Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme

At present it is generally accepted that plasma prekallikrein (PPK) is synthesized in the liver and secreted into the bloodstream. Surprisingly, it has recently been shown that PPK mRNA is present also in RNA from the kidney, adrenal gland and placenta. In spite of its novelty and possible important physiological implications this finding has been neglected. Here we report that PPK mRNA is expressed also in the human brain, heart, lung, trachea, endothelial cells and leukocytes as well as in a variety of fibroblast and epithelial cell lines. Expression of PPK mRNA in fibroblasts, endothelial cells and leukocytes suggests that PPK mRNA detected in RNA preparations from whole tissue may originate solely from these ubiquitously occurring cells. However, PPK mRNA expression in various epithelial cell lines demonstrates that tissue-specific cells also transcribe the PPK gene. The presence of PPK mRNA in nonhepatic tissues and cells indicates that they have the capacity to synthesize the protein. The physiological role of PPK synthesized in extrahepatic tissue is unknown. It may participate in local actions within tissues as well as contributing to the PPK pool in blood plasma. Cultured cells will provide a valuable model for exploring the physiological significance of extrahepatic PPK expression

Molekulare Analyse der mRNA-Expression von Plasma-Prokallikrein

Plasma-Kallikrein (PK) ist eine Serinprotease, die als inaktives Zymogen Plasma- Prokallikrein (PPK) in der Leber gebildet und in den Blutstrom sezerniert wird. Dort erfolgt die Konvertierung zum aktiven Enzym im Kontaktphasen-System der Blutgerinnung. Neben der Funktion im Kontakt-System spielt diese Protease sowohl in der Fibrinolyse als auch im Kallikrein-Kinin-System eine zentrale Rolle. Neuere Arbeiten haben gezeigt, dass PPKmRNA auch außerhalb der Leber exprimiert wird. Daraus ist zu schließen, dass dort ebenfalls PPK-Protein gebildet wird und dass diesem extrahepatischen, gewebeständigen PPK bzw. PK spezielle lokale Funktionen zukommen. Dieser Befund bildet die Grundlage dieser Dissertation. Mit dem langfristigen Ziel die physiologische und pathophysiologische Rolle des extrahepatisch gebildeten PPK aufzuklären, sollte in dieser Arbeit zunächst mittels einer quantitativen PCR-Technik (TaqMan) untersucht werden, in welchem Umfang PPK-mRNA und die mRNAs der übrigen Komponenten des Kontaktphasen-Systems in humanen Zellen bzw. Geweben exprimiert werden. Aufgrund dieser Ergebnisse sollte im zweiten Teil der Arbeit untersucht werden, ob bzw. inwieweit das PPK-Gen gewebespezifisch reguliert werden kann. Hierzu sollten die regulatorisch bedeutsamen Regionen des PPK-Gens charakterisiert werden. Mittels der TaqMan-Technologie konnte die PPK-mRNA-Expression in allen untersuchten humanen Geweben quantifiziert werden. In neun Geweben liegt die mRNA-Expression im Vergleich zur Leber zwischen 1 % und 68 % und in sechs Geweben zwischen 0,1 % und 1 %. Diese Ergebnisse sprechen für eine ubiquitäre, aber teils sehr unterschiedliche Expression der PPK-mRNA in humanen Geweben. Die mRNAs von hochmolekularem Kininogen (HK), niedermolekularem Kininogen (LK), Faktor XI (FXI) und Faktor XII (FXII) werden dagegen nur in wenigen Geweben exprimiert. Die Transkripte von HK, LK und FXI findet man außerhalb der Leber nur noch in Nierengewebe in signifikanter Menge, während FXII-mRNA nahezu ausschließlich in der Leber synthetisiert wird. Ein gemeinsames Merkmal aller Transkripte ist, dass sie in Lebergewebe am stärksten expimiert werden. Zellstimulationsversuche mit HepG2-Zellen ergaben, dass die PPK-mRNA-Synthese mittels Lipopolysacchariden (LPS) kurzfristig induziert werden kann, was für eine wesentliche Funktion von PPK bzw. PK bei Entzündungsreaktionen spricht. Im Gegensatz hierzu bewirkt Phorbol-12-myristat-13-acetat (PMA) eine Herabregulation der PPK-Transkription. Untersuchungen zur Stabilität der mRNAs von PPK und dem Haushaltsgen GAPDH mit den Transkriptionsinhibitoren Actinomycin D, α-Amanitin und DRB zeigten, dass die mRNAAbbaurate in HepG2-Zellen vom jeweils eingesetzten Transkriptionsinhibitor abhängt. Bei allen Transkriptionsinhibitoren wurde das PPK-Transkript im Vergleich zu GAPDH deutlich schneller (Faktor 3-5) abgebaut. Dies weist darauf hin, dass die PPK-mRNA-Spiegel in den Zellen durch ständige Neusynthese aufrechterhalten werden, um eine kontinuierliche Produktion von PPK zu gewährleisten. Mittels der RLM-RACE Methode wurden in Leber-, Pankreas-, Nieren- und Testisgewebe die PPK-Transkriptionsstartpunkte (TS) identifiziert. Während in der Leber und im Pankreas nur TSs in Exon 1 des PPK-Gens benutzt werden, findet man in Nieren- und Testisgewebe TSs sowohl in der upstream-Region als auch in Intron 1. Weiterhin wurden in Nierengewebe drei am 5´-Ende verkürzte PPK-Transkripte detektiert, die aufgrund dieser Deletion keine Signalpeptid- Sequenz enthalten und dadurch eine intrazelluläre Lokalisation der entsprechenden PPK-Isoform bedingen. Die Consensussequenz-Analyse für die Transkriptionsinitiation zeigt, dass jedem experimentell ermittelten Transkriptionsstart eine TATA-Box oder/und ein Initiator-Element assoziiert ist. Außerdem konnte durch die RLM-RACE-Analysen die Existenz eines weiteren untranslatierten Exons im 5´-Bereich des PPK-Gens ausgeschlossen werden. Unter Anwendung der Genome Walker Technik wurde die Promotorregion (P-1729) stromaufwärts von Exon 1 kloniert und mittels Reportergen-Studien analysiert. In HepG2-Zellen zeigt dieses Gensegment eindeutig transkriptionsaktivierende Kapazität. Durch die Generierung von neun Verkürzungsvarianten konnten der Core-Promotor (-158 bis +22), Enhancerbereiche (-1675 bis -1494) und Silencerregionen (-1304 bis -674) charakterisiert werden. Die Transkriptionsfaktoren-Analyse ergab, dass bei der Regulation durch die P-1729 Region neben ubiquitär exprimierten auch streng gewebespezifische Transkriptionsfaktoren eine zentrale Rolle spielen. Reportergen-Untersuchungen des Intron-1-Bereichs in HEK 293-Zellen zeigten, dass auch diese Region des PPK-Gens transkriptionsaktivierende Kapazität besitzt. Ebenso zeigt aber auch der P-1729 Bereich in HEK 293-Zellen Promotoraktivität. Somit können innerhalb eines Zelltyps beide Promotorbereiche funktionell aktiv sein, wodurch eine alternative Regulation der PPK-Transkription ermöglicht wird

T-SP1: a novel serine protease-like protein predominantly expressed in testis

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends ( RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue

Dissecting the impact of Frizzled receptors in Wnt/beta-catenin signaling of human mesenchymal stem cells

Wnt/beta-catenin signaling is of fundamental importance in the regulation of self-renewal, migration/invasion, and differentiation of human mesenchymal stem cells (hMSCs). Because little information is available about the function of Frizzled receptors (Fzds) as the main receptors of Wnt proteins in hMSCs, we first performed comparative Fzd mRNA expression profiling. Fzd9 and Fzd10 were not expressed in hMSCs. While Fzd3 was expressed at low levels in hMSCs, the other Fzds exhibited high expression rates. Activation and repression of Wnt signaling in hMSCs revealed that the expression levels of Fzd1, Fzd6, and Fzd7 are positively correlated with the Wnt/beta-catenin activation status, whereas Fzd8 exhibited an inverse relation. For studying the functional relevance of Fzds in Wnt/beta-catenin signaling, RNA interference, ectopic expression studies, and rescue approaches were performed in hMSCs carrying a highly sensitive TCF/LEF reporter gene system (Gaussia luciferase). We found that, Fzd1, Fzd5, Fzd7, and Fzd8 are largely involved in Wnt/beta-catenin signaling of hMSCs. Moreover, the knockdown of Fzd5 can be compensated by the ectopic expression of Fzd7. Conversely, the ectopic expression of Fzd5 in Fzd7-knockdown hMSCs resulted in a rescue of Wnt/beta-catenin signaling, pointing to a functional redundancy of Fzd5 and Fzd7

Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways

WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the beta-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced beta-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the beta-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways

Validation of STAT1 variants found in patients with primary immunodeficiencies and evaluation of the effect of JAK inhibition using an in vitro model

Regulation of cellular responses to interferons, cytokines, growth factors and hormones is mediated by signal transducer and activator of transcription (STAT) proteins. In the immune system binding of a cytokine (e.g. IFN) to the corresponding surface receptor, Janus kinase (JAK) molecules are phosphorylated, resulting in the docking and phosphorylation of the associated STAT proteins. The STATs will form homo or heterodimers and translocate to regulate transcription of pro-inflammatory target genes. Mutations in STAT1 are known to result in immunodeficiency and/or immune dysregulation syndromes. In this project, the functional impact of variants in the STAT1 gain-of-function gene (STAT1 GOF) will be analyzed on a protein level. The effect of a directed treatment approach targeting the JAK-STAT pathway (JAK inhibitors) will be evaluated in an in vitro model. Freshly isolated PBMCs or whole blood samples from patients and healthy controls were obtained. The cells were stimulated with IFNg and the treated with the JAK inhibitor Ruxolitinib. Extra and intracellular staining with anti-human fluorochrome conjugated antibodies was performed in order to determine the expression of STAT1 and pSTAT1 on monocytes by means of flow cytometry. Two pediatric patients and one related adult patient were studied and the pathogenicity of the variants was confirmed as STAT1 and pSTAT1 levels in the patients at baseline as well as after IFNg stimulation were markedly increased, when compared with healthy controls. The in vitro administration of different concentrations of the JAK inhibitor Ruxolitinib resulted in the normalization of pSTAT1 levels in the cells obtained from patients with STAT1 GOF mutations. Patients with STAT1 GOF mutations show a severe clinical phenotype with recurrent bacterial and fungal infections. Currently no specific treatment options are available. However recent case reports suggested the benefit of JAK inhibition. Therefore we studied the effect of this drug using primary cells in an in vitro model on a molecular level. Importantly two patients have been started on this medication and have achieved a significant clinical response. We are currently evaluating the capacity of our protocol to identify patients with alterations of the JAK-STAT pathway eligible for this targeted treatment approach

Automatic data quality control for understanding extreme climate event

The understanding of extreme events strongly depends on knowledge gained from data. Data integration of mul-tiple sources, scales and earth compartments is the fo-cus of the project Digital Earth, which also join efforts on the quality control of data. Automatic quality control is embedded in the ingest component of the O2A, the ob-servation-to-archive data flow framework of the Alfred-Wegener-Institute. In that framework, the O2A-Sensor provides observation properties to the O2A-Ingest, which delivers quality-flagged data to the O2A-dash-board. The automatic quality control currently follows a procedural approach, where modules are included to implement formulations found in the literature and other operational observatory networks. A set of plausibility tests including range, spike and gradient tests are cur-rently operational. The automatic quality control scans the ingesting data in near-real-time (NRT) format, builds a table of devices, and search - either by absolute or derivative values - for correctness and validity of obser-vations. The availability of observation properties, for in-stance tests parameters like physical or operation ranges, triggers the automatic quality control, which in turn iterates through the table of devices to set the qual-ity flag for each sample and observation. To date, the quality flags in use are sequential and qualitative, i.e. it describes a level of quality in the data. A new flagging system is under development to include a descriptive characteristic that will comprise technical and user inter-pretation. Within Digital Earth, data on flood and drought events along the Elbe River and methane emissions in the North Sea are to be reviewed using automatic qual-ity control. Fast and scalable automatic quality control will disentangle uncertainty raised by quality issues and thus improve our understanding of extreme events in those cases

Colchicine treatment in children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome: A multicenter study in Spain

Objective: To evaluate the efficacy of colchicine therapy in pediatric patients with PFAPA syndrome who present with an incomplete response to the standard treatment or with frequent episodes (an interval of less than 14 days between two disease flares). Methods: A multicenter cohort study of children diagnosed with PFAPA syndrome and treated with colchicine was performed in three separate hospitals located in Spain. The patients clinical and laboratory data were reviewed by accessing their medical records. Response to colchicine was evaluated after 12 months of treatment for frequency, duration, and intensity of PFAPA episodes. Results: A total of 13 children were included in our study, 43% of whom were boys. Median age of the colchicine therapy initiation was 6 years (interquartile range (IQR)=3-9.5). Following a 12-month period of colchicine therapy (median dosage of 0.02 mg/kg/day; IQR=0.02-0.03), a significant decrease in the median number of flares (median 8; IQR=7-14 vs 3; IQR=2-4; p=0.005) and the duration of disease episodes (median 4 days; IQR=3.25-5.125 vs 1 day; IQR=1-2; p=0.003) was observed. Furthermore, the highest degree of fever during disease flares was reduced from median 40ºC (IQR=39.5-40) to 38.5ºC (IQR=37.7-38.9) (p=0.002). Conclusion: Colchicine therapy decreased the frequency and intensity of PFAPA. The use of colchicine could be an effective treatment in pediatric patients with PFAPA syndrome who present with frequent or severe relapses

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/β-catenin signaling

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/β-catenin signaling pathway as indicated by the increased stability and nuclear localization of β-catenin in TIMP-1–deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced β-catenin transcriptional activity, determined by Wnt/β-catenin target gene expression analysis and a luciferase-based β-catenin– activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on β-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1–mediated effects on Wnt/β-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of β-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1–depleted hMSCs and demonstrably reduced axin 2, an antagonist of β-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/β-catenin activity

O2A - Data Flow Framework from Sensor Observations to Archives

The Alfred Wegener Institute coordinates German polar research and is one of the most productive polar research institutions worldwide with scientists working in both Polar Regions – a task that can only be successful with the help of excellent infrastructure and logistics. Conducting research in the Arctic and Antarctic requires research stations staffed throughout the year as the basis for expeditions and data collection. It needs research vessels, aircrafts and long-term observatories for large-scale measurements as well as sophisticated technology. In this sense, the AWI also provides this infrastructure and competence to national and international partners. To meet the challenge the AWI has been progressively developing and sustaining an e-Infrastructure for coherent discovery, visualization, dissemination and archival of scientific information and data. Most of the data originates from research activities being carried out in a wide range of sea-, airand land-based operating research platforms. Archival and publishing in PANGAEA repository along with DOI assignment to individual datasets is a pursued end-of-line step. Within AWI, a workflow for data acquisition from vessel-mounted devices along with ingestion procedures for the raw data into the institutional archives has been well established. However, the increasing number of ocean-based stations and respective sensors along with heterogeneous project-driven requirements towards satellite communication, sensor monitoring, quality control and validation, processing algorithms, visualization and dissemination has recently lead us to build a more generic and cost-effective framework, hereafter named O2A (observations to archives). The main strengths of our framework (https://www.awi.de/en/data-flow) are the seamless flow of sensor observation to archives and the fact that it complies with internationally used OGC standards and assuring interoperability in international context (e.g. SOS/SWE, WMS, WFS, etc.). O2A comprises several extensible and exchangeable modules (e.g. controlled vocabularies and gazetteers, file type and structure validation, aggregation solutions, processing algorithms, etc.) as well as various interoperability services. We are providing integrated tools for standardized platform, device and sensor descriptions following SensorML (https://sensor.awi.de), automated near-real time and “big data” data streams supporting SOS and O&M and dashboards allowing data specialists to monitor their data streams for trends and early detection of malfunction of sensors (https://dashboard.awi.de). Also in the context of the “Helmholtz Data Federation” with outlook towards the European Open Science Cloud we are developing a cloud-based workspace providing user-friendly solutions for data storage on petabyte-scale and state-of-the-art computing solutions (Hadoop, Spark, Notebooks, rasdaman, etc.) to support scientists in collaborative data analysis and visualization activities including geo-information systems (http://maps.awi.de). Our affiliated repositories offer archival and long-term preservation as well as publication solutions for data, data products, publications, presentations and field reports (https://www.pangaea.de, https://epic.awi.de)