Molecular Genetics of Alopecia Areata in Dundee Experimental Bald Rats and in Humans

Alopecia areata (AA) (MIM 104000) ist eine chronisch entzündliche Erkrankung des aktiv wachsenden Haarfollikels in der Anagenphase mit einer starken genetischen Komponente. Sie ist im Allgemeinen durch einen kreisrunden Haarverlust am Kopf oder aber auch an anderen Körperstellen charakterisiert. Die Krankheitsentstehung ist nach wie vor unklar wobei ein gewebespezifischer Autoimmunmechanismus vermutet wird. In dieser Studie wurde der Stamm Dundee Experimental Bald Rat (DEBR) als Nagetiermodell für AA verwendet. In vorhergehenden Arbeiten wurde eine Kreuzung von DEB mit PVG Ratten angesetzt aus der 320 weibliche F2 Tiere entstanden. Diese Tiere wurden genomweit mit Mikrosatelliten auf Kopplung getest, was zu einem hoch signifikanten Lokus auf Chromosom 19 mit einem lod score von 20 führte. In dieser Arbeit folgte daher eine Sättigungskartierung für dieses Chromosom mit weiteren Mikrosatellitenmarkern die eine Kandidatenregion bei 33 bis 36.5 Mb (rn4) identifizierte. Exone der meisten Gene innerhalb dieser Region wurden sequenziert wobei keine Mutationen, als potentielle Ursache für Haarausfall, identifiziert werden konnten. Daher wurde die komplette Kandidatenregion erneut mittels Next Generation Sequencing (NGS) sequenziert. Mutationen konnten damit weiterhin nicht identifiziert werden. Einige intergenetische Varianten suggerieren jedoch das Vorkommen von bisher unbekannten Genen innerhalb der Kandidatenregion oder es könnte sich um Mutationen in regulatorischen Elementen wie beispielsweise in Promotor- oder Enhancerregionen handeln. Dies muss in weiteren bioinformatischen Berechnungen geklärt werden. Zusätzlich zur Sequenzierung wurde ein weiterer Ansatz über die Expressionsanalyse mittels Affymetrix GeneChip Rat Gene 1.0 ST Arrays verfolgt. Diese ließ Abweichungen in der Expression von verschiedenen (Haar-) Keratingenen, Genen von weiteren strukturellen Komponenten sowie immunregulatorische Gene wie Chemokine und H2 Gene, die ortholog zu HLA sind, erkennen. Weitergehende Auswertungen der Expressionsdaten von Genen der Kandidatenregion auf Chromosom 19 mit dem Programm „Network Explorer“ von Ingenuity Pathways Analysis verwiesen auf eine wichtige Rolle von Cadherinen. Daher wurde ein Set aus Cadherinen, Cateninen und Desmogleinen immunohistochemisch in Hautproben gefärbt. Dieses Experiment zeigte eine Reduzierung in der Konzentration von Catenin gamma in Korrespondenz zum Phänotyp. In Proben von Ratten mit einem ausgeprägten Haarverlust war Catenin gamma nicht mehr nachweisbar. Zusätzlich zu Catenin gamma zeigte M- und P-Cadherin ebenfalls eine abnormale Proteinlokalisierung in der humanen Epidermis der Haut. Die Resultate der Expressionsanalyse von Kandidatengenen wurden schließlich in Rattenhaut und teilweise auch in Herzproben mittels qRT-PCR (Light Cycler 480 System von Roche) validiert und verfeinert. Die Ergebnisse der Expressionsanalyse und der Färbungen weisen auf eine Beteiligung des Wnt/Catenin beta Signalweges und einem Defekt in den Zell-Zell-Verbindungsstrukturen in der Krankheitsentwicklung von Alopezie hin. Im nächsten Schritt werden immunohisto-chemische Färbungen in Rattenhaut in verschiedener Stadien von Haarverlust wiederholt und elektronenmikroskopisch untersucht, wobei der Fokus auf Desmosomen der Haarfollikel und der Epidermis liegt, um ein tiefergehendes Verständnis in die strukturellen Defekte der Zell-Zell-Verbindungskomplexe und dem Zeitpunkt der Zerstörung zu bekommen. Eine genomweite Kopplungsanalyse mit SNP Marker wurde im Vorfeld zu dieser Studie mit humanen Proben durchgeführt, die auf einen signifikanten Lokus auf Chromosom 19 verwies. Im Rahmen dieser Studie wurde eine Feinkartierung dieses Lokus auf Chromosom 19 in 301 Familien (1131 Individuen) mittels SNPstream, Taqman und Pyrosequencing durchgeführt. Eine Kopplungsanalyse identifizierte eine Kandidatenregion bei 37 bis 38 Mb (GRCH37), welche mehrere Zinkfingergene beinhaltet. Die höchsten nichtparametrischen lod scores wurden für SNPs in oder nahe der Gene ZNF567 und ZNF568 erzielt. Daher wurden diese Gene auf Mutationen mittels Schmelzkurvenanalyse und Sequenzierung untersucht. Es konnten keine Mutationen gefunden werden, da alle aufgetretenen Varianten auf bekannte SNPs zurückzuführen sind. Zusätzlich wurden für eine SNP basierte genomweite Assoziationsstudie (GWAS) mit 357 Fällen und 2534 Kontrollen weitere Proben gesammelt und mit Affymetrix Genome-Wide Human SNP Array 6.0 genotypisiert. Signifikante assoziierte Loci wurden für die Chromosomen 5, 6 (welches die HLA Region beinhaltet) und 16 (CLEC16A) ausfindig gemacht. Außerdem resultierte eine Kopplungsanalyse mit 259 Familien mit 855 Individuen in einer signifikanten Region auf Chromosom 10 und 19 (Zinkfingerregion). Mit einer definierten Auswahl an SNP Marker wurde eine Kandidatenregion bei 35.9 Mb bis 36.5 Mb (GRCH37) auf Chromosom 19 identifiziert. In einem nächsten Schritt wird die kombinierte Region von 35.9 Mb bis 38 Mb in Gänze mit NGS sequenziert und nach Mutationen untersucht. Abschließend weist die vorliegende Studie auf eine starke Assoziation von zellulären Defekten in der Haut mit der Krankheit hin, was in weiterführenden Experimenten mit dem Rattenmodell DEBR in der Dermatogenetikgruppe des CCG weitergehend untersucht werden wird. Zusätzlich zu den bereits bekannten immunoregulatorischen Genen, die mit AA assoziiert sind, konnte diese Studie signifikante Kopplungsbefunde für Loci auf Chromosom 10 und besonders für Chromosom 19, welche ein Zinkfingercluster beinhaltet, ausfindig machen. Diese Resultate machen erneut deutlich, dass mehrere komplexe Mechanismen zu der Suszeptibilität der Krankheit beitragen

Working Around the Withdrawal Agreement: Statutory Evidentiary Safeguards Negate the Need for a Withdrawal Agreement in Collaborative Law Proceedings

This Comment will proceed by: (I) comparing state collaborative law statutes; (II) evaluating the current ethical climate surrounding withdrawal agreements in collaborative law; (III) considering the purpose of the withdrawal agreement and how evidentiary safeguards can provide the same incentives; and finally, concluding that statutory evidentiary safeguards eliminate the need for a mandatory withdrawal agreement in the collaborative law setting

Matrix-based assessment of spatial correlations between marine uses and ecosystem service supply in German marine areas

Marine ecosystems are highly dynamic and complex and contribute immensely to human well-being. Spatiotemporal overlaps of marine uses and human activities are constantly pressuring ecosystems, and that in turn impacts the supply quantity and quality of various ecosystem services (ES). This study is the first attempt to use the ES matrix to link marine uses and the capacity to supply ES. Combining expert- and literature-based evaluations, we assessed the relationships of twelve marine uses and five selected ecosystem services in the German North and Baltic Sea and mapped their spatial distribution. Despite a limited data availability and a higher need for simplification, the matrix approach proved to be applicable for the marine realm. Areas used for tourism and those that provide coastal safeguarding show high values of ES supply in comparison to areas used for sediment extraction and areas previously used as ammunition dumping areas. Nature conservation areas tend to have the highest capacity to supply ES. Differences in the ES supply pattern between the North Sea and Baltic Sea were identified. The results show the influence of anthropogenic activities on the spatial distribution of ES supply and can support future marine planning

A rapid method for an offline glycerol determination during microbial fermentation

Background: The purpose of this work was to find a rapid method for glycerol detection during microbial fermentations. The method requirements were, first, to avoid sample pretreatment, and second, to measure glycerol precisely especially out of fermentation broth. Results: This was achieved by combining two reaction principles — the Malaprade reaction and the Hantzsch reaction. In the Malaprade reaction, glycerol is converted into formaldehyde. This forms a dye in the Hantzsch reaction after which adsorption is than detected. The subsequent assay was investigated with two different fermentation media, a chemically undefined and a chemically defined media, used for Pichia pastoris fermentation. In both media, as well as in real fermentation samples, glycerol content could be reproducibly detected with the method. Moreover, measurements were more precise than using a standard glycerol detection kit. Conclusions: With this rapid assay, glycerol could be detected easily in microbial fermentation broth. It is reliable over a wide concentration range including advantages such as an easy assay set-up, a short assay time and no sample pretreatment.Citation: Kuhn J, H Müller, D Salzig, P Czermak:A rapid method for an offline glycerol determination during microbial fermentation, Electronic Journal of Biotechnology 18 (2015) 3, 252-255 http://dx.doi.org/10.1016/j.ejbt.2015.01.00

Review of Current Neuroimaging Studies of the Effects of Prenatal Drug Exposure: Brain Structure and Function

Neuroimaging tools have provided novel methods for understanding the impact of prenatal drug exposure on brain structure and function and its relation to development and behavior. Information gained from neuroimaging studies allows for the investigation of how prenatal drug exposure alters the typical developmental trajectory. The current prevalence and characteristics of prenatal drug exposure and its implications for vulnerable periods of brain development are reviewed. Structural and functional neuroimaging methods are introduced with examples of how study results from prenatal alcohol, cocaine, marijuana, and tobacco exposure further our understanding of the neurodevelopment impact of prenatal drug exposure. Prenatal drug neuroimaging studies have advanced our understanding of mechanisms and functional deficits associated with prenatal drug exposure. Studies have identified brain circuits associated with the default mode network, inhibitory control, and working memory that show differences in function as a result of prenatal drug exposure. The information gained from studies of prenatal drug exposure on brain structure and function can be used to make connections between animal models and human studies of prenatal drug exposure, identify biomarkers of documented effects of prenatal drug exposure on behavior, and inform prevention and intervention programs for young children

Matrix-based assessment of spatial correlations between marine uses and ecosystem service supply in German marine areas

Marine ecosystems are highly dynamic and complex and contribute immensely to human well-being. Spatiotemporal overlaps of marine uses and human activities are constantly pressuring ecosystems, and that in turn impacts the supply quantity and quality of various ecosystem services (ES). This study is the first attempt to use the ES matrix to link marine uses and the capacity to supply ES. Combining expert- and literature-based evaluations, we assessed the relationships of twelve marine uses and five selected ecosystem services in the German North and Baltic Sea and mapped their spatial distribution. Despite a limited data availability and a higher need for simplification, the matrix approach proved to be applicable for the marine realm. Areas used for tourism and those that provide coastal safeguarding show high values of ES supply in comparison to areas used for sediment extraction and areas previously used as ammunition dumping areas. Nature conservation areas tend to have the highest capacity to supply ES. Differences in the ES supply pattern between the North Sea and Baltic Sea were identified. The results show the influence of anthropogenic activities on the spatial distribution of ES supply and can support future marine planning.

Cytomorphology of Circulating Colorectal Tumor Cells:A Small Case Series

Several methodologies exist to enumerate circulating tumor cells (CTCs) from the blood of cancer patients; however, most methodologies lack high-resolution imaging, and thus, little is known about the cytomorphologic features of these cells. In this study of metastatic colorectal cancer patients, we used immunofluorescent staining with fiber-optic array scanning technology to identify CTCs, with subsequent Wright-Giemsa and Papanicolau staining. The CTCs were compared to the corresponding primary and metastatic tumors. The colorectal CTCs showed marked intrapatient pleomorphism. In comparison to the corresponding tissue biopsies, cells from all sites showed similar pleomorphism, demonstrating that colorectal CTCs retain the pleomorphism present in regions of solid growth. They also often retain particular cytomorphologic features present in the patient's primary and/or metastatic tumor tissue. This study provides an initial analysis of the cytomorphologic features of circulating colon cancer cells, providing a foundation for further investigation into the significance and metastatic potential of CTCs

A low cost high flux solar simulator

A low cost, high flux, large area solar simulator has been designed, built and characterized for the purpose of studying optical melting and light absorption behavior of molten salts. Seven 1500 W metal halide outdoor stadium lights are used as the light source to simulate concentrating solar power (CSP) heliostat output. Metal halide bulbs and ballasts are far less costly per-watt than typical xenon arc lamp solar simulator light sources. They provide a satisfactory match to natural sunlight; although ‘unfiltered’ metal halide lights have irradiance peaks between 800 and 1000 nm representing an additional 5% of measured energy output as compared to terrestrial solar irradiance over the same range. With the use of a secondary conical concentrator, output fluxes of approximately 60 kW/m[superscript 2] (60 suns) peak and 45 kW/m[superscript 2] (45 suns) average are achieved across a 38 cm diameter output aperture. Unique to the design of this simulator, the tilt angle and distance between the output aperture and the ground are adjustable to accommodate test receivers of varying geometry. Use of off-the-shelf structural, lighting and electrical components keeps the fabrication cost below $10,000

Chronic prenatal depression and neonatal outcome

Four hundred and thirty pregnant women were recruited at approximately 22 weeks gestation at prenatal clinics. Of these, 86 (20%) were diagnosed as depressed. The women were seen again at approximately 32 weeks gestation and after delivery. Chronicity of depression was evidenced by continuing high depression scores in those women diagnosed as depressed. Comorbid problems were chronically high anxiety, anger, sleep disturbance, and pain scores. Less optimal outcomes for the depressed women included lower gestational age and lower birthweight of their newborns

A rapid method for an offline glycerol determination during microbial fermentation

Background: The purpose of this work was to find a rapid method for glycerol detection during microbial fermentations. The method requirements were, first, to avoid sample pretreatment, and second, to measure glycerol precisely especially out of fermentation broth. Results: This was achieved by combining two reaction principles \u2014 the Malaprade reaction and the Hantzsch reaction. In the Malaprade reaction, glycerol is converted into formaldehyde. This forms a dye in the Hantzsch reaction after which adsorption is than detected. The subsequent assay was investigated with two different fermentation media, a chemically undefined and a chemically defined media, used for Pichia pastoris fermentation. In both media, as well as in real fermentation samples, glycerol content could be reproducibly detected with the method. Moreover, measurements were more precise than using a standard glycerol detection kit. Conclusions: With this rapid assay, glycerol could be detected easily in microbial fermentation broth. It is reliable over a wide concentration range including advantages such as an easy assay set-up, a short assay time and no sample pretreatment