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PubMed2006

Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project.

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).

Animals↗

PubMed1995

[Ultrasound blood flow measurement in malignant tumors is a possible new prognostic factor].

OBJECTIVE: The aim of the present study was to find a possible relationship between biological behavior of breast cancer and sonographically detectable blood flow. METHODS: 157 patients with ductal invasive breast cancer were examined by means of a new sonographic procedure, the MEM (Maximum Entropy Method), able to detect considerably lower blood flow velocities than doppler sonography. In the absence of objectifyable quantification methods, findings were allotted to 3 classes, depending on the visual color information obtained. Blood flow was correlated to tumor size, lymph node and receptor status, ploidy and S-phase-fraction. RESULTS: Most patients with small tumors, no lymph node metatases, positive receptors, diploid genome and low S-phase found to have low blood flow. CONCLUSION: This close relation between established prognostic factors and results of sonographic blood flow examination with the MEM might indicate a new preoperative prognostic factor, which, however, will have to be proven by larger studies.

Biomarkers, Tumor↗

PubMed1996

A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors.

Adeno-associated virus (AAV) vectors are being developed for in vivo and ex vivo gene transfer to human cells. At present, widespread usage of AAV vectors is limited primarily by difficulties in generating recombinant virions on a scale sufficient for in-depth preclinical and clinical trials. However, recent work in several laboratories suggests that this technical obstacle should be overcome in the near future. As a result, it can be anticipated that the interest in AAV vectors will expand, Thus, it becomes important to develop assay systems that will permit accurate quantification of the infectivity of AAV vectors derived from a variety of sources. We have developed an assay using a cell line that expresses AAV helper functions (rep and cap) upon induction by adenovirus infection. This assay system is based on the replication of input rAAV genomes rather than transgene expression (transduction). Thus, infectivity titrations in this system yield an estimation of rAAV infectious particles irrespective of the promoter or transgene present in the vector genome. Moreover, this assay method is more sensitive than conventional methods being used in other laboratories.

Adenoviridae↗

PubMed2000

Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR.

Oocytes, in general, are greatly enriched in mitochondria to support higher rates of macromolecular synthesis and critical physiological processes characteristic of early development. An inability of these organelles to amplify and/or to accumulate ATP has been linked to developmental abnormality or arrest. The number of mitochondrial genomes present in mature mouse and human metaphase II oocytes was estimated by fluorescent rapid cycle DNA amplification, which is a highly sensitive technique ideally suited to quantitative mitochondrial DNA (mtDNA) analysis in individual cells. A considerable degree of variability was observed between individual samples. An overall average of 1.59 x 10(5) and 3.14 x 10(5) mtDNA molecules were detected per mouse and human oocyte, respectively. Furthermore, the mtDNA copy number was examined in polar bodies and contrasted with the concentration in their corresponding oocytes. In addition, the density of mtDNA in a cytoplasmic sample was estimated in an attempt to determine the approximate number of mitochondria transferred during clinical cytoplasmic donation procedures as well as to develop a clinical tool for the assessment and selection of oocytes during in vitro fertilisation procedures. However, no correlation was identified between the mtDNA concentration in either polar bodies or cytoplasmic samples and their corresponding oocyte.

Animals↗

PubMed2003

An overview of quantitative PCR assays for biologicals: quality and safety evaluation.

Applications of real time quantitative PCR (Q-PCR) to the evaluation of biologicals quality and safety are discussed in the following areas: (i) endogenous retrovirus load quantification in production cell cultures, (ii) viral clearance studies, (iii) lot release testing, (iv) detection of specific viral contaminants for raw material screen and in-process control, (v) mycoplasma and bioburden detection, (vi) host cell DNA quantification, and (vii) genetic stability characterization of production cell lines.

Animals↗

PubMed1997

Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans.

V(D)J recombinase is normally involved in the highly regulated rearrangement of immunoglobulin and T-cell-receptor gene segments (in B and T cells, respectively) to form functional antibody genes and T-cell-receptor genes. Occasionally, this tightly controlled process acts on inappropriate places in the genome and results in deletions and translocations. Some of these illegitimate V(D)J recombinase-mediated events have been implicated in the genetic changes associated with several forms of leukemia and lymphoid malignancy. We have developed a sensitive, specific polymerase chain reaction (PCR)-based assay to quantify such events in the peripheral blood cells of humans. This assay detects a V(D)J recombinase-mediated deletion in the hprt gene, which codes for a housekeeping enzyme and is not implicated in cancer development. Alterations in this gene serve as a surrogate indicator for these illegitimate events, which may be occurring throughout the genome. The assay involves a hemi-nested PCR with two sets of primers. Multiple replicates of genomic DNA (each representing 4 x 10(5) cells) are amplified with specific primers under conditions in which a single copy will give a detectable PCR product. Poisson statistics are then used to estimate the deletion mutant frequency. The frequency of cells with the hprt deletion among 20 healthy young adults ranged from <1.3 x 10(-7) to 4.1 x 10(-7) and was compared with the frequency of t(14;18) previously determined in these same individuals. No correlation was found between the frequencies of these two measures of genomic rearrangement. The DNA sequences at the deletion junctions were determined and provided evidence for multiple independent mutations in some individuals. This assay may serve as a biomarker for the level of illegitimate V(D)J recombination occurring in peripheral blood cells of humans.

Adolescent↗

PubMed1995

High nm23-H1/NDPK-A expression in Ewing tumors: paradoxical immunohistochemical reactivity and lack of prognostic significance.

Expression of nm23-H1/NDPK-A has been reported to correlate inversely with metastasizing potential of rodent experimental cells and some human tumors. In the search for reliable molecular prognostic indicators for Ewing tumors (ET), a group of aggressive presumably neuroectodermal malignancies in children and adolescents, we studied nm23-H1/NDPK-A expression. Northern-blot and RT-PCR analyses were employed to semi-quantificatively measure nm23-H1 mRNA levels in ET cell lines and tissue extracts. A panel of monoclonal antibodies (MAbs) were used to evaluate protein abundance by Western blotting and immunohistochemistry. The nm23-H1/NDPK-A gene was also investigated on the DNA level to define possible genomic alterations. Our results revealed neither nm23-H1 allelic loss nor gene amplification and failed to show any significant variation in nm23-H1 mRNA or NDPK-A protein levels of primary or metastatic ET. NDPK-A protein levels were high and comparable to those of MCF-7 breast-cancer cells and to aggressive stage-IV neuroblastoma cell lines. nm23-H2/NDPK-B expression in ET was slightly more variable but generally lower than in MCF-7 cells. In the immunohistochemical analysis, however, discrepancies in the reactivity patterns with different antibodies were observed. Differential sensitivity to various fixation methods and heat treatment pointed to a structurally polymorphic NDPK-A protein. nm23-H1 expression studies using immunohistochemistry for prognostic counselling should thus be interpreted with caution.

Adolescent↗

PubMed2002

Gene-sequence-tag expression analyses of 1,800 genes related to chloroplast functions.

Quantification of the expression levels of nuclear genes encoding plastid proteins under different genetic or environmental conditions can contribute to the genetic dissection of plastid functions. To facilitate such measurements, a set of 1,827 Arabidopsis thaliana genes coding for plastid proteins was PCR-amplified from genomic DNA and spotted on nylon membranes to generate an array of chloroplast-specific gene-sequence-tags (GSTs). The sensitivity and reliability of the experimental system was evaluated and a procedure was developed for detecting differential gene expression. The GST array was found to serve as a reliable monitor of changes in gene expression induced by environmental and genetic alteration of chloroplast functions. Based on comparisons of dark- versus light-grown seedlings, and wild-type versus prpl11-1 plants, lists of differentially expressed genes are provided which include 193/7 and 25/42 up/down-regulated genes, respectively. The cut-off values for differential expression were 2.5-times (up) and 0.40 (down). Additional up-regulated genes with relatively low expression ratios (from 1.5- to 2.5-times) or down-regulated with relatively high ratios (0.4-0.67) can be accessed at the website: http://www.mpiz-koeln.mpg.de/~richly/GST-array.html. A sample of genes analysed by quantitative reverse transcription PCR confirmed the expression profiles monitored by the GST array. Differential hybridisation experiments with the prpl11-1 mutant revealed the existence of regulatory networks sensing the protein state of the chloroplast and transmitting the signal to the nucleus.

Arabidopsis↗

PubMed2024

Bioinformatic approaches for accurate assessment of A-to-I editing in complete transcriptomes.

A-to-I RNA editing is an RNA modification that alters the RNA sequence relative to the its genomic blueprint. It is catalyzed by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes, and contributes to the complexity and diversification of the proteome. Advancement in the study of A-to-I RNA editing has been facilitated by computational approaches for accurate mapping and quantification of A-to-I RNA editing based on sequencing data. In this chapter we review some of the main computational approaches currently used, describe potential hurdles, challenges and pitfalls, and discuss possible ways to mitigate them.

RNA Editing↗

PubMed2005

Real-time in vivo imaging of stem cells following transgenesis by transposition.

Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.

Animals↗

PubMed2003

Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA.

BACKGROUND: Tick borne encephalitis virus (TBEV), is a human flavivirus causing tick borne encephalitis (TBE), a viral infection of the central nervous system endemic in Europe and Asia. OBJECTIVES: To develop a reverse transcription polymerase chain reaction (RT-PCR) assay based on quantitative real-time RT-PCR technology (TaqMan) for detection and quantification of TBEV RNA. The test includes an internal control (IC) to avoid false negative results. STUDY DESIGN: The system was established and validated using wild-type (WT) non-infectious synthetic RNA representing a fragment of the 3' non-coding region of the TBEV genome. In addition, synthetic RNA differing from the WT synthetic RNA by a unique probe binding region was used as IC to monitor the overall efficiency of the RT-PCR. RESULTS: The analytical sensitivity of the assay was at least ten copies of the TBEV synthetic transcript in presence of 50 copies of the IC. Successful amplification was obtained for different strains within the TBEV complex (Hypr, Hochosterwitz, Laibach, Elsass=Alsace, ZZ9, Wladiwostok). Among 14 serum and 21 cerebrospinal fluid (CSF) samples obtained from 28 patients with clinical suspicion of TBEV 1 CSF sample tested positive for TBEV RNA. In addition, no TBEV RNA could be detected in blood samples obtained from three vaccinated people 1 and 3 days post-vaccination. Thus indicating that a positive result is unlikely to be caused by recent vaccination. CONCLUSIONS: A quantitative, highly sensitive and specific real-time RT-PCR assay has been developed for the detection of TBEV RNA. Inclusion of an IC is important to monitor the possible occurrence of false-negative results caused by the presence of inhibitory factors. This assay should be an important asset for the routine laboratory detection of TBEV RNA.

3' Untranslated Regions↗

PubMed2006

Impaired expression of CYP2D6 in intermediate metabolizers carrying the *41 allele caused by the intronic SNP 2988G>A: evidence for modulation of splicing events.

We investigated the molecular basis for low expression and activity of CYP2D6 associated with the CYP2D6*41 allele in about 10-15% of Caucasians with intermediate metabolizer phenotype. With respect to two previously described polymorphisms in the promoter (-1584C>G) and in intron 6 (2988G>A; c.985+39G>A), the three most frequent functional alleles have the distinct haplotypes 2D6*1[CG], 2D6*2[GG] and 2D6*41[CA], respectively. Reporter gene analyses in transiently transfected HepG2 and Huh7 hepatoma cells did not indicate changes in transcription rate by these polymorphisms. By reverse-transcription polymerase chain reaction analysis of liver RNA of genotyped patients, however, we discovered that the 2988G>A change was associated with increased levels of a nonfunctional splice variant lacking exon 6. Quantification by denaturing high-performance liquid chromatography revealed up to 7.3-fold increased levels of the splice variant and up to 2.9-fold less functional transcript in carriers of 2D6*41, in good concordance with concomitant changes in immunoquantified CYP2D6 protein. Recombinant expression of the entire genomic sequence coding for 2D6*41, 2D6*2 and 2D6*1 alleles but lacking the upstream region in COS-1 and Huh7 cell lines resulted in two-fold to five-fold reduced levels of CYP2D6 mRNA containing exon 6, apoprotein and enzyme activity of 2D6*41. These experiments establish the causal relationship between the intron 6 single-nucleotide polymorphism 2988G>A and the low expression phenotype associated with allele 2D6*41. These data improve the CYP2D6 genotype-phenotype relationship and they demonstrate that major phenotype changes occurring in large population subgroups can be caused by intronic polymorphisms outside of splice site consensus sequences.

Alleles↗

PubMed2006

Proteomic analysis of seed filling in Brassica napus. Developmental characterization of metabolic isozymes using high-resolution two-dimensional gel electrophoresis.

Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 nonredundant proteins. These proteins were classified into 14 functional categories based upon the Arabidopsis (Arabidopsis thaliana) genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins, respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to acetyl-coenzyme A [corrected] is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a systematic analysis of metabolic processes operating in developing B. napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) that is accessible to the public domain.

Arabidopsis↗

PubMed2006

Golli-MBP copy number analysis by FISH, QMPSF and MAPH in 195 patients with hypomyelinating leukodystrophies.

The inherited disorders of CNS myelin formation represent a heterogeneous group of leukodystrophies. The proteolipoprotein (PLP1) gene has been implicated in two X-linked forms, Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2, and the gap junction protein alpha12 (GJA12) gene in a recessive form of PMD. The myelin basic protein (MBP) gene, which encodes the second most abundant CNS myelin protein after PLP1, presents rearrangements in hypomyelinating murine mutants and is always included in the minimal region deleted in 18q- patients with an abnormal hypomyelination pattern on cerebral MRI. In this study, we looked at the genomic copy number at the Golli-MBP locus in 195 patients with cerebral MRI suggesting a myelin defect, who do not have PLP1 mutation. Although preliminary results obtained by FISH suggested the duplication of Golli-MBP in 3 out of 10 patients, no abnormal gene quantification was found using Quantitative Multiplex PCR of Short Fluorescent fragments (QMPSF), Multiplex Amplifiable Probe Hybridization (MAPH), or another FISH protocol using directly-labelled probes. Pitfalls and interest in these different techniques to detect duplication events are emphasised. Finally, the study of this large cohort of patients suggests that Golli-MBP deletion or duplication is rarely involved in inherited defects of myelin formation.

DNA Primers↗

PubMed2003

Reconstruction of the central carbon metabolism of Aspergillus niger.

The topology of central carbon metabolism of Aspergillus niger was identified and the metabolic network reconstructed, by integrating genomic, biochemical and physiological information available for this microorganism and other related fungi. The reconstructed network may serve as a valuable database for annotation of genes identified in future genome sequencing projects on aspergilli. Based on the metabolic reconstruction, a stoichiometric model was set up that includes 284 metabolites and 335 reactions, of which 268 represent biochemical conversions and 67 represent transport processes between the different intracellular compartments and between the cell and the extracellular medium. The stoichiometry of the metabolic reactions was used in combination with biosynthetic requirements for growth and pseudo-steady state mass balances over intracellular metabolites for the quantification of metabolic fluxes using metabolite balancing. This framework was employed to perform an in silico characterisation of the phenotypic behaviour of A. niger grown on different carbon sources. The effects on growth of single reaction deletions were assessed and essential biochemical reactions were identified for different carbon sources. Furthermore, application of the stoichiometric model for assessing the metabolic capabilities of A. niger to produce metabolites was evaluated by using succinate production as a case study.

Aspergillus niger↗

PubMed1994

Sequence spectra of spontaneous lacZ gene mutations in transgenic mouse somatic and germline tissues.

As a critical step in determining whether transgenic mouse gene mutation systems are suitable models for the detection and quantification of induced gene mutations in vivo, spontaneous mutant frequencies and mutation spectra have been characterized for liver, bone marrow, and male germ cells of the lacZ transgenic mouse strain 40.6. The lacZ transgene is carried on a recombinant bacteriophage lambda shuttle vector that is recovered from mouse genomic DNA, and analysed in vitro for mutations that occurred in the mouse tissues. Mutations are detected visually as clear or pale blue plaques when X-gal is the substrate for beta-galactosidase; whereas, the wild-type plaques are dark blue. There was no statistical difference in the mutant frequency among the three tissues studied, the pooled mutant frequency being 2.23 +/- 0.41 per 10(5) pfu. The predominant type of mutation was GC-->AT transitions, with most occurring in 5'-CpG dinucleotides, suggesting that the deamination of 5-methylcytosine is the main mechanism of mutagenesis. There was, however, a statistically significant difference in the base pair substitution mutation spectrum for the liver and bone marrow when mutations were grouped according to GC or AT base-pairs. The proportion of transition versus transversion mutations was also statistically different among the three tissues, resulting mainly from the fact that germ cells were different from both bone marrow and liver. A lower number of spontaneous transitions in male germ cells was accompanied by an increase in transversions, with the proportion of GC-->AT transitions in 5'-CpG sites also declining.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

Mitochondrial DNA quantity increases with histopathologic grade in premalignant and malignant head and neck lesions.

PURPOSE: Mitochondria are highly susceptible to oxidative damage. Although mitochondrial function decreases with oxidative damage, overall mitochondrial DNA (mtDNA) content increases to compensate for general mitochondrial dysfunction. We performed quantitative polymerase chain reaction for genes specific to mitochondrial and nuclear genomes to investigate relative mitochondrial abundance in a spectrum of dysplastic head and neck lesions. EXPERIMENTAL DESIGN: DNA from mild, moderate, and severe dysplasias, as well as invasive tumors and normal mucosal cells, was extracted. Using quantitative polymerase chain reaction, mitochondrial to nuclear DNA ratios were determined by quantification of cytochrome c oxidase subunit 1 (CoxI) and beta-actin genes. RESULTS: Mean CoxI/beta-actin DNA ratios for mild, moderate, and severe premalignant lesions were 0.0529, 0.0607, and 0.1021, respectively. The mean ratio for the normal mucosal cells contained in saliva was 0.0537, whereas the mean ratio for tumors was 0.1667. As a whole, our experimental model demonstrated significance (P = 0.0358). Comparisons between individual categories showed borderline significance when compared with the normal group, with P values of 0.0673, 0.0747, and 0.0824 for moderate and severe dysplasia and invasive tumor, respectively. CONCLUSIONS: Head and neck squamous cell carcinomas arise through premalignant intermediates and may be merely morphologic manifestations of accumulated genetic alterations. In keeping with this molecular tumor progression model, our study shows that mtDNA increases according to histopathologic grade, a phenomenon that may be a feedback mechanism that compensates for a generalized decline in respiratory chain function. Therefore, high mtDNA content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathologic grade.

Adult↗

PubMed2007

Hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect.

Worker termites of the genus Reticulitermes are temporally-arrested juvenile forms that can terminally differentiate into adultsoldier- or reproductive-caste phenotypes. Soldier-caste differentiation is a developmental transition that is induced by high juvenile hormone (JH) titers. Recently, a status quo hexamerin mechanism was identified, which reduces JH efficacy and maximizes colony fitness via the maintenance of high worker-caste proportions. Our goal in these studies was to investigate more thoroughly the influences of the hexamerins on JH-dependent gene expression in termite workers. Our approach involved RNA interference (RNAi), bioassays and quantification of gene expression. We first investigated the expression of 17 morphogenesis-associated genes in response to RNAi-based hexamerin silencing. Hexamerin silencing resulted in significant downstream impacts on 15 out of the 17 genes, suggesting that these genes are members of a JH-responsive genomic network. Next, we compared gene-expression profiles in workers after RNAi-based hexamerin silencing to that of (i) untreated workers that were held away from the colony; and (ii) workers that were also held away from the colony, but with ectopic JH. Here, although there was no correlation between hexamerin silencing and colony-release effects, we observed a significant correlation between hexamerin silencing and JH-treatment effects. These findings provide further evidence supporting the hypothesis that the hexamerins modulate JH availability, thus limiting the impacts of JH on termite caste polyphenism. Results are discussed in a context relative to outstanding questions on termite developmental biology, particularly on regulatory gene networks that respond to JH-, colony- and environmental-cues.

Animals↗