Identification of allele-specific KIV-2 repeats and impact on Lp(a) measurements for cardiovascular disease risk.

The abundance of Lp(a) protein holds significant implications for the risk of cardiovascular disease (CVD), which is directly impacted by the copy number (CN) of KIV-2, a 5.5 kbp sub-region. KIV-2 is highly polymorphic in the population and accurate analysis is challenging. In this study, we present the DRAGEN KIV-2 CN caller, which utilizes short reads. Data across 166 WGS show that the caller has high accuracy, compared to optical mapping and can further phase ~50% of the samples. We compared KIV-2 CN numbers to 24 previously postulated KIV-2 relevant SNVs, revealing that many are ineffective predictors of KIV-2 copy number. Population studies, including USA-based cohorts, showed distinct KIV-2 CN, distributions for European-, African-, and Hispanic-American populations and further underscored the limitations of SNV predictors. We demonstrate that the CN estimates correlate significantly with the available Lp(a) protein levels and that phasing is highly important.

Nano-LC-MS/MS for the quantitation of ceramides in mice cerebrospinal fluid using minimal sample volume.

A new nano-liquid chromatography-ESI-MS/MS method has been developed for the sensitive quantitation of C8:0, C16:0, C18:0, C18:1, C20:0, C24:1 and C24:0 ceramide in cerebrospinal fluid of mice using minimal sample volume. Volumes of 2 µL CSF were undertaken a simple, fast extraction procedure involving protein precipitation with methanol and dilution. Ceramides were separated by nano-liquid chromatography with a reversed phase C8 column and detected with a triple quadrupole mass spectrometer. C17:0 ceramide was used as internal standard. The method has been validated in terms of linearity, lower limit of quantitation, precision, accuracy and autosampler stability. Calibration curves covered a range of 2.25-120 pg/µL for most ceramides (7.5-120 pg/µL for C24:0 ceramide). The lower limits of quantitation determined for C8:0, C16:0, C18:1, C18:0, C20:0 and C24:1 ceramide were 0.225 pg on column (2.25 pg/µL) and that for C24:0 ceramide 0.750 pg on column (7.5 pg/µL). Intra- and interday precision and accuracy values, expressed as relative standard deviation and relative error, respectively, were lower than 15% in all cases. Autosampler stability for calibration standards and CSF samples was proven for at least 24h for all analytes. The suitability of the method has been demonstrated by quantifying the analytes, except the non-endogenous C8:0 ceramide, in cerebrospinal fluid samples of 12 mice. Calculated concentrations ranged from 3 to 120 pg/µL in cerebrospinal fluid for all analytes, except for C24:0 ceramide, which could not be detected in any of the analyzed samples.

Positron emission tomography-computed tomography evaluation for recurrent differentiated thyroid carcinoma.

OBJECTIVES: Recurrence is observed in 15-20% of patients under surveillance following treatment of differentiated thyroid cancer (DTC). However, due to cell dedifferentiation, the recurrence may be iodine-negative, thereby compromising detection. For this reason, new methods of exploration are indispensable to enable localization of such recurrences. The purpose of this work is to review the contribution of positron emission tomography-computed tomography (PET-CT) in the exploration of iodine-negative recurrent DTC. METHOD: A comprehensive review and discussion of the medical literature was carried out. RESULTS: Depending on the report, the sensitivity of PET-CT ranged from 70% to 85%, with up to 90% specificity. However, the large number of false negatives, which can reach 40%, is the disadvantage of this examination. PET-CT results lead to change in the therapeutic strategy in approximately 50% of patients with isolated raised serum thyroglobulin levels, and surgical exploration of a precise anatomical area in the neck. CONCLUSION: As post-treatment recurrence of a DTC can affect patient survival, a thorough diagnostic work-up is required in these cases. Where thyroglobulin levels are elevated with no uptake on 131-iodine scans, PET-CT can be a useful complementary exploration, especially for localizing the site of recurrence.

Dual-color fluorescence cross-correlation spectroscopy on a planar optofluidic chip.

Fluorescence cross-correlation spectroscopy (FCCS) is a highly sensitive fluorescence technique with distinct advantages in many bioanalytical applications involving interaction and binding of multiple components. Due to the use of multiple beams, bulk optical FCCS setups require delicate and complex alignment procedures. We demonstrate the first implementation of dual-color FCCS on a planar, integrated optofluidic chip based on liquid-core waveguides that can guide liquid and light simultaneously. In this configuration, the excitation beams are delivered in predefined locations and automatically aligned within the excitation waveguides. We implement two canonical applications of FCCS in the optofluidic lab-on-chip environment: particle colocalization and binding/dissociation dynamics. Colocalization is demonstrated in the detection and discrimination of single-color and double-color fluorescently labeled nanobeads. FCCS in combination with fluorescence resonance energy transfer (FRET) is used to detect the denaturation process of double-stranded DNA at nanomolar concentration.

Assessment of liver metastases from colorectal adenocarcinoma following chemotherapy: SPIO-MRI versus FDG-PET/CT.

PURPOSE: This study compared superparamagnetic iron-oxide-enhanced magnetic resonance imaging (SPIO-MRI) and combined fluorodeoxyglucose positron emission tomography and computed tomography (FDG-PET/CT) in evaluating liver metastases from colorectal adenocarcinoma following chemotherapy. MATERIALS AND METHODS: Nineteen patients were included in this retrospective study. SPIO-MRI and PET/CT results were compared with surgery, intraoperative ultrasound and pathology results in 11 patients and with the follow-up in eight patients. RESULTS: SPIO-MRI and PET/CT identified 125 and 71 metastases, respectively. False negative lesions were 11 for SPIO-MRI and 65 for PET/CT. In the whole study population, the per-lesion analysis of SPIO-MRI and PET/CT showed a sensitivity of 92% and 52% (p<0.001) and the per-segment analysis a sensitivity of 99% and 79% (p<0.001), respectively. In patients who underwent surgery, the per-lesion analysis of SPIO-MRI and PET/CT showed a sensitivity of 85% and 58% (p<0.05) and the per-segment analysis a sensitivity of 97% and 63% (p<0.05), respectively. In patients who underwent follow-up, the per-lesion analysis of SPIO-MRI and PET/CT showed a sensitivity of 97% and 47% (p<0.001) and the per-segment analysis a sensitivity of 100% and 63% (p<0.007), respectively. For lesions ≥15 and <30 mm and for lesions <15 mm, SPIO-MRI demonstrated a higher sensitivity than PET/CT (p<0.001). CONCLUSIONS: SPIO-MRI appears superior to PET/CT in evaluating liver metastases from colorectal adenocarcinoma following chemotherapy.

Functional T lymphocytes infiltrate implanted polyvinyl alcohol foams during surgical wound closure therapy.

Vacuum-assisted closure involving the implantation of polyvinyl alcohol foam is a technique recently developed for the treatment of patients suffering from either wound infection or chronic wounds. This method has been shown to improve and accelerate wound healing. However, little is known about the cell populations that infiltrate the foam, and their potential role in resolving the infection and promoting granulation tissue formation. Our study demonstrates that wound-implanted foams are mainly infiltrated with granulocytes, but that mononuclear cells, including macrophages and minor populations of T, B and natural killer lymphocytes, are also present. We show that foam-infiltrating T cells, especially CD4(+) T cells, constitute a phenotypically and functionally heterogeneous population influenced by wound-infecting bacteria. Thus, T lymphocytes could play a role in wound cleansing. In addition, our data indicate that implanted polyvinyl alcohol foams might be suitable microenvironments for manipulating T cell-mediated immune responses in patients.

Lower-than-expected linkage disequilibrium between tightly linked markers in humans suggests a role for gene conversion.

Understanding the pattern of linkage disequilibrium (LD) in the human genome is important both for successful implementation of disease-gene mapping approaches and for inferences about human demographic histories. Previous studies have examined LD between loci within single genes or confined genomic regions, which may not be representative of the genome; between loci separated by large distances, where little LD is seen; or in population groups that differ from one study to the next. We measured LD in a large set of locus pairs distributed throughout the genome, with loci within each pair separated by short distances (average 124 bp). Given current models of the history of the human population, nearly all pairs of loci at such short distances would be expected to show complete LD as a consequence of lack of recombination in the short interval. Contrary to this expectation, a significant fraction of pairs showed incomplete LD. A standard model of recombination applied to these data leads to an estimate of effective human population size of 110,000. This estimate is an order of magnitude higher than most estimates based on nucleotide diversity. The most likely explanation of this discrepancy is that gene conversion increases the apparent rate of recombination between nearby loci.

Sequence variation and linkage disequilibrium in the human T-cell receptor beta (TCRB) locus.

The T-cell receptor (TCR) plays a central role in the immune system, and > 90% of human T cells present a receptor that consists of the alpha TCR subunit (TCRA) and the beta subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations-African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r(2) > 0.1) over distances of approximately 30 kb in TCRB, and, in general, the distribution of r(2) as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in "blocks" of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association.

Routine measurements of left and right ventricular output by gated blood pool emission tomography in comparison with thermodilution measurements: a preliminary study.

The aim of this preliminary study was to evaluate the accuracy of left and right ventricular output computed from a semi-automatic processing of tomographic radionuclide ventriculography data (TRVG) in comparison with the conventional thermodilution method. Twenty patients with various heart diseases were prospectively included in the study. Thermodilution and TRVG acquisitions were carried out on the same day for all patients. Analysis of gated blood pool slices was performed using a watershed-based segmentation algorithm. Right and left ventricular output measured by TRVG correlated well with the measurements obtained with thermodilution (r = 0.94 and 0.91 with SEE = 0.38 and 0.46 l/min, respectively, P < 0.001). The limits of agreement for TRVG and thermodilution measurements were -0.78-1.20 l/min for the left ventricle and -0.34-1.16 l/min for the right ventricle. No significant difference was found between the results of TRVG and thermodilution with respect to left ventricular output (P = 0.09). A small but significant difference was found between right ventricular output measured by TRVG and both left ventricular output measured by TRVG (mean difference = 0.17 l/min, P = 0.04) and thermodilution-derived cardiac output (mean difference = 0.41 l/min, P = 0.0001). It is concluded that the watershed-based semi-automatic segmentation of TRVG slices provides non-invasive measurements of right and left ventricular output and stroke volumes at equilibrium, in routine clinical settings. Further studies are necessary to check whether the accuracy of these measurements is good enough to permit correct assessment of intracardiac shunts.

An update on the science and therapy of obesity and its relationship to osteoarthritis.

Obesity and osteoarthritis are two commonly encountered clinical problems that can lead to significant physical and emotional disability. This report examines the association between obesity and osteoarthritis, and discusses potential mechanisms by which obesity influences osteoarthritis. Special attention is devoted to reviewing the molecular and genetic mechanisms that underlie the development of clinical obesity. Improved understanding of obesity will hopefully lead to improved treatment and subsequent amelioration of this important risk factor for osteoarthritis.

An analysis of strategies for discovery of single-nucleotide polymorphisms.

Strategies for the discovery of single-nucleotide polymorphisms (SNPs) can be characterized by the number of individuals in the discovery sample, and by the minimal required number of observations of each allele. We examine the effect of different strategies on two key properties of the resulting SNP collection: (1) the probability that a SNP with a given population allele frequency is detected; and (2) the allele-frequency distribution of the discovered SNPs. We show that strategies that accept all polymorphic sites lead to collections with a high fraction of SNPs with rare minor alleles, particularly in expanded populations. Such SNPs have a low probability of replication in a second sample. We discuss how to tailor a discovery strategy to the desired properties of a SNP collection.

[Ganglioglioma of optic nerve in neurofibromatosis type 1. Case report and review of the literature]. / Gangliogliom des Nervus opticus bei Neurofibromatose Recklinghausen Typ 1: Fallbericht und Literaturüberblick.

BACKGROUND: Gangliogliomas are rare tumors of the central nervous system. In only seven cases they have been found within the optic nerve. Two of these cases were associated with neurofibromatosis (NF), but a pathogenic link between gangliogliomas of the optic nerve and NF still remains controversial. PATIENT: Here we report on a 71-year-old patient with a ganglioglioma of the optic nerves and NF type 1. Post mortem examination revealed multiple neurofibromas of the vagal and left femoral nerves, multiple schwannomas of the small bowel and Lisch noduli of both irides. In addition, a spindle shaped thickening of the right optic nerve was observed in its intracranial portion. The left optic nerve was normal on gross inspection. RESULT: Histologically, tumoral tissue was found in both optic nerves. The tumor was composed of two cell types: highly differentiated, partly stellate, partly pilocytic astrocytes and, at the rim of the axon bundles of the optic nerve, fully developed synaptophysin- and neurofilament-antigen-positive ganglion-cells with short corkscrew-shaped processes. No mitosis could be found in the neuronal or in the glial cell population. In spite of the tumoral involvement of the optic nerves, there has been no evidence of visual disturbance. CONCLUSION: Optic nerve tumors in NF most often are of glial origin (astrocytomas, pilocytic astrocytomas). Our case illustrates the rare condition of a glioneuronal tumor associated with neurofibromatosis type 1.

Blood speed imaging with an intraluminal array.

In applications in which Doppler processing is not possible, such as side-looking intravascular imaging systems, decorrelation methods can be used to estimate blood speed. Here, a method is presented measuring relative blood speed using an FIR filter bank to estimate temporal decorrelation rates. It can be implemented in a modern commercially available ultrasound imaging system with no additional hardware. Both simulations and experiments using an intraluminal scanner appropriate for coronary artery applications have tested the system. In this study, the FIR filter bank is contrasted with previous methods, and its utility is further demonstrated with real-time color flow images from a pig model.

[Dermatitis and conjunctivitis after contact with Euphorbia myrsinites (wolf's milk extract)--a case report]. / Dermatitis und Konjunktivitis nach Kontakt mit dem Saft der Euphorbia myrsinites (Wolfsmilchsaft)--Eine Kasuistik.

BACKGROUND: Fresh sap of euphorbiaceae leads to a toxic burn of the skin and the eyes. Since years the sap of euphorbiaceae has been used in the treatment of different kinds of verrucas. PATIENTS: After contact with the sap of Euphorbia myrsinites three children developed a toxic dermatitis. In addition, the youngest girl showed a conjunctivitis and an occlusion of the right eye. Phorbolesters are considered to be responsible for the toxicity of the euphorbiaceae. All three children have resulted in a restitutio ad integrum. CONCLUSION: This case report is demonstrating the danger of toxic burn of this kind of plant.

Further diversification of the HLA-B locus in Central American Amerindians: new B*39 and B*51 alleles in the Kuna of Panama.

Several new HLA-B locus alleles have been discovered in South American Amerindians. By contrast, analysis of the MHC class I alleles of North American native populations has revealed few new HLA-B alleles. This suggests that the HLA-B locus is evolving rapidly in South American populations. Here we describe the HLA-B locus alleles present in individuals from a Central American tribe, the Kuna of Panama. Using a sequence-based typing technique that separates alleles by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we determined the HLA-B alleles from eight Kunas. Two of the HLA-B alleles present in the Kuna have been previously described in other South American Amerindian populations; one allele has been characterized in a Mexican-American. We characterized two new HLA-B alleles in the Kuna, HLA-B*3911 and HLA-B*5110. HLA-B*3911 differed from HLA-B*3905 by only a single nucleotide substitution in exon 3. This substitution resulted in an amino acid replacement of leucine by arginine at residue 156 in the alpha 2 domain. Such a change may affect the repertoire of peptides that are bound by this molecule. HLA-B*5110 differed significantly from other HLA-B*51 alleles in that it is the result of an unusually large intra-locus recombination event of minimally 216 nucleotides. This recombination results in an allele that is part HLA-B*51 and part HLA-B*40. Thus, more dramatic recombination events may also play a role in the rapid evolution of the HLA-B locus in Amerindians.

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing.

High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. Here we describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB-specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.

HLA-B typing by allele separation followed by direct sequencing.

Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous molecular typing method for the alleles at this locus. Here we describe a technique for the direct sequencing of HLA-B alleles. Initially, HLA-B alleles were PCR-amplified after locus-specific reverse transcription of RNA. Alleles were then separated using denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplified and directly sequenced. The derived sequences were aligned to a database of published HLA-B sequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 known HLA-B alleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE-separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood samples from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities.

Identification of a new HLA-B*08 variant, HLA-B*0804.

The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA-B*08 variant, HLA-B*0804, found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B*51 in JH and HLA-B*4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B*08 variant, HLA-B*0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B*0804 differed from HLA-B*0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR-SSP. This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles.

The latest in electronic imaging.

Development of new transducer and system technologies has led to major advances in image quality for electronic imaging catheters. New, 64-element arrays have increased sensitivity and optimized image resolution. System technology has advanced to include high speed reconstruction using 'complete data sets' of information. The incorporation of personal computer technology has enabled new user interfaces and digital image archiving. New, combined imaging and therapy catheters allow for efficient usage of devices. The debut of intravascular colour flow imaging technology promises a new dimension in the treatment of coronary artery disease.