Christian Gausterer

TYK2 kinase activity is required for functional type I interferon responses in vivo.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

Effects assessment: Boron compounds in the aquatic environment

In previous studies, boron compounds were considered to be of comparatively low toxicity in the aquatic environment, with predicted no effect concentration (PNEC) values ranging around 1 mg B/L (expressed as boron equivalent). In the present study, we describe an evaluation of toxicity data for boron available for the aquatic environment by different methods. For substances with rich datasets, it is often possible to perform a species sensitivity distribution (SSD). The typical outcome of an SSD is the Hazardous Concentration 5% (HC5), the concentration at which 95% of all species are protected with a probability of 95%. The data set currently available on the toxic effects of boron compounds to aquatic organisms is comprehensive, but a careful evaluation of these data revealed that chronic data for aquatic insects and plants are missing. In the present study both the standard assessment factor approach as well as the SSD approach were applied. The standard approach led to a PNEC of 0.18 mg B/L (equivalent to 1.03 mg boric acid/L), while the SSD approach resulted in a PNEC of 0.34 mg B/L (equivalent to 1.94 mg boric acid/L). These evaluations indicate that boron compounds could be hazardous to aquatic organisms at concentrations close to the natural environmental background in some European regions. This suggests a possible high sensitivity of some ecosystems for anthropogenic input of boron compounds. Another concern is that the anthropogenic input of boron could lead to toxic effects in organisms adapted to low boron concentration.

Rapid genetic detection of ingested Amanita phalloides

Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fools mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning.

Octamer-binding factor 6 (Oct-6/Pou3f1) is induced by interferon and contributes to dsRNA-mediated transcriptional responses

BackgroundOctamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family. POU proteins regulate key developmental processes and have been identified from a diverse range of species. Oct-6 expression is described to be confined to the developing brain, Schwann cells, oligodendrocyte precursors, testes, and skin. Its function is primarily characterised in Schwann cells, where it is required for correctly timed transition to the myelinating state. In the present study, we report that Oct-6 is an interferon (IFN)-inducible protein and show for the first time expression in murine fibroblasts and macrophages.ResultsOct-6 was induced by type I and type II IFN, but not by interleukin-6. Induction of Oct-6 after IFNβ treatment was mainly dependent on signal transducer and activator of transcription 1 (Stat1) and partially on tyrosine kinase 2 (Tyk2). Chromatin immunopreciptitation experiments revealed binding of Stat1 to the Oct-6 promoter in a region around 500 bp upstream of the transcription start site, a region different from the downstream regulatory element involved in Schwann cell-specific Oct-6 expression. Oct-6 was also induced by dsRNA treatment and during viral infections, in both cases via autocrine/paracrine actions of IFNα/β. Using microarray and RT-qPCR, we furthermore show that Oct-6 is involved in the regulation of transcriptional responses to dsRNA, in particular in the gene regulation of serine/threonine protein kinase 40 (Stk40) and U7 snRNA-associated Sm-like protein Lsm10 (Lsm10).ConclusionOur data show that Oct-6 expression is not as restricted as previously assumed. Induction of Oct-6 by IFNs and viruses in at least two different cell types, and involvement of Oct-6 in gene regulation after dsRNA treatment, suggest novel functions of Oct-6 in innate immune responses.

In Vivo Target Validation: Methodology and Case Studies on the Janus Kinase Tyk2

Dysregulated protein kinase activity can cause severe human diseases. Consequently, there is a growing number of kinases that constitute candidate targets for pharmaceutical intervention. However, target validation is critical, since not all kinases are “druggable”, i.e. suitable as a selective drug target. In this review, we briefly introduce major strategies for generating mouse models to analyse and control the expression and function of distinct genes, to confirm specific inhibition of intended drug targets and to identify undesirable secondary effects in vivo. Focussing on tyrosine kinase 2 (Tyk2) and other Janus kinases (Jaks) as case studies we follow the path of investigations from in vitro towards in vivo experimentation. We give examples for the sometimes surprising consequences of the systemic absence of a distinct kinase; consequences that can not be easily deduced solely based on results from in vitro data. We discuss aspects of kinase functions that are distinct from their catalytic activity and give examples for cell-type specific functions. Potential pitfalls (e.g. embryonic lethality, species differences) of using mouse models as experimental systems for studying human diseases are discussed and strategies for improvements to deal with such complications are exemplified.

First genetic evidence of leprosy in early medieval Austria

SummaryLeprosy used to be a widespread, dreaded disease in Europe during the middle ages, and it still remains an important health problem in some parts of the world today. Herein, we present data on the earliest ‘Austrian’ (an adult female from the early medieval period) proven to have suffered from leprosy. Manifestations of the disease were first identified during a systematic screening of pathological changes in skeletons recovered from an archaeological site in Pottenbrunn (Lower Austria). In the present study, DNA extracts from selected cranial and postcranial bone samples were investigated using polymerase chain reaction primers specific to the Mycobacterium leprae (M. leprae) repetitive element (RLEP). M. leprae traces were detected in extracts from nasal and palatine bones. Sequence analysis of informative polymorphic sites supports previous reports indicating that European M. leprae strains fall into single nucleotide polymorphism group 3. In summary, these findings put Austria on the map of confirmed leprosy cases in ancient Europe.ZusammenfassungLepra war während des Mittelalters eine in Europa weit verbreitete, gefürchtete Volkskrankheit, und gilt auch noch heute in manchen Teilen der Welt als ein wichtiges öffentliches Gesundheitsproblem. In diesem Artikel berichten wir über den ersten bestätigten Leprafall aus dem frühmittelalterlichen Österreich.Die Symptome der Krankheit sind erstmals während eines systematischen Screenings der pathologischen Veränderungen an einem Skelett aus dem Gräberfeld von Pottenbrunn (Niederösterreich) identifiziert worden. In der vorliegenden Studie wurde DNA aus ausgewählten Schädel- und postcranialen Resten extrahiert und mittels Polymerase-Kettenreaktion untersucht. Spezifische DNA Spuren des Lepra-Erregers Mycobacterium leprae (M. leprae) wurden in Proben von Nasen- und Gaumenknochen eindeutig nachgewiesen und mittels Sequenzanalyse typisiert. Unsere Ergebnisse unterstützen frühere Hinweise, dass europäische M. leprae-Stämme zur Einzelnukleotid-Polymorphismen Gruppe 3 gehören.Zusammenfassend erscheint Österreich aufgrund dieser Befunde erstmals auf der Landkarte bestätigter Leprafälle im alten Europa.

Molecular identification of traces from the White-tailed Sea Eagle

PurposeOver the preceeding decades, after periods of dramatic decline and extinction in many parts of Europe, the White-tailed Sea Eagle (Haliaeetus albicilla) has re-colonized traditional breeding areas. However, this large apex predator remains threatened, not only by the bioaccumulation of environmental pollutants, but also by targeted poisoning and poaching. In connection with a forensic case, a novel PCR assay was developed for the sensitive and specific detection of sea eagle DNA traces in questioned samples of unknown origin.MethodsThe assay amplifies a fragment of the popular phylogenetic marker gene cytochrome b. Primers were designed to bind sites with relatively high variability between homologous sequences from H. albicilla and other related European birds of prey.ResultsAssay sensitivity was sufficient for single cell analysis. Specificity was tested in vitro and the primers did not cross-detect DNA from humans, chicken and the following raptors: Common Buzzard (Buteo buteo), Northern Goshawk (Accipiter gentilis), Red Kite (Milvus milvus) and Black Kite (Milvus migrans). Applicability for the analysis of poor quality samples was demonstrated with extracts from field-collected small molted down feathers that did not contain detectable amounts of sea eagle nuclear DNA. Amplicons of the expected size were generated, purified and sequenced. Sequence data were subjected to Basic Local Alignment Search Tool analysis and affiliated with cytochrome b from H. albicilla.ConclusionsThe novel PCR primers allowed for the correct assignment of traces from H. albicilla, even in mixed samples and in cases with limited and degraded biological material.