The consumption of shellfish has been associated with viral infections even in cases where shellfish complied with the current regulation, which is based on bacterial analysis. In this study, depuration rates of potential indicators and human viruses have been analysed in order to study the use of complementary parameters for evaluating the microbiological quality of depurated shellfish. Depuration of naturally highly polluted mussels has been evaluated and analyses for Escherichia coli, Clostridium perfringens, somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis RYC2056 and HSP40, human adenovirus, enterovirus have been done. Seawater of the depuration tank was disinfected by UV irradiation, ozone and passed through a skimmer and a biological filter. The correct functioning of the depuration tank was monitored by the quantification of total organic carbon (TOC), NH4+ and total aerobic bacteria in the seawater. To study the relation between the bacteriophages and the human viruses analysed, a logistic regression model was applied. F-RNA phages are significantly related to human adenoviruses and enteroviruses. Thus, they can be used as a complementary parameter for evaluating the efficiency of the depuration treatment. Somatic coliphages are also significantly associated with enteroviruses. Bacteriophages infecting B. fragilis HSP40 were analysed by the double-agar-layer (DAL) method, which quantifies infectious viruses, and by nested PCR, which detects the presence of the genome of these phages. The highest sensitivity of the molecular techniques was demonstrated and the results obtained are an indicator of a close relation between positive results by PCR and the presence of infectious viral particles in shellfish. All shellfish samples were negative for human viruses by PCR after 5 days of depuration treatment and the results obtained applying a regression model also showed negative results or nearly for F-RNA phages and bacteriophages infecting B. fragilis RYC2056. Thus, in this specific depuration treatment, 5 days may be necessary to assess the sanitary quality of shellfish.

Adenoviridae↗
PubMed2003

Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients.

PURPOSE AND EXPERIMENTAL DESIGN: Using real-time quantitative methylation-specific PCR (RTQ-MSP), we quantified methylated p16INK4a sequences and determined the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells collected preoperatively, intraoperatively, and postoperatively from 49 patients with hepatocellular carcinoma (HCC). RESULTS: RTQ-MSP was sufficiently sensitive to detect down to 10 genome-equivalents of methylated p16INK4a sequences. Quantitative MSP data were expressed in terms of the methylation index, which was the percentage of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of methylated p16INK4a sequences. Quantities of methylated p16INK4a sequences were detected in peripheral circulation of 80% (23 of 29) of HCC patients. No significant difference was seen in the detectability and concentrations of methylated p16INK4a sequences (range: 10-4046 genome-equivalents/ml) between preoperative plasma and serum samples from HCC patients. Preoperatively, the p16INK4a methylation indices ranged from 0.2 to 100% and from 0.012 to 0.075% in the patients' plasma and buffy coat samples, respectively. After surgical resection, the median p16INK4a methylation indices in plasma and buffy coat concordantly decreased 12- and 15-fold, respectively. These results demonstrated the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring HCC after treatment. Furthermore, none of the intraoperative plasma samples and only two of the intraoperative buffy coat samples were p16INK4a methylation positive. CONCLUSIONS: Quantification of epigenetic changes in peripheral blood by RTQ-MSP is useful for the detection and monitoring of HCC.

Apoptosis↗
PubMed2003

Primer on medical genomics. Part IX: scientific and clinical applications of DNA microarrays--multiple myeloma as a disease model.

Multiple myeloma (MM) is a poorly understood and uniformly fatal malignancy of antibody-secreting plasma cells. Although several key molecular events in disease initiation or progression have been confirmed (eg, 14q32 translocations) or implicated (eg, chromosome 13 deletion), a unifying mechanism of myelomagenesis has eluded investigators. Furthermore, although MM is generally indistinguishable morphologically, it exhibits a tremendous degree of variability clinically with some patients surviving only months and others many years, suggesting that MM is composed of distinct clinical entities. Given that abnormal gene expression lies at the heart of most, if not all, cancers, high-throughput global gene expression profiling has become a powerful tool for investigating the molecular biology and clinical behavior of cancer. DNA microarray technology has facilitated the simultaneous quantification of thousands of cellular messenger RNAs (ie, gene expression). This review discusses progress made in the development of molecular-based diagnostics and prognostics for MM through the dissection of the transcriptome of plasma cells from healthy individuals and patients with MM and other plasma cell dyscrasias.

B-Lymphocytes↗
PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (∼150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ∼20% for AlphaMissense underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular protein trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗
PubMed2004

Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry.

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.

Arabidopsis↗
PubMed1991

[Genesis, progression and prevention of human cancers].

Long-term studies of in vivo kinetics of growth and proliferation of human cancers in vivo have revealed that carcinomas become manifest in patients some 25 years after carcinogenetic process has begun (Fujita '78). During this long period of cancer growth, do cancer cells remain unchanged, in morphology and function? So far, few pathologists have attempted to answer the question. At late stages of cancer growth, human carcinoma cells show high degree of atypism so that correct diagnoses can always be established by microscopic examination of the atypism. By DNA quantification or karyotype analyses of individual cancer cells, however, we found that incipient cancers show much less DNA abnormalities than the advanced ones. It is now clear, from molecular studies (Fialkow '76) that, in principle, a human cancer starts from one mutated host cell. Histochemical studies revealed that the newborn cancer cell and their direct progeny, at least during the earliest stage of carcinogenesis, are diploid in DNA content and seem to show very little abnormalities. Obviously during the progression of cancer growth from the single cell to the final stage, gross changes are accumulated in the cancer cell genome so that highly atypical cells are constant final outcome of the human cancer growth in vivo. We analyzed abnormalities of chromosomal morphology together with DNA contents in advanced human cancer cells and found that, commonly, 10-20 chromosomal changes accumulate in individual cancer cells, thereby marked atypisms are produced in morphology and DNA contents in the cancer cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important, promiscuous divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (~150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ~20% underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗
PubMedDate not supplied

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

Amino Acid Sequence↗
PubMed2006

The real-time polymerase chain reaction.

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.

Animals↗
PubMed2007

The levels of PCV2 specific antibodies and viremia in pigs.

Porcine circovirus 2 quantification by real-time PCR and determination of virus specific antibodies using a peptide based ELISA test was performed on a total of 400 serum samples. Samples for antibody measurement and virus quantification were obtained from a conventional pig farm with a clinical form of post-weaning multisystemic wasting syndrome. Samples were taken during the post-weaning period. Seven litters of weaned piglets (6-25 weeks) were systematically sampled at 2-3 week intervals. Porcine circovirus 2 specific antibodies were detected using the peptide based ELISA test. IgM antibodies were first detected at week 8 and reached their highest levels at week 12; IgG antibody appeared at week 10 and peaked at week 16 with an average titer at 1:3500. Although, the viral load peaked at week 10 (7x10(7) genomes copy/ml of sera), viral infection persisted through to adult age (10(5) genomes copy/ml of sera).

Animals↗
PubMed2001

Quantification of polyoma BK viruria in hemorrhagic cystitis complicating bone marrow transplantation.

Polyoma BK virus (BKV) is frequently identified in the urine of bone marrow transplantation (BMT) patients with hemorrhagic cystitis (HC). However, viruria is common even in asymptomatic patients, making a direct causative role of BKV difficult to establish. This study prospectively quantified BK viruria and viremia in 50 BMT patients to define the quantitative relationship of BKV reactivation with HC. Adenovirus (ADV) was similarly quantified as a control. More than 800 patient samples were quantified for BKV VP1 gene with a real-time quantitative polymerase chain reaction. Twenty patients (40%) developed HC, 6 with gross hematuria (HC grade 2 or higher) and 14 with microscopic hematuria (HC grade 1). When compared with asymptomatic patients, patients with HC had significantly higher peak BK viruria (6 x 10(12) versus 5.7 x 10(7) genome copies/d, P <.001) and larger total amounts of BKV excreted during BMT (4.9 x 10(13) versus 7.7 x 10(8) genome copies, P <.001). There was no detectable increase in BK viremia. Binary logistic regression analysis showed that BK viruria was the only risk factor, with HC not related to age, conditioning regimen, type of BMT, and graft-versus-host disease. Furthermore, the levels of ADV viruria in patients with or without HC were similar and comparable with those of BK viruria in patients without HC, suggesting that the significant increase in BK viruria in HC patients was not due to background viral reactivation or damage to the urothelium. BK viruria was quantitatively related to the occurrence of HC after BMT.

Adenoviridae↗
PubMed2003

Real-time quantitative PCR for analysis of genetically mixed infections of malaria parasites: technique validation and applications.

A technique that can distinguish and quantify genetically different malaria parasite clones in a mixed infection reliably and with speed and accuracy would be very useful for researchers. Many current methods of genotyping and quantification fall down on a number of aspects relating to their ease of use, sensitivity, cost, reproducibility and, not least, accuracy. Here we report the development and validation of a method that offers several advantages in terms of cost, speed and accuracy over conventional PCR or antibody-based methods. Using real-time quantitative PCR (RTQ-PCR) with allele-specific primers, we have accurately quantified the relative proportions of clones present in laboratory prepared ring-stage mixtures of two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi. Accurate and reproducible measurement of the amount of genomic DNA representing each clone in a mixture was achieved over 100-fold range, corresponding to 0.074% parasitised erythrocytes at the lower end. To demonstrate the potential utility of this method, we include an example of the type of application it could be used for. In this case, we studied the growth rate dynamics of mixed-clone infections of P. chabaudi using an avirulent/virulent clone combination (AS (PYR) and AJ) or two clones with similar growth rate profiles (AQ and AJ). The modification of the technique described here should enable researchers to quickly extract accurate and reliable data from in-depth studies covering broad areas of interest, such as analyses of clone-specific responses to drugs, vaccines or other selection pressures in malaria or other parasite species that also contain highly polymorphic DNA sequences.

Alleles↗