Kinetic study of an on-chip isocyanate derivatization reaction by on-line nano-esi ms

A high-throughput method is presented for the study of reaction kinetics by nano- electrospray ionization mass spectrometry (nano-ESI MS). The reaction of propyl isocyanate (2), benzyl isocyanate (3), and toluene-2,4-diisocyanate (4) with 4-nitro-7- piperazino-2,1,3-benzoxadiazole (NBDPZ) (1) to yield the corresponding urea derivatives (5) was carried out in a continuous flow glass microchip. Real-time monitoring of the reactions was done by nano-ESI MS. Rate constants of 1.6 ␣ 104 M-1 min-1, 5.2 ␣ 104 M-1 min-1, and 2.5 ␣ 104 M-1 min-1 were determined for isocyanate 2, 3 and 4, respectively

Recombinant factorVIII Fc fusion protein for the prevention and treatment of bleeding in children with severe hemophilia A

This work was supported by funding from Biogen, including funding for the editorial and writing support in the the development of this paper

Thermal plasticity of the kelp Laminaria digitata: latitudinal and transgenerational effects

In temperate and polar rocky coastal ecosystems, kelps form the base of complexly structured and highly diverse species associations. In recent years, kelp forests worldwide have faced extinction at their equatorward limits and models have predicted a poleward shift of kelp ecosystems during climate change. To gain an understanding of local thermal adaptation and response plasticity in a forest-forming kelp species, we assessed populations of Laminaria digitata along its entire European distribution range from Brittany to Spitsbergen for their capacity to withstand high temperature stress, and connected physiological results to population genetics. Although the overall heat response was similar across the distribution range, we identified subtle adaptions reflecting the long-term local temperature history in one northern and one southern population. Additionally, we investigated the potential role of transgenerational plasticity (TGP, i.e. effects of parental environment on offspring traits) as a means of fast response to a warming environment between haploid gametophyte parents and diploid sporophyte offspring of L. digitata. Our results show TGP for the first time in a kelp, in that not the 15°C, but the 5°C gametophyte parent treatment enhanced juvenile sporophyte offspring growth. In this talk, I will conceptualize the thermal response of L. digitata and discuss our results within the framework of global warming

Mutational analysis of PhiC31 integrase to improve gene therapeutic applications

Die Bakteriophagen Integrase PhiC31 stellt ein viel versprechendes Werkzeug zur Integration genetischen Materials im nicht viral-basierten Gentransfer dar. Die PhiC31 Integrase vermittelt die Rekombination von spezifische Erkennungssequenz attB enthaltenden Plasmiden mit natürlich vorkommenden attP Erkennungssequenzen innerhalb des Zielgenoms mit unterschiedlicher Integrationsfrequenz und -spezifität. Nebeneffekte der Integration in Form von insertioneller Mutagenese, wie z.B. große Deletionen und chromosomale Veränderungen im Genom der Zielzelle konnten beobachtet werden. Ziel dieser Dissertation war, die PhiC31 vermittelte Effizienz zu verbessern und die Spezifität zu adressieren. Als Ansatz wurde die Mutagenese der DNA-Bindungsdomäne der Integrase basierend auf Punktmutanten zur verbesserten Integrationseffizienz gewählt. Integrationsassays wurden in verschiedenen humanen Zelllinien durchgeführt. Etablierung von Doppelmutanten, sowie Dosisoptimierung des Integrase kodierenden Plasmids verbesserten die Integrationseffizienz mehr als dreifach, verglichen mit der Wildtypintegrase in den Zelllinien HeLa und HCT. Weitere Assays verglichen die Exzisionsaktivität der Integrasemutanten mit dem Wildtyp. Bei fünf Mutanten wurde eine etwa zweifach erhöhte Exzision gefunden. Die Beurteilung der Spezifität der Integrasemutanten erfolgte durch Substitution der Wildtyp-attP Sequenz mit drei Pseudo attP Erkennungssequenzen des Reporterplasmids, deren erhöhte Spezifität bereits dokumentiert war. Einzelne Mutanten zeigten eine zweifach erhöhte Exzisionsaktivität. Die Rekombinationsaktivität von Integrasemutanten wurde im Kontext chromosomaler DNA mittels einer stabil GFP-exprimierenden Reporterzelllinie, in der die eGFP Expression mittels Integrase-vermittelter „Raus-Rekombination“ eines polyA Stoppsignals angeschaltet wird, untersucht. Auf chromosomaler Ebene wurde keine verbesserte Ausschneidungsaktivität erreicht. Zur Evaluierung der in vivo Effizienz zweier ausgewählter PhiC31 Integrasemutanten, die in vitro erhöhte Integrationsaktivität aufwiesen, wurden zwei Plasmide am C57BL/6 Mausmodell getestet. Reportergen war ein für den humanen Koagulationsfaktor IX kodierendes Gen. In Abhängigkeit von der Integrationseffizienz der Mutanten und des Wildtyps, wurden im Zeitraum von einhundert Tagen ähnliche Expressionslevel gefunden. Die Mutanten zeigten keine Verbesserung der Langzeitexpression von humanem Faktor IX. Die hier durchgeführten Studien zur Mutationsanalyse der Phagenintegrase PhiC31 zeigten einen wirksamen Ansatz zur Verbesserung der PhiC31 Integrase-vermittelten Integrationseffizienz in vitro.The PhiC31 integrase system represents an attractive tool for somatic integration of extrinsic genes or specific excision in applications such as therapeutic gene transfer or genetic engineering and transgenesis. In its most prominent role, the integrase mediates integration of plasmids carrying a therapeutic gene of interest and a specific attB site into pseudo attP sites. As a non-viral integrating vector, the PhiC31 integrase suffers from insufficient gene transfer and low integration efficiency. Additionally, aberrant events such as chromosomal rearrangements and deletions are known to occur at a given frequency of about 15 % within the host genome. Therefore, the goal of this study was to first optimise the PhiC31 integrase-mediated integration efficiency and second to optimise the specificity by performing site-directed alanine scanning mutagenesis for the first time within the DNA binding domain. In the course of this work, 22 single amino acid mutations were generated, which were then evaluated in antibiotic-selective integration assays in different human cell lines, based on cotransfection of integrase encoding plasmid and substrate plasmid with attB and neomycin resistance marker. Initial screening revealed one mutant with 1.7-fold higher integration efficiency. Upon optimisation of the plasmid ratio and with the creation of double mutants, the integration efficiency of two double mutants could be enhanced above threefold in HeLa cells. A nearly twofold improvement of one particular mutant could be achieved in Huh7 and in HCT cells, indicating that integration efficiency is cell line dependent. Intramolecular plasmid-based excision assays were established leading to gene activation of the reporter. At native attB ×attP recombination sites, five mutants with about twofold enhanced integration efficiency compared to wt integrase were identified, whereas recombination activity in chromosomal context of a stably integrating GFP reporter cell line showed no improvement. Increasing integrase plasmid dose fivefold and replacing the native attP site within the substrate plasmid by three preferentially targeted pseudo attP sites found in mammalian genomes did not result in any improvement of the integrase-mediated excision activity of a polyA termination site beyond twofold. To evaluate integration efficacy of PhiC31 integrase in vivo in murine liver a human coagulation Factor IX (hFIX) encoding substrate plasmid and the respective integrase encoding plasmid were co-injected via high-pressure tail vein injection into C57BL/6 mice. Two integrase mutants, K457A and D470A, showed a similar hFIX expression profile compared to wt integrase as determined by serum hFIX levels

Patterns of bruising in preschool children with inherited bleeding disorders: a longitudinal study

Objective The extent that inherited bleeding disorders affect; number, size and location of bruises in young children <6 years. Design Prospective, longitudinal, observational study. Setting Community. Patients 105 children with bleeding disorders, were compared with 328 without a bleeding disorder and classified by mobility: premobile (non-rolling/rolling over/ sitting), early mobile (crawling/cruising) and walking and by disease severity: severe bleeding disorder factor VIII/IX/XI <1 IU/dL or type 3 von Willebrand disease. Interventions Number, size and location of bruises recorded in each child weekly for up to 12 weeks. Outcomes The interventions were compared between children with severe and mild/moderate bleeding disorders and those without bleeding disorders. Multiple collections for individual children were analysed by multilevel modelling. Results Children with bleeding disorders had more and larger bruises, especially when premobile. Compared with premobile children without a bleeding disorder; the modelled ratio of means (95% CI) for number of bruises/ collection was 31.82 (8.39 to 65.42) for severe bleeding disorders and 5.15 (1.23 to 11.17) for mild/moderate, and was 1.81 (1.13 to 2.23) for size of bruises. Children with bleeding disorders rarely had bruises on the ears, neck, cheeks, eyes or genitalia. Conclusions Children with bleeding disorder have more and larger bruises at all developmental stages. The differences were greatest in premobile children. In this age group for children with unexplained bruising, it is essential that coagulation studies are done early to avoid the erroneous diagnosis of physical abuse when the child actually has a serious bleeding disorder, however a blood test compatible with a mild/moderate bleeding disorder cannot be assumed to be the cause of bruising

Verkörperungen von Glitch

Die Arbeit ‚Verkörperungen von Glitch: Fiktive Figuren, Alter Egos und Avatare in der Gegenwartskunst‘ behandelt die Frage, wie Glitch in den bildenden Künsten ‘verkörpert’ wird und wie das Prinzip des Glitchs von sich als nicht-binär indentifizierenden, rassifizierten und weiteren marginalisierten Gruppen aufgegriffen wird, um eine Nicht-Identität im Bereich zwischen Online-Welt und ‘realer’ Welt zu kreieren. Neben umfangreicher Besprechung der Terminologien Glitch, Glitch Art und Glitch Feminismus werden potenzielle Verkörperungen von Glitch in der zeitgenössischen Kunst erforscht, insbesondere die Verwendung fiktiver Figuren, Alter Egos und Avatare als künstlerische Strategien der Selbstdarstellung. Anhand der Arbeiten verschiedener selbstgewählter beispielhafter KünstlerInnen wird ergründet, ob und, wenn ja, wie die Glitch-Ästhetik genutzt werden kann, um normative Annahmen über Identität, Verkörperung und Repräsentation zu hinterfragen. Der vorgestellte künstlerische Kanon beschränkt sich im Sinne Legacy Russels Glitch Feminismus: Ein Manifest (2020) bewusst auf queere, Schwarze und weibliche Positionen der Gegenwartskunst: Juliana Huxtable, Kapwani Kiwanga, E. Jane und Jayson Musson, die den Glitch mittels fiktiver Figuren und Alter Egos verkörpern; Lil Miquela, LaTurbo Avedon, American Artist und Sondra Perry, welche anhand der Erschaffung oder Verkörperung von (KI-generierten) Avataren die Grenzen von Online- und Offline-Welt verwischen und diskriminierende Technologien untergraben, und die Glitch-PerformerInnen Sin Wai Kin, Cibelle Cavalli Bastos und boychild, die mit ihrer Arbeit die Bedeutung des Internets als Kabarett und Ort der Gemeinschaftsbildung hervorheben. Abschließend soll festgestellt werden, ob die Verkörperung von Glitch als wirksame Strategie der Untergrabung von Kontrolle über Geschlecht, Rasse und Technologie zu legitimieren ist

Cold thermal priming of Laminaria digitata (Laminariales, Phaeophyceae) gametophytes enhances gametogenesis and thermal performance of sporophytes

Thermal characteristics of kelp species have been studied in many ways, but potentially persistent effects of temperature across generations are yet poorly understood. In this context, the effect of thermal priming on fertility and growth of the N-Atlantic kelp species Laminaria digitata was investigated within and across life cycle generations in a two-step common garden experiment. Using vegetative clonal gametophytes from cold (5°C) and warm (15°C) pre-experimental cultivation (3 years), we first quantified gametogenesis and recruitment over two weeks at a common temperature of 10°C. Then, recruited sporophytes were transferred to a temperature gradient spanning the tolerance range of the species from 0°C to 20°C. We hypothesized that a warm gametophyte preexperimental cultivation promotes performance of sporophytes at warm temperatures and vice versa. Interestingly, gametogenesis speed and sporophyte recruitment were higher in gametophytes following cold compared to warm pre-experimental cultivation, which indicates carry-over effects of temperature within the gametophyte generation. Compared to warm pre-experimental cultivation of gametophytes, a cold preexperimental cultivation enhanced growth of juvenile Laminaria digitata sporophytes by more than 69% at the extreme low and high temperatures of 0 and 20°C. This is the first evidence for a cross-generational effect between gametophyte parents and offspring sporophytes. As cold gametophyte cultivation increased the trait performance of gametogenesis, recruitment and thermal tolerance of juvenile sporophytes, priming of early life cycle stages may be used to increase resilience and productivity of kelps in marine forest restoration efforts and kelp mariculture

How private is private? Considerations on the public funding of private educational providers

Der folgende Beitrag geht der Frage nach, welchen Charakter der aktuelle Privatisierungstrend im Bildungsbereich hat. Im Mittelpunkt stehen dabei die Privatschulen in Deutschland, deren Schülerzahl seit den 1990er-Jahren kontinuierlich ansteigt. Zunächst wird in internationaler Perspektive der politische und ökonomische Kontext dieser Entwicklung skizziert. Anschließend geht es um die Finanzierung und die Nutznießer des Privatschulbooms. Dass private Ersatzschulen zwar eng an staatliche Vorgaben gebunden sind, sie dafür aber im Gegenzug massiv durch die öffentliche Hand gefördert werden, ist dann Anlass für eine ausblickende Diskussion: Welche neuen Privatschulformen zeichnen sich ab? Und welche gesellschaftlichen Effekte könnten diese haben? (DIPF/Orig.)This article deals with the question how the topical trend towards privatization in the field of education can be characterized. A key focus is on private schools in Germany. The number of their students has been continuously increasing since the nineties. Firstly, the political and economic context of this development is described in an international perspective. Secondly, the financing of this private school boom and the beneficiaries of this development are depicted. The fact that private schools are tightly tied to government regulations on the one hand, but are largely supported by public funding on the other, gives cause for a prospective discussion: What kind of new types of private schools are looming? And which societal effects might they have? (DIPF/Orig.