Genome-wide single-generation signatures of local selection in the panmictic European eel.

Next-generation sequencing and the collection of genome-wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD-sequenced European eel individuals (glass eels) from eight locations between 34 and 64(o) N, we examined the patterns of genome-wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using F(ST) -based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (F(ST) = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single-generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand-receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.

Phase I and II metabolism and MRP2-mediated export of bosentan in a MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 quadruple-transfected cell line.

BACKGROUND AND PURPOSE: Hepatic uptake (e.g. by OATP1B1), phase I and II metabolism (e.g. by CYP3A4, UGT1A1) and subsequent biliary excretion (e.g. by MRP2) are key determinants for the pharmacokinetics of numerous drugs. However, stably transfected cell models for the simultaneous investigation of transport and phase I and II metabolism of drugs are lacking. EXPERIMENTAL APPROACH: A newly established quadruple-transfected MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 cell line was used to investigate metabolism and transcellular transport of the endothelin receptor antagonist bosentan. KEY RESULTS: Intracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) was significantly lower in all cell lines expressing MRP2 compared to cell lines lacking this transporter (P < 0.001). Accordingly, considerably higher amounts of bosentan equivalents were detectable in the apical compartments of cell lines with MRP2 expression (P < 0.001). HPLC and LC-MS measurements revealed that mainly unchanged bosentan accumulated in intracellular and apical compartments. Furthermore, the phase I metabolites Ro 48-5033 and Ro 47-8634 were detected intracellularly in cell lines expressing CYP3A4. Additionally, a direct glucuronide of bosentan could be identified intracellularly in cell lines expressing UGT1A1 and in the apical compartments of cell lines expressing UGT1A1 and MRP2. CONCLUSIONS AND IMPLICATIONS: These in vitro data indicate that bosentan is a substrate of UGT1A1. Moreover, the efflux transporter MRP2 mediates export of bosentan and most likely also of bosentan glucuronide in the cell system. Taken together, cell lines simultaneously expressing transport proteins and metabolizing enzymes represent additional useful tools for the investigation of the interplay of transport and metabolism of drugs.

[Assessment of quality of life in health services research - conceptual, methodological and structural prerequisites]. / Die Erfassung von Lebensqualität in der Versorgungsforschung - konzeptuelle, methodische und strukturelle Voraussetzungen.

On July 1, 2009, the German Network for Health Services Research [Deutsches Netzwerk Versorgungsforschung e. V. (DNVF e. V.)] approved the Memorandum III "Methods for Health Services Research", supported by the member societies mentioned as authors and published in this Journal [Gesundheitswesen 2009; 71: 505-510]. This is an in-depth publication on "quality-of-life assessment in health services research". Within the context of the health sciences, quality of life (QL) encompasses the subjective well-being and functioning in the physical, psychological and social domains. QL informs about the aspects of health care that "actually get to the patient". QL is what patients primarily experience, what they talk about and what to a large degree affects the acceptance of health-care services and processes in the society. Therefore, QL can be considered as a highly important endpoint within health services research. The importance of the construct quality of life is also emphasised in German treaties on social law and utility analyses. This paper is the first account on the relations between health services research and the concept and assessment of QL. Our working group has specified key criteria for QL assessment within studies on health services research. (1) Assessment instruments need to comply with standard quality criteria (reliability, validity, sensitivity, interpretability) and the decision for a particular instrument has to be reasonably justified. (2) Study design and study population have to match with the scientific research question and the sample size has to be biometrically sound. (3) QL assessment including time points over the course of the study has to follow a standardized protocol. (4) Criteria for analysis and interpretation have to be prospectively specified. (5) Studies focusing on diagnostic/therapeutic issues need to specify standards for diagnostic criteria and related therapeutic interventions.

[Measurement of the diameter of segments of retinal branch vessels in digital fundus images - An experimental study of the method and reproducibility]. / Durchmesserbestimmung von Netzhautgefässabschnitten in digitalen Fundusfotografien - Eine klinische Studie zur Methodik und Reproduzierbarkeit.

PURPOSE: The aim of this study was the evaluation of new algorithms to measure diameter of segments of retinal branch vessels offline in local dependence and independent by observer. Methods 360 flashed fundus images (camera FF 450 ZEISS Germany, Visualis IMEDOS GmbH Weimar/Germany) of 12 eyes of healthy volunteers (10 independent sessions containing 3 images for every eye) were analysed. Algorithms detect the vessel diameter along the vessel course automatically. Corresponding segments of a retinal artery and vein were examined (mean length of the segment 2.5 mm) in every image of one eye. Results The marked arterial segment was detected automatically in 359 pictures and the venous segment in all pictures. The mean vessel diameter was detected in single pictures with a mean coefficient of variation (CV) for arteries of 3.4 % and for veins of 2.7 %. The differences of arterial and venous diameter between images were not significant. Analyzing sessions the CV of the mean vessel diameter were reduced for arteries to 2.7 % and for veins to 2.5 %. The standard deviation of the mean vessel diameter was independent of the vessel diameter (branch vessels with diameter of 120 to 200 micrometer). The mean CV of the vessel diameter at single locations were 5.7 % for arteries and 3.8 % for veins. Conclusions The new algorithms are useful for retinal vessel analysis, if there are no questions concerning the dynamic of vessel behaviour.

Use of simple and complex in vitro models for multiparameter characterization of human blood-material/device interactions.

Medical devices, intended for blood contacting applications, undergo extensive in vitro testing followed by animal and clinical feasibility studies. Besides the use of materials known to be intrinsically blood-compatible, the surface of such devices is often modified with a coating in order to improve the performance characteristics during blood exposure. In vitro evaluation of blood-device interactions accompanies the product development cycle from the early design phase using basic material geometries until final finished-product testing. Specific test strategies can vary significantly depending on the end application, the particular study objectives and variables of interest, and cost. To examine the degree to which findings derived from two different in vitro approaches complement one another, this report contrasts findings from a simple multipass loop model with findings from a simulated cardiopulmonary bypass (CPB) model. The loop model consists of tubular test materials, with and without surface modification, formed into valved Chandler loops. The CPB model has an oxygenator with and without surface modification connected to a reservoir and a blood pump. The surface modifications studied in this report are the Carmeda BioActive Surface and Duraflo II heparin coatings. Common blood parameters in the categories of coagulation, platelets, hematology, and immunology were monitored in each model. Ideal models employ the optimal level of complexity to study the design variables of interest and to meet practical cost considerations. In the case of medical device design studies, such models should also be predictive of performance. In the more complex and realistic simulated CPB model, experimental design and cost factors prevented easy/optimum manipulation of critical variables such as blood donor (use of paired samples) and heparin level. Testing in the simpler loop model, on the other hand, readily offered manipulation of these variables, and produced findings which overlapped with observations from the more complex CPB model. Thus, the models described here complimented one another. Moreover, conclusions from consistent findings, such as favorable responses associated with the heparin coatings, between the two models were considered to be more robust.

MEN1 gene mutations in 12 MEN1 families and their associated tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited tumor syndrome characterized by the development of multiple endocrine tumors. The gene responsible for the disease, termed MEN1 gene. has recently been isolated and germline mutations have been described in affected MEN1 individuals. Twelve unrelated (German MEN1 families and their associated tumors (5 parathyroid tumors, 1 vipoma, 1 gastrinoma, 1 insulinoma) were characterized for MEN1 gene mutations by single-strand conformational variant (SSCV) analysis and DNA sequence analysis as well as for loss of heterozygosity on chromosome 11q13. We identified nine different heterozygous germline mutations (6 frameshift, 2 missense, 1 nonsense), eight of them were novel. Four of five informative MEN1-associated tumors revealed deletion of the second MEN1 allele, supporting the concept of a tumor suppressor gene. Furthermore. SSCV analysis proved an effective and sensitive method for the detection of menin mutations providing a reliable genetic screening approach supporting genetic counseling and clinical management of MEN1 family members.

Frequent abnormalities of the putative tumor suppressor gene FHIT at 3p14.2 in pancreatic carcinoma cell lines.

The FHIT gene is localized on chromosome 3p14, a region including a tumor cell-specific, commonly deleted region. To determine the role of the FHIT gene in pancreatic carcinogenesis, 14 pancreatic carcinoma cell lines were analyzed by reverse transcription-PCR and exon-specific PCR amplification of genomic DNA. The full-length FHIT transcript was lost in 70% of the pancreatic carcinoma cell lines analyzed, while 66% also revealed intragenic homozygous deletions of exons 3, 4, and 5. Truncated FHIT transcripts lacking a variable number of exons most likely represented alternative splicing products. Fhit protein expression was dependent on a full-length FHIT transcript. The results suggest that the FHIT gene may be a target tumor suppressor gene involved in pancreatic carcinogenesis.

[Germline mutations in the MEN1 gene: basis for predictive genetic screening and clinical management of multiple endocrine neoplasia type 1 (MEN1) families]. / Keimbahnmutationen im MEN1-Gen: Basis für prädiktives genetisches Screening und klinisches Management von MEN1-Familien.

BACKGROUND AND OBJECTIVE: Mutations in the MEN 1 gene were recently discovered as the causative genetic defect of the autosomal dominantly inherited multiple endocrine neoplasia type 1. It was the aim of this study to evaluate the spectrum of MEN 1 mutations in our own series of patients in order to obtain a basis for predictive family screening. PATIENTS AND METHODS: Genomic DNA from peripheral blood of 21 patients with MEN 1, members of 14 non-related MEN 1 families, was examined for MEN 1 germ-line mutations by means of single-strand conformation variant analysis (SSCP) and direct DNA sequencing. In addition, blood from 20 asymptomatic family members of five families was tested for its predictive value. RESULTS: Eleven different heterozygotic germ-line mutations, among them eight frameshift, two missense and one nonsense mutations, were identified. In four of the 20 asymptomatic members from five MEN 1 families who had been tested after appropriate genetic counselling, the MEN 1 mutation characteristic for the particular family was found. Clinical screening programme in three mutation carriers revealed abnormal findings in all three: one primary hyperparathyroidism, one prolactinoma and one nonfunctioning pancreatic tumour each. The 16 family members without MEN 1 mutation were spared further unnecessary screening investigations. CONCLUSION: Although the function of the MEN 1 gene is not yet known, molecular genetic tests provide a basis for genetic counselling, predictive genetic screening and clinical management of MEN 1 families.

Impact of increased intraperitoneal fill volume on tolerance and dialysis effectiveness in children.

The known relationship between peritoneal fill volume (IVP) and dialysis efficiency favors the use of an optimal IVP to enhance peritoneal dialysis (PD). Therefore, we have studied the effects of an increased IVP in consecutive stages [800, 1400, and 2000 mL/m2 of body surface area (BSA), respectively] in 8 children on chronic PD (mean age: 9 years 6 months; range: 2-16 years). Each prescribed IVP was maintained for 60 minutes of dwell time, allowing a short peritoneal equilibration test. Tolerance was assessed clinically and by intraperitoneal pressure (IPP) measurements at the end of each dwell test. Determination of dialysate-to-plasma ratios, and calculation of mass transfer area coefficients (K0A) using the Henderson method for urea, creatinine, and phosphate, were used to assess the impact of an increased IVP on dialytic efficiency. Increasing IVP from 800 to 1400 and thereafter to 2000 mL/m2 induced an IPP increment, respectively, from 8.4 +/- 1.4 cm (of water) to 12.1 +/- 1.4 cm and thereafter to 18.3 +/- 1.4 cm, with a positive strong linear correlation (r = 0.92; P = 0.001; IPP = 1.46 +/- 8.17(-3) IVP). In the same manner increasing IVP induced K0A increments for urea of 10.6 +/- 1.2 mL/min per m2 to 15.3 +/- 1.6 mL/min per m2 and 17.1 +/- 1.9 mL/min per m2; for creatinine of 7.9 +/- 0.09 mL/min per m2 to 11.2 +/- 0.18 mL/min per m2, and 12.3 +/- 0.21 mL/min per m2; and for phosphate of 5.2 +/- 0.08 mL/min per m2 to 6.7 +/- 0.09 mL/min per m2 and 6.6 +/- 0.07 mL/min per m2, respectively. When K0A values were normalized to the values achieved at the IVP of 1400 mL/m2, the K0A gain obtained increasing IVP from 1400 to 2000 mL/m2 was only significant for urea, peaked for creatinine, and even slowly decreased for phosphate. Moreover, a fill volume over 1400 mL/m2, which appears to be the optimal volume in terms of dialysis efficiency, was only barely tolerated with clinical signs of discomfort and an increased IPP. Therefore, in our opinion, the maximal IVP in children over the age of 2 years should be nearly 1400 mL/m2, both in terms of abdominal tolerance and in terms of urea, creatinine, and phosphate peritoneal membrane purification capacities.

RAP1-like binding activity in islet cells corresponds to members of the Sp1 family of transcription factors.

Deletion and mutational analyses of the gastrin promoter have identified a binding site for the yeast transcription factor RAP1 relevant for transcriptional activation in islet cells. We here report that the mammalian transcription factors binding to this site in islet cells are the Sp transcription factor members Sp1 and Sp3. Furthermore, functional analyses revealed Sp1- and Sp3-mediated transcriptional activation of gastrin. These data reveal that the zinc finger proteins Sp1 and Sp3 do have similar binding specificities as the multifunctional yeast RAP1 protein.

Servoregulation of centrifugal pumps. A new technical approach to improve patient safety during long-term extracorporeal life support.

The objective of this study was to evaluate the principle technical requirements to servoregulate a centrifugal pump (Bio-Pump) and to discuss the potential risks and benefits of the potential use of such a device during long-term extra-corporeal support in an experimental laboratory study. A pressure control module (PCM) for the Bio-Pump was developed and a pressure measurement chamber to indirectly measure pressure in the venous limb of an extracorporeal circuit was constructed. The performance of the PCM combined with the pressure measurement chamber was evaluated in an experimental test circuit by recording pressure changes after sudden clamping of the venous line with and without servoregulation. Without the PCM pressure dropped from baseline to approximately -200 mm Hg after clamping and remained at that level. With the PCM active the pump speed was automatically and immediately reduced and the preclamping pressure level (+/- 10 percent) was restored within 500 msec. In this laboratory setting the Bio-Pump could effectively and rapidly be servoregulated with a conventional controller and an indirect pressure monitoring system. A potential clinical use of this system could help to improve the safety without imposing additional risks such as air embolism or backflow.

Two-dimensional temperature determination in sooting flames by filtered Rayleigh scattering.

We present what to our knowledge are the first filtered Rayleigh scattering temperature measurements and use them in sooting f lame. This new technique for two-dimensional thermography in gas combustion overcomes some of the major disadvantages of the standard Rayleigh technique. It suppresses scattered background light from walls or windows and permits detection of two-dimensional Rayleigh intensity distributions of the gas phase in the presence of small particles by spectral filtering of the scattered light.

[Adaptive algorithm for automatic measurement of retinal vascular diameter]. / Adaptive Algorithmen zur automatischen Messung retinaler Gefässdurchmesser.

A new adaptive computer-aided method for the measurement of blood vessel diameters has been developed. Within areas of interest in the image, the algorithm detects, line-wise, the edges of the vessels, which are then used for image-wise approximation and noise filtration. A high level of adaptivity with respect to numerous measuring parameters ensures its use in a wide range of applications. Thus, it has been shown to significantly improve clinically relevant reproducibility in the area of follow-up observations. The standard deviation for vessel diameter was (2.2 +/- 0.7)% in the case of arteries and (1.8 +/- 0.5)% in the case of veins. Testing the algorithm in images of poor quality revealed its high level of reliability and sensitivity.

[Adaptive procedures for measuring arterial blood flow velocity in retinal vessels using indicator technique]. / Adaptive Verfahren zur Messung arterieller Blutgeschwindigkeiten in Netzhautgefässen mittels Indikatortechnik.

There are highly significant differences in the measuring results of arterial blood velocity between the indicator and laser-Doppler techniques (up to 800%). A new measuring procedure for the analysis of indicator dilution curves was developed based on indicator model and experimental results. The use of this new measuring procedure results in reduced mean systematic error between the indicator and laser-Doppler techniques to values around 10%. With the introduction of adaptive measuring arrays for the creation of indicator dilution curves and the application of adaptive algorithms for centering and spectral normalizing of the dilution curves, improved reproducibility can be expected.

An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA.

The product of the ie1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS-PAGE, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as beta-galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions.

The core histone-binding region of the murine cytomegalovirus 89K immediate early protein.

The gene regulatory immediate early protein, pp89, of murine cytomegalovirus interacts with both DNA-associated and isolated histones in vitro. We characterized the histone-binding region of pp89 and its cellular localization during cell division to examine the possible interaction between pp89 and chromatin. pp89 expressed constitutively in cell line BALB/c 3T3 IE1 does not interact with condensed chromatin. As observed in infected cells, pp89 is localized within the nucleus of cells during interphase but spreads throughout the cell plasma following degradation of the nuclear membrane during early mitosis. In late telophase, pp89 is reorganized within the nucleus. Analysis of pp89 deletion mutants and of fragments generated by cleavage at pH 2.5 revealed that the regions responsible for association with histone are located between amino acids 71 and 415, and are not identical with the domain that shows homology to histone H2B or the highly acidic carboxy-terminal region. A potential gene-activating role of the high affinity of pp89 for isolated histones and the low affinity for DNA-associated histones is discussed.

Impact of culture and cryopreservation on MHC class II antigen expression in canine and porcine islets.

Canine and porcine islets were either cultured for 10 days at 37 degrees C or cryopreserved. The effect of these treatments on MHC class II antigen expression was examined by indirect immunofluorescence test using the class II monoclonal antibody 2MC3. Within untreated canine islets exclusively round shaped cells as leukocytes and monocyte/macrophage like cells with dendritic branches were positive with 2MC3. Porcine islets additionally exhibited strong immunofluorescence of the vascular endothelium. Tissue culture significantly reduced the class II antigen expression in both species. The majority of the untreated canine islets had between 5 and 19 2MC3 positive cells. Cell culture reduced the number of class II positive cells to a maximum of 3 and even 84.9% of the islets were completely negative for class II antigens. Porcine islets showed a total class II antigen loss of the vascular endothelium, whereas leukocytes and monocytes/macrophages remained class II antigen positive. Cryopreservation did not have clear-cut effects on the MHC expression in both species.

Presentation of CMV immediate-early antigen to cytolytic T lymphocytes is selectively prevented by viral genes expressed in the early phase.

The regulation of antigen processing and presentation to MHC class I-restricted cytolytic T lymphocytes was studied in cells infected with murine cytomegalovirus. Recognition by cytolytic T lymphocytes of the phosphoprotein pp89, the immunodominant viral antigen expressed in the immediate-early phase of infection, was selectively prevented during the subsequent expression of viral early genes. The surface expression of MHC class I glycoproteins and their capacity to present externally added pp89-derived antigenic peptides were not affected. Because recognition of several other antigens occurred during the early phase, a general failure in processing and presentation was excluded. Since neither rate of synthesis, amount, stability, nor nuclear transport of pp89 was modified, the failure in recognition indicates a selective interference with pp89 antigen processing and presentation.