Photolysis of cell-permeant caged inositol pyrophosphates controls oscillations of cytosolic calcium in a ß-cell line.

Among many cellular functions, inositol pyrophosphates (PP-InsPs) are metabolic messengers involved in the regulation of glucose uptake, insulin sensitivity, and weight gain. However, their mechanisms of action are still poorly understood. So far, the influence of PP-InsPs on cellular metabolism has been studied by overexpression or knockout/inhibition of relevant metabolizing kinases (IP6Ks, PPIP5Ks). These approaches are, inter alia, limited by time-resolution and potential compensation mechanisms. Here, we describe the synthesis of cell-permeant caged PP-InsPs as tools to rapidly modulate intracellular levels of defined isomers of PP-InsPs in a genetically non-perturbed cellular environment. We show that caged prometabolites readily enter live cells where they are enzymatically converted into still inactive, metabolically stable, photocaged PP-InsPs. Upon light-triggered release of 5-PP-InsP5, the major cellular inositol pyrophosphate, oscillations of intracellular Ca2+ levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP5, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca2+]i oscillations, a key element in triggering exocytosis and secretion in ß-cells.

Quantifying variant differences in DNA melting curves: Effects of length, melting rate, and curve overlay.

High resolution DNA melting of PCR products is a simple technique for sequence variant detection and analysis. However, sensitivity and specificity vary and depend on many factors that continue to be defined. We introduce the area between normalized melting curves as a metric to quantify genotype discrimination. The effects of amplicon size (51-547 bp), melting rate (0.01-0.64 °C/s) and analysis method (curve shape by overlay vs absolute temperature differences) were qualitatively and quantitatively analyzed. To limit experimental variance, we studied a single nucleotide variant with identical predicted wild type and homozygous variant stabilities by nearest neighbor thermodynamic theory. Heterozygotes were easier to detect in smaller amplicons, at faster melting rates, and after curve overlay (superimposition), with some p-values <10-20. As heterozygote melting rates increase, the relative magnitude of heteroduplex contributions to melting curves increases, apparently the result of non-equilibrium processes. In contrast to heterozygotes, the interplay between curve overlay, PCR product size, and analysis method is complicated for homozygote genotype discrimination and is difficult to predict. Similar to temperature cycling in PCR, if the temperature control and temperature homogeneity of the solution are adequate, faster rates improve melting analysis, just like faster rates improve PCR.

Detection of chromosomal translocations in formalin-fixed paraffin-embedded (FFPE) leukemic specimens by digital expression profiling.

INTRODUCTION: Detection of chromosomal translocations in formalin-fixed paraffin-embedded (FFPE) leukemic samples is important for confirmation of histopathological findings, classification, prognostication, and therapeutic decisions. Herein, we aim to determine whether digital expression profiling could detect chromosomal translocations in FFPE leukemic samples identified by RT-PCR, FISH, and/or karyotyping. METHODS: RNA was extracted from 28 FFPE bone marrow specimens from 19 patients diagnosed with leukemia. Eight patients were translocation t(9;22) positive, three inv(16)/t(16/16) positive, five translocation t(15;17) positive, and one translocation t(8;21) positive. Two patients (four specimens) were normal. The extracted RNA was hybridized to DNA reporter probes overnight at 65 °C, followed by purification of the labeled translocations. Six hundred fields of view were counted to enumerate the number of translocations. RESULTS: Digital expression profiling had 100% concordance with RT-PCR, FISH, or karyotyping analysis in the two normal individuals, eight translocation t(9;22) samples, five translocation t(15;17) samples, and one translocation t(8;21) sample. None of the inv(16) positive samples were detected. Digital expression profiling detected translocations with 0.014 p190 allelic burden. CONCLUSION: Digital expression profiling can be used to measure translocation t(9;22), t(15;17), and t(8;21) in FFPE samples and is useful when a confirmatory test from a FFPE sample is necessary.

Cytokeratin 19-fragments (CYFRA 21-1) as a novel serum biomarker for response and survival in patients with advanced pancreatic cancer.

BACKGROUND: CYFRA 21-1 serves as biomarker in several epithelial malignancies. However, its role in pancreatic cancer (PC) has not yet been investigated. METHODS: Within a prospective single-centre study serial blood samples were collected from patients with confirmed advanced PC. Pre-treatment values and weekly measurements of CYFRA 21-1, carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (assessed by Elecsys 2010, Roche Diagnostics) during palliative first-line chemotherapy were obtained. Biomarker data were correlated with objective response (determined by RECIST) as well as time to progression (TTP) and overall survival (OS) using uni- and multivariate analyses. RESULTS: Seventy-eight patients were included, 45% of these received treatment in prospective clinical trials. Median TTP was 3.9 months, median OS 7.7 months. Pre-treatment CYFRA 21-1 levels were significantly associated with performance status (P=0.0399) and stage of disease (P=0.0001). Marker values before chemotherapy and at the 2-month staging of all three markers were considered significant predictors for objective treatment response. Pre-treatment CYFRA 21-1 levels, as well as CA 19-9 values, could be applied to define subgroups (categorised by tertiles) with a different OS outcome (CYFRA: 14.8 vs 7.1 vs 4.8 months, CA 19-9: 14.2 vs 7.1 vs 5.2 months; P<0.0001). CYFRA 21-1 and CA 19-9 (both as categorised and as continuous variables) showed a highly significant correlation with TTP and OS at nearly all-time points assessed in univariate analysis. In multivariate analysis, only CYFRA 21-1 and performance status were independent predictors for OS. CONCLUSIONS: CYFRA 21-1 may serve as a valuable tool for monitoring treatment response and assessing prognosis in advanced PC.

A study to demonstrate freedom from Trichinella spp. in domestic pigs in Switzerland.

Trichinellosis is a food-borne zoonotic disease caused by the nematode Trichinella spp. Many omnivorous and carnivorous animal species can act as host for this parasite, including domestic pigs. To protect public health, it should be ensured that pork should not contain infective Trichinella larvae. Surveillance for Trichinella spp. can be done using direct (larval detection) and indirect (antibody detection) diagnostic techniques. The aim of this study was to demonstrate the absence of infection in Swiss domestic pigs. An ELISA was used as the initial screening test, and sera reacting in ELISA were further investigated using both a Western blot for serology and an artificial digestion test with 20 g of diaphragm tissue for larval detection. A total of 7412 adult pigs, 9973 finishing pigs and 2779 free-ranging pigs were tested. Samples from 17 (0.23%) adult pigs, 16 (0.16%) finishing pigs and nine (0.32%) free-ranging pigs were ELISA-positive, but all of these sera were subsequently negative by Western blot and by the artificial digestion method. Based on these findings, an absence of Trichinella infections in adult pigs (target prevalence 0.04%) and finishing pigs (target prevalence 0.03%) can be concluded. The results also demonstrated that the prevalence of Trichinella infections does not exceed 0.11% in free-ranging pigs, the group with the highest risk of exposure.

Genetic parameters for the prevalence of osteochondrosis in the limb joints of South German Coldblood horses.

Heritabilities were estimated for osteochondrosis (OC) in fetlock and hock joints and palmar/plantar osseous fragments in fetlock joints of South German Coldblood (SGC) horses using Residual Maximum Likelihood (REML) under a linear animal model. The analyses were based on the results of a standardized radiographic examination of 167 SGC horses with a mean age of 14 months. The heritabilities linearly estimated and transformed onto the liability scale were for OC in fetlock joints 0.16 and for OC in hock joints 0.04. Considering fetlock and hock OC together, results in a heritability of 0.17. Palmar/plantar osseus fragments of the fetlock joints showed a heritability of 0.48. We concluded that there is most likely a genetic component in the variation of the development of osteochondrosis in fetlock and hock joints as well as for palmar/plantar osseus fragments of fetlock joints of the investigated population of SGC horses.

Mapping quantitative trait loci for osteochondrosis in fetlock and hock joints and palmar/plantar osseus fragments in fetlock joints of South German Coldblood horses.

The aim of this study was to identify quantitative trait loci (QTL) for osteochondrosis (OC) and palmar/plantar osseous fragments (POF) in fetlock joints in a whole-genome scan of 219 South German Coldblood horses. Symptoms of OC and POF were checked by radiography in 117 South German Coldblood horses at a mean age of 17 months. The radiographic examination comprised the fetlock and hock joints of all limbs. The genome scan included 157 polymorphic microsatellite markers. All microsatellite markers were equally spaced over the 31 autosomes and the X chromosome, with an average distance of 17.7 cM and a mean polymorphism information content (PIC) of 63%. Sixteen chromosomes harbouring putative QTL regions were further investigated by genotyping the animals with 93 additional markers. QTL that had chromosome-wide significance by non-parametric Z-means and LOD scores were found on 10 chromosomes. This included seven QTL for fetlock OC and one QTL on ECA18 associated with hock OC and fetlock OC. Significant QTL for POF in fetlock joints were located on equine chromosomes 1, 4, 8, 12 and 18. This genome scan is an important step towards the identification of genes responsible for OC in horses.

Prevalence of osteochondrosis in the limb joints of South German Coldblood horses.

This study aimed to quantify the factors associated with the prevalence of the radiological signs of osteochondrosis (OC) and osteochondrosis dissecans (OCD) in South German Coldblood (SGC) horses. The prevalence of OC and OCD in fetlock and hock joints was analysed in 167 young coldblood horses with a mean age of 14 months. The presence of at least one osteochondrotic lesion (OC) in fetlock or hock joints was documented for 61.7% of the horses and 26.9% of the horses had osseus fragments. Osteochondrotic findings at the dorsal aspect of the sagittal ridge of the third metacarpal/metatarsal bone were seen in 53.9% of the horses and palmar/plantar osseous fragments in fetlock joints in 16.2% of the horses. Hock joint OC was found in 40.1% of the horses and hock OCD in 0.6%. Osteochondrotic findings in the distal part of the tibia were prevalent in 28.1% and in the lateral trochlea tali in 17.4% of the horses. The sex of the investigated horses significantly influenced the prevalence of OC in fetlock and hock joints, as well as the findings in the distal part of the tibia and lateral trochlea tali. Age at radiological examination was significant for the prevalence of OC in hock joints, palmar/plantar osseous fragments in fetlock joints and osteochondrotic findings in the distal part of the tibia. Female horses showed a 2-fold higher risk for OC in fetlock and hock joints than male horses. The distribution of the affected horses by age classes showed that radiographic signs of OC in fetlock and hock joints significantly increased at an age of about 1 year. We can conclude from our study that fetlock and hock OC is a prevalent radiographic finding in more than 1-year-old female and male SGC horses.

High-resolution DNA melting curve analysis to establish HLA genotypic identity.

High-resolution melting curve analysis is a closed-tube fluorescent technique that can be used for genotyping and heteroduplex detection after polymerase chain reaction. We applied this technique at the HLA-A locus and suggest that this method can be used as a rapid, inexpensive screen between siblings prior to living-related transplantation. At any locus, there are seven general cases of shared alleles among two individuals, ranging from identical homozygous genotypes (all alleles shared) to two heterozygous genotypes that share no alleles. We studied each case using previously typed cell lines to show that identity or non-identity can be determined in all cases by high-resolution melting curve analysis. HLA genotype identity is suggested when two individuals have the same melting curves. Identity is confirmed by comparing the melting curve of a 1:1 mixture with the individual melting curves. Non-identity at the amplified locus changes the heteroduplexes formed in the mixture compared with the original samples and alters the shape of the melting curve. The technique was tested on DNA from a 17-member CEPH family. High-resolution melting curve analysis revealed six different genotypes in the family. The genotype clustering was confirmed by sequence-based typing. Although this technique does not sequence or determine specific HLA alleles, it does rapidly establish identity at highly polymorphic HLA loci. The technique may also prove useful for confirmation of HLA genotypic identity between unrelated individuals prior to allogeneic hematopoietic stem-cell transplantation.

Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas.

BACKGROUND: Many techniques in molecular biology depend on the oligonucleotide melting temperature (T(m)), and several formulas have been developed to estimate T(m). Nearest-neighbor (N-N) models provide the highest accuracy for T(m) prediction, but it is not clear how to adjust these models for the effects of reagents commonly used in PCR, such as Mg(2+), deoxynucleotide triphosphates (dNTPs), and dimethyl sulfoxide (DMSO). METHODS: The experimental T(m)s of 475 matched or mismatched target/probe duplexes were obtained in our laboratories or were compiled from the literature based on studies using the same real-time PCR platform. This data set was used to evaluate the contributions of [Mg(2+)], [dNTPs], and [DMSO] in N-N calculations. In addition, best-fit coefficients for common empirical formulas based on GC content, length, and the equivalent sodium ion concentration of cations [Na(+)(eq)] were obtained by multiple regression. RESULTS: When we used [Na(+)(eq)] = [Monovalent cations] + 120(square root of ([Mg2+]-[dNTPs])) (the concentrations in this formula are mmol/L) to correct DeltaS(0) and a DMSO term of 0.75 degrees C (%DMSO), the SE of the N-N T(m) estimate was 1.76 degrees C for perfectly matched duplexes (n = 217). Alternatively, the empirical formula T(m) ( degrees C) = 77.1 degrees C + 11.7 x log[Na(+)(eq)] + 0.41(%GC) - 528/bp - 0.75 degrees C(%DMSO) gave a slightly higher SE of 1.87 degrees C. When all duplexes (matched and mismatched; n = 475) were included in N-N calculations, the SE was 2.06 degrees C. CONCLUSIONS: This robust model, accounting for the effects of Mg(2+), DMSO, and dNTPs on oligonucleotide T(m) in PCR, gives reliable T(m) predictions using thermodynamic N-N calculations or empirical formulas.

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

Fluorescein-labeled oligonucleotides for real-time pcr: using the inherent quenching of deoxyguanosine nucleotides.

Fluorescein-labeled oligonucleotide probes can be used to continuously monitor the polymerase chain reaction. Depending on the sequence, the fluorescence intensity of the probe is either increased or decreased by hybridization. The greatest effect is probe quenching by hybridization to amplicons containing deoxyguanosine nucleotides (Gs), giving a sequence-specific decrease in fluorescence as product accumulates. Quenching of the probes by Gs is position dependent. A 25% decrease in fluorescence of 5'-labeled probes was observed with a G at the first position of the 3'-dangling end. Additional Gs can increase quenching to about 40%. This change in fluorescence with hybridization allows real-time quantification and mutation detection with a simple single labeled probe. Quantification of the initial template copy number is possible by monitoring fluorescence at each cycle at a constant temperature. Mutation detection by Tm estimates from melting curve analysis for factor V Leiden, hemoglobin C, hemoglobin S, the thermolabile mutation of methylenetetrahydrofolate reductase, and the cystic fibrosis-associated deletion F508del is demonstrated. By using the inherent quenching of deoxyguanosine nucleotides in the amplicon, complicated probe designs involving internal quenching can be avoided.

Real-time multiplex PCR assays.

The ability to multiplex PCR by probe color and melting temperature (T(m)) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05-0.2 degrees C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and T(m) creates a "virtual" two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.

Developing the options for managing marine pests: specificity trials on the parasitic castrator, Sacculina carcini, against the European crab, Carcinus maenas, and related species.

The impacts of introduced marine pests are becoming increasingly apparent, prompting interest in the possibility of their biological control. We undertook laboratory and field experiments on host selection of one potential control agent (the endoparasitic barnacle, Sacculina carcini) against its natural host (the widely invasive European shore crab, Carcinus maenas) and several confamilial and more distantly related crustaceans. For comparison, we also tested host specificity in a related parasitic barnacle, Heterosaccus lunatus. The results confirm indistinct behavioral host selection in S. carcini, indicate very different mechanisms for host selection by S. carcini and H. lunatus (which could be related to differences between the two species in attachment points), and suggest host specificity in S. carcini depends on interactions between the parasite and the host's physiology. Development of convincing safety trials for marine parasites like S. carcini, in which the infective stage is a planktonic larva, will be more difficult than for many terrestrial parasites and will require detailed knowledge of the parasite's behavior and physiological interaction with its hosts.

PCR amplification using electrolytic resistance for heating and temperature monitoring.

An alternative method of rapid-cycle PCR for DNA amplification is demonstrated using electrolyte resistance for heating and temperature monitoring. The PCR amplification solution is electrically conductive and can be heated by passing an alternating current through the sample. The temperature of the solution is evaluated by monitoring its electrical resistance. Cooling is accomplished by forced air convection at ambient temperature. Heating and cooling rates of up to 20 degrees C/s were achieved. The 35 cycles of PCR were completed in less than 12 min with product yields equivalent to conventional temperature cycling. Electrolyte resistance provides a method for both direct heating and monitoring the temperature of PCR samples.

Flow cytometry: principles and clinical applications in hematology.

The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory.

Comparison of automated short tandem repeat and manual variable number of tandem repeat analysis of chimerism in bone marrow transplant patients.

Hematopoietic chimerism can be monitored in bone marrow transplant patients at DNA polymorphic sites. In this study, allele detection and quantification by ethidium bromide-stained agarose gels were compared with automated fluorescent sizing on an artificially mixed system and on chimeric post-transplant whole blood and sorted cell populations. A panel of five variable number of tandem repeats (VNTRs) were amplified and quantified visually on an ethidium bromide-stained gel. The ten short tandem repeats (STRs) were amplified as a multiplex polymerase chain reaction (PCR) and fluorescently detected on a DNA sequencer. Fluorescent band intensities were converted to fluorescent peak areas for allele quantification. Using mixed DNA of different proportions, both STRs and VNTRs showed linearity and appeared equally sensitive. However, case studies showed STRs to be more sensitive (<5%) than VNTRs (<10%). The STRs more accurately quantified the minor DNA component at low concentrations.

Monitoring hybridization during polymerase chain reaction.

The polymerase chain reaction (PCR) is usually analyzed by gel electrophoresis for size separation of PCR products. Additional separation techniques, such as single-stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and denaturing high-performance liquid chromatography (DHPLC), can also be used to scan for sequence alterations. These techniques are all based on the effect of PCR product hybridization on mobility. Hybridization can also be monitored with fluorescence during PCR without chromatographic or electrophoretic separation. Continuous monitoring of PCR allows the detection, quantification and sequence specificity of PCR products to be assessed, often without any need for further analysis. In such a closed system, PCR quantification with sensitivity to the single copy level can be achieved using either double-stranded DNA binding dyes or fluorescently labeled allele-specific oligonucleotide (ASO) probes. Melting curve analysis with ASO probes can be used to genotype various alleles, including single base alterations. The integration of rapid cycle PCR and ASO probes in an automated system greatly facilitates research and clinical applications of nucleic acid analysis in genetics, oncology, and infectious disease.