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PubMed2005

Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR.

Oral infectious diseases, including dental caries, various forms of periodontitis and oral malodor, are not caused by a single pathogen. The etiology of these diseases is known to be associated with bacterial accumulation and plaque composition on the hard and soft tissues of the oral cavity. Therefore, the quantitative, as well as qualitative, analysis of the microorganisms present in oral biofilms, namely dental plaque, subgingival plaque and tongue debris, is important for diagnosis and rational treatment decisions. The quantitative microbial analysis of oral multi-species biofilms also provides useful information for establishing the etiology of oral infectious diseases. Recently, a 5' fluorogenic, nuclease-based, real-time polymerase chain reaction (PCR) technique has been increasingly employed for the quantitative microbial assessment of the human oral cavity. We review the development and use of TaqMan real-time PCR for quantifying oral bacteria, its role in the diagnosis of oral infectious diseases and their microbial etiology.

Bacteria↗

PubMed1994

Quantitative analysis of the degree of irreversible deformation of F cells and non-F cells and its relationship to cell density in sickle cell disease.

To study the effect of fetal hemoglobin (Hb F) on the formation of dense and irreversibly deformed cells in patients with sickle cell disease (SCD), we investigated the distribution of Hb F and Hb F cells (FC, cells having HbF) and the difference in the degree of irreversible deformation between FC and non-Hb F cells (NFC) in relation to their cell density. We used immunofluorescence to define FC and image analysis to quantify the degree of deformation. Blood samples from 16 patients with Hb F levels ranging from 4 to 24% were separated into less dense [top (T), d < 1.11 g/mL] and dense [bottom (B), d > or = 1.11 g/mL] fractions. We found higher percentages of Hb F and FC in top fractions, suggesting that FC are hydrated more than NFC. Quantitative shape analysis demonstrated that the irreversible deformation of FC in the top fraction [FC(T)] was not significantly different from that of NFC in the fraction [NFC(T)] (p = 0.163), but rather that the irreversible deformation of FC in the bottom fraction [FC(B)] was much milder than for NFC(B) (p < 0.001). The order of the degree of irreversible deformation was NFC(B) > FC(B) > NFC(T) = FC(T). These results clearly demonstrated that FC are less susceptible to both irreversible deformation and dehydration than NFC, suggesting that in circulation, the sickling process plays a major role in cell dehydration. Additionally, we found that the ratio of NFC(B) to FC(B) decreased as a patient's Hb F increased (r = 0.84) and that NFC(B) = FC(B) when Hb F was about 20%.

Adolescent↗

PubMed1982

Lymphocyte proteins in Huntington's disease: quantitative analysis by use of two-dimensional electrophoresis and computerized densitometry.

We used quantitative two-dimensional electrophoresis to study lymphocyte proteins in Hungtington's disease. Three hundred and six polypeptides from 14C-labeled, phytohemagglutinin-stimulated lymphocytes were measured for variation in relative spot density and 186 for variation in spot position by use of a computer program requiring operator interaction. Each polypeptide was measured in a total of 30 electrophoretograms from 28 individuals, including 13 with Huntington's disease, 2 at risk for it, and 13 controls. The study included two sets of identical twins and, as neurological controls, individuals with neurofibromatosis, Alzheimer's disease, or Shy-Drager syndrome. Seven protein polymorphisms were identified among the 186 most dense polypeptides of each gel, corresponding to a minimum average heterozygosity of 1.4%. Stringent criteria were used to define polymorphic proteins, including observation of at least one individual with each of two homozygous phenotypes and one with the heterozygous phenotype, demonstration of the expected gene dosage relationship by quantitative densitometry, consistency with genetic relationships, and reproducibility. One polymorphic protein showed three electrophoretically variant alleles. Our identification of seven polymorphisms among the 186 proteins measured on a single electrophoretogram illustrates the potential of this technique for performing linkage analysis in diseases of genetic origin. However, we observed no quantitative or positional protein variations that were characteristic of (i.e. specific for) Huntington's disease.

Blood Proteins↗

PubMed2004

Quantitative analysis of immunohistologic features of very early rheumatoid synovitis in disease modifying antirheumatic drug- and corticosteroid-naïve patients.

OBJECTIVE: To describe the immunohistochemical features of very early rheumatoid synovitis in disease modifying antirheumatic drug- and corticosteroid-naïve patients. METHODS: Eight patients presenting with oligoarthritis or polyarthritis, who later met American Rheumatism Association criteria for rheumatoid arthritis (RA), underwent needle synovial biopsies of a knee joint within the first 6 weeks after onset of disease symptoms. Using antibodies to CD3, CD8, L26, CD68, and von Willebrand factor, a detailed quantitative immunohistochemical analysis was done. RESULTS: CD3+ T lymphocytes, CD8+ T lymphocytes, L26+ B lymphocytes, and CD68+ macrophages were seen in 8/8 (100%), 7/8 (87%), 4/8 (50%), and 6/6 (100%) of synovial biopsies stained with the respective marker. There was a wide variation in number of positive cells between patients. CD3+ and CD8+ T cells were seen predominantly in perivascular areas, less often in a diffuse distribution, and not in aggregates. L26+ B lymphocytes were found in much smaller numbers compared with T lymphocytes. A mean of 67 vessels/mm2 was noted. No lymphoid aggregates were seen. In all cases, infiltration of macrophages and lymphocytes was limited mainly to relatively superficial parts of synovium, i.e., within 1 to 2 high power fields of the surface. CONCLUSION: An absence of lymphoid aggregates or dramatic vascularity and a predominantly superficial infiltrate consisting mainly of perivascular T cells with few B cells characterized our patients with very early RA of < 6 weeks' duration. Thus, there appear to be some potentially important differences from findings reported in well established disease.

Adrenal Cortex Hormones↗

PubMed1996

Usefulness of a quantitative analysis of the recovery phase patterns of the ST-segment depression in the diagnosis of coronary artery disease.

PURPOSE: To assess the diagnostic value of the recovery phase patterns of the ST-segment depression in patients referred for chest pain. PATIENTS AND METHODS: Continuous plots of ST-segment depression against heart rate during exercise and recovery were constructed within a population of 160 consecutive symptomatic patients who all had undergone catheterization (80 with > or = 1 stenosis > or = 50%). We used a new quantitative method of measurement allowing all kinds of rate recovery loops (even the so-called "intermediate" loops) to be considered for analysis. The measurements of the heart rate (HR)-adjusted ST-segment depression were performed at 20 and 60 ms from the J point, providing two different values of a quantified recovery loop index (RLI): RLI 20 and RLI 60. RESULTS: Both RLI showed a higher specificity (0.81 +/- 0.04 and 0.74 +/- 0.05, respectively) than did the standard criterion (0.65 +/- 0.10), but the difference was significant regarding RLI 20 only (P = 0.011). As to the sensitivity, no significant differences were found among all of the criteria (0.74 +/- 0.05, 0.80 +/- 0.04, 0.76 +/- 0.05, respectively). The timing of measurements of the RLI within the repolarization phase did not affect their overall accuracy (0.77 +/- 0.03 for both RLI). The values of the receiver-operating characteristic (ROC) curve areas were significantly greater for both RLI (0.83 +/- 0.06 and 0.84 +/- 0.06 respectively) than for the standard criterion (0.75 +/- 0.07; P < 0.02). Finally, both RLI allowed to differentiate accurately the study subjects according to the number of diseased vessels, whereas the standard criterion could only distinguish between CAD patients and subjects with normal angiograms. CONCLUSION: The quantitative analysis of the rate recovery phase patterns appears to be useful for the diagnosis of coronary heart disease and the assessment of its severity in symptomatic patients.

Adult↗

PubMed2005

Quantitative analysis of protein dynamics during asymmetric cell division.

In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis.

Animals↗

PubMed2005

A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples.

BACKGROUND: There is increasing interest in DNA methylation and in its implication in transcriptional gene silencing, a phenomenon commonly seen in human cancer. AIMS: To develop a new method that would allow quantitative DNA methylation analysis in a large range of clinical samples, independently of the processing protocol. METHODS: A methylation sensitive dot blot assay (MS-DBA) was developed, which is quantitative and combines bisulfite modification, PCR amplification using primers without CpG sites, and dot blot analysis with two probes specific for methylated and unmethylated DNA. RESULTS: The established method was used to study methylation of the hTERT, APC, and p16 promoter regions in microdissected, formalin fixed and paraffin wax embedded tissues. CONCLUSIONS: MS-DBA is a sensitive, specific, and quantitative approach to analyse DNA methylation in a variety of frozen or fixed tissues. Moreover, MS-DBA is rapid, easy to perform, and permits the screening of a large panel of samples in one experiment. Thus, MS-DBA can facilitate the routine analysis of DNA methylation in all types of clinical samples.

Adenocarcinoma↗

PubMed2002

Quantitative analysis of chiral compounds from unresolved peaks in capillary electrophoresis using multivariate calibration with experimental design and artificial neural networks.

Quantitation of optical isomers can be achieved even from incompletely resolved peaks with a multivariate calibration applying a combination of experimental design and artificial neural networks (ANN). Using the proposed approach, method development can be more efficient and analysis time shortened while quantitation of optical isomers with acceptable precision (+/-1-3%) can be achieved.

Journal Article↗

PubMed1997

Quantitative analysis of neurofibromatosis type 2 gene transcripts in meningiomas supports the concept of distinct molecular variants.

Meningiomas frequently show mutational inactivation of the neurofibromatosis type 2 tumor suppressor gene (NF2 gene). In a previous study, mutations were preferentially observed in the fibroblastic and transitional subtypes (75%), whereas the meningothelial variant was significantly less affected (25%). To study a potential role of the NF2 gene on the transcriptional level, we have analyzed NF2 transcripts in 67 meningiomas of different subtypes. A competitive reverse transcriptase-PCR assay with an external NF2 gene standard was used for quantitative mRNA analysis. Fibroblastic and transitional meningiomas exhibited significantly lower levels of NF2 mRNA compared with meningothelial variants (p = 0.001, unpaired t test). These data support the concept of a distinct molecular pathway in the formation of meningothelial meningiomas independent from the NF2 gene or its gene product merlin/schwannomin. In addition, in these tumors, NF2 expression was reduced by a factor of 10 (p < 0.001, unpaired t test) in those meningiomas with NF2 gene mutations suggesting decreased stability or impaired transcription of mutated NF2 mRNA. In conclusion, our data provide further evidence for molecular differences between subtypes of meningiomas and support an NF2-independent pathogenesis of meningothelial meningiomas.

Genes, Neurofibromatosis 2↗

PubMed1999

Rapid Quantitative Analysis of Individual (18)FDG-PET Scans.

To evaluate an objective, quantitative, semi-automatic method of single (18)FDG-PET scan analysis. Scans of 60 normal controls was compared to scans from individuals with known pathology using two methods of visual analysis and Statistical Parametric Mapping with two different smoothing filters. We analyzed the sensitivity, specificity, and accuracy of each technique and evaluated the agreement between the four methods using a kappa statistic. When corrected for false positives, which were due to anatomical distortions, all techniques had a sensitivity and accuracy of 0.5 to 0.6. Specificity was highest, 1.0, using SPM with a 16 mm smoothing filter, followed by visual analyses. Good agreement between all techniques was found. A computerized expert which makes use of a normal data base and SPM to analyze single (18)FDG-PET scans can aid in the routine interpretation of scans and provide rapid, quantification of abnormalities for research or clinical purposes.

Journal Article↗

PubMed2001

Evaluation of cerebral maturation by visual and quantitative analysis of resting electroencephalography in children with primary nocturnal enuresis.

This study was undertaken to evaluate resting electroencephalographic (EEG) changes and their relations to cerebral maturation in children with primary nocturnal enuresis. Cerebral maturation is known to be important in the pathogenesis of this disorder. Twenty-five right-handed patients with primary nocturnal enuresis, aged 6 to 14 years, and 23 age- and sex-matched healthy children were included in this cross-sectional case-control study. The abnormalities detected using such techniques as hemispheral asymmetry, regional differences, and hyperventilation response in addition to visual and quantitative EEG analysis were examined statistically by multivariate analysis. A decrease in alpha activity in the left (dominant hemisphere) temporal lobe and in the frontal lobes bilaterally and an increase in delta activity in the right temporal region were observed. We concluded that insufficient cerebral maturation is an important factor in the pathogenesis of primary nocturnal enuresis, and EEG, as a noninvasive and inexpensive method, could be used in evaluating cerebral maturation.

Adolescent↗

PubMed1993

Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status.

Lesch-Nyhan syndrome is an X-linked recessive disorder caused by molecular defects within the HPRT gene. Deletional forms of this syndrome, most of which are inherited, account for 15% of the cases. In addition, a large percentage of cases are due to de novo point mutations. We have used complementary fluorescence-based PCR assays to analyse disease-causing mutations in three unrelated families: (1) inheritance of dye-labelled PCR products of linked polymorphic loci mapping within and flanking the HPRT gene; (2) dye-labelled exon dosage analysis and (3) automated fluorescence-based DNA sequence analysis. Our results using fluorescent, dye-tagged PCR products show that inheritance of two polymorphic small tandem repeats, HPRTB [AGAT]n, mapping within intron 3 of the HPRT gene, and the CA-repeat at DXS294 can be used to establish linkage to the disease. In addition, we modified a previously described PCR protocol to use fluorescent dye-labelled oligoprimers and an ABI Gene Scanner in order to rapidly quantitate deletional forms of Lesch-Nyhan syndrome. Quantitative PCR analysis of individual exons followed by dosage analysis confirmed a deletion encompassing exon 9. A similar approach was used to confirm a previously described HPRT gene duplication involving exons 2 and 3. In this analysis, we co-amplified the HPRTB [AGAT]n and HUMARA [AGC]n repeats and confirmed increased exon dosage in carriers for the duplication. DNA sequence analysis remains the method of choice for delineating new disease-causing mutations, most of which are non-deletional forms of Lesch-Nyhan syndrome. We have also used a cycle-sequencing strategy employing dye-labelled dideoxy terminators and a laser-activated, fluorescence-emission DNA sequencer in order to define carrier status in 10 family members at risk for Lesch-Nyhan syndrome due to a splice donor mutation in intron 7. Our DNA sequence analyses corroborate small tandem repeat (STR) inheritance patterns in this family. Multiple fluorescence-based strategies should facilitate rapid diagnosis of the various Lesch-Nyhan disease-causing mutations.

Adolescent↗

PubMed1999

Quantitative analysis of synovial membrane inflammation: a comparison between automated and conventional microscopic measurements.

OBJECTIVE: The objective of this study was to quantify selected features of chronic synovial tissue inflammation by computerised image analysis and to validate the results by comparison with conventional microscopic measurements. METHODS: Synovial biopsy samples were obtained from the knee joints of patients with chronic arthritis and prepared for immunohistochemical analysis using standard techniques. Following the development of special software, four parameters of chronic synovial inflammation were evaluated: intimal layer thickness, CD3+ cell infiltration, CD8+ cell infiltration and vascularity. Intimal layer thickness was expressed in microns. The intensity of CD3+ and CD8+ cell infiltration was expressed as the percentage area of the tissue section occupied by positively stained cells. Vascularity was expressed as the percentage area occupied by blood vessels. Conventional quantitative microscopic analysis was also undertaken and the results from both methods compared. RESULTS: Seventy eight tissue sections were selected for study. Measurements of intimal layer thickness by both techniques correlated strongly: r = 0.85, p = 0.0006. Measurements of CD8+ cell infiltration, usually widely dispersed, also correlated well: r = 0.64, p = 0.005. Measurements of CD3+ cell infiltration, often densely aggregated, correlated less well: r = 0.55, p = 0.02. Measurements of vascularity demonstrated no statistically significant correlation: r = 0.41, p = 0.07. Proficiency in the use of computerised image analysis was readily acquired. CONCLUSION: Computerised image analysis was successfully applied to the measurement of some features of synovial tissue inflammation. Further software development is required to validate measurement of blood vessels of variable size.

Arthritis↗

PubMed1988

Quantitative analysis of synaptic input to gonadotropin-releasing hormone neurons in normal mice and hpg mice with preoptic area grafts.

Mutant hypogonadal (hpg) mice with a truncated gene for the precursor to gonadotropin-releasing hormone (GnRH) show certain aspects of recovery of reproductive function after receiving grafts of normal preoptic area into the third ventricle. We have previously shown that GnRH neurons from within the grafts can innervate the appropriate neural-hemal target in the host. To determine if in turn these exogenously derived neurons receive a synaptic input comparable to the GnRH neurons in the normal animal we have now carried out a quantitative ultrastructural analysis to compare the synaptic input to GnRH neurons in the normal preoptic area and in the grafts. In almost all cases GnRH cells or dendrites in normal brains and within the grafts received a synaptic input. In normal animals, input to GnRH dendritic profiles was significantly greater (P less than 0.001) than to the somatic plasma membrane and this trend was also observed within the grafts though the difference was not statistically significant. In addition, no statistically significant difference was found between the input to GnRH structures within the grafts and in normal preoptic area. However, a substantial variability in input among grafted animals was evident which was not observed in normal animals. The sources of variability within the grafts are discussed and we suggest that the deficiencies and differences that exist in regulation of gonadotropin secretion among grafted hpg animals may be reflected in aberrant synaptic input.

Animals↗

PubMed2004

[Study of net analyte signal with near-infrared spectra for quantitative analysis].

Near-infrared spectroscopy is a rapid, high-efficiency, non-destructive and low-cost analytical technique, which has found widespread use for quantitative and qualitative analysis. Because of the complex nature of NIR spectra, multivariate calibration model plays a key role in the spectroscopic analysis. Further understandings of the model can be helpful for optimizing the model's performance. This paper introduces a convenient definition of net analyte signal, which is one of the figures of merit charactering multivariate calibration model. The absorbance data of the glucose aqueous solution are used for the calculation of net signal and explanation of the qualitative relationships between net analyte signal of glucose and absorptivity coefficients of other components. Qualitative changes of net signal with the complexity of samples are also given. Finally a simple expression is proposed for estimating the prediction error of concentration, which is instructive for further study of the robustness and accuracy of the multivariate calibration model.

English Abstract↗

PubMed2001

[Polyphenol constituents from Salacia species: quantitative analysis of mangiferin with alpha-glucosidase and aldose reductase inhibitory activities].

Mangiferin, three catechins, and two catechin dimers were isolated from the roots of Salacia reticulata (SRE), and examined their inhibitory activities against several carbohydrate metabolize enzymes (sucrase, maltase, isomaltase, alpha-amylase, and aldose reductase). Among them, mangiferin was found to inhibit sucrase, isomaltase, and aldose reductase from rat with IC50 values of 87, 216 and 1.4 micrograms/ml, respectively. The inhibitory activities of mangiferin are competitive for sucrase and isomaltase with inhibitor constant (Ki) 55 micrograms/ml and 70 micrograms/ml, respectively. In order to determine the mangiferin contents in the water extracts from the roots of S. reticulata, a quantitative analytical method by means of HPLC was developed and the mangiferin contents in SRE were determined to be in the range of 0.9-2.3% by the application of this method. A high linear correlation (r = 0.934) was observed between the mangiferin contents and the sucrase inhibitory activity. In addition, this analytical procedure of mangiferin was found to be applicable for other Salacia species (S. oblonga, S. chinensis, and S. prinoides). Thus, the quantitative HPLC analysis of mangiferin was supposed to be suitable for the quality control of Salacia species and its products.

Aldehyde Reductase↗

PubMedDate not supplied

Quantitative analysis of motion control in long term microgravity.

In the frame of the 179-days EUROMIR '95 space mission, two in-flight experiments have foreseen quantitative three-dimensional human movement analysis in microgravity. For this aim, a space qualified opto-electronic motion analyser based on passive markers has been installed onboard the Russian Space Station MIR and 8 in flight sessions have been performed. Techhology and method for the collection of kinematics data are described, evaluating the accuracy in three-dimensional marker localisation. Results confirm the suitability of opto-electronic technology for quantitative human motion analysis on orbital modules and raise a set of "lessons learned", leading to the improvement of motion analyser performance with a contemporary swiftness of the on-board operations. Among the experimental program of T4, results of three voluntary posture perturbation protocols are described. The analysis suggests that a short term reinterpretation of proprioceptive information and re-calibration of sensorimotor mechanisms seem to end within the first weeks of flight, while a continuous long term adaptation process allows the refinement of motor performance, in the frame of never abandoned terrestrial strategies.

Adaptation, Physiological↗

PubMed1979

Quantitative analysis of monosialogangliosides by high-performance liquid chromatography of their perbenzoyl derivatives.

A quantitative high-performance liquid chromatographic method for the analysis of monosialogangliosides as their perbenzoyl derivatives has been devised. Samples containing as little as 3 nmol were converted to their perbenzoyl derivatives by reaction with 0.1 ml of 10% benzoyl chloride in pyridine at 60 degrees C for 1 hr. The products were purified by silicic acid chromatography and analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis was performed with a 50 cm X 2.1 mm LiChrosphere SI 4000 column and a linear gradient of 7-23% dioxane in hexane in 18 min. Detection was at 230 nm. The detector response was found to be proportional to the amount of monosialoganglioside analyzed. As little as 50 pmol of injected material could be conveniently quantitated. The overall yield from derivatization and chromatography, as determined with radiolabeled GM1, was found to be 86%. To take advantage of the high sensitivity of the HPLC, a small-scale isolation method for gangliosides was devised. This method coupled with HPLC isotope dilution analysis was used to analyze the GM3 content of 1 ml of human plasma.

Benzoates↗