Facilitating trypanosome imaging.

Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton.ME is supported by DFG grants EN305 and SPP1726. ME is a member of the Wilhelm Conrad Röntgen Center for Complex Material Systems (RCCM). Work in MC's lab was funded by the a Wellcome Trust award (093008/Z10/Z)

How can signaling in authentic classroom videos support reasoning on how to induce learning strategies?

Classroom videos are a viable means to implement evidence-informed reasoning in teacher education in order to establish an evidence-informed teaching practice. Although learning with videos relieves pre-service teachers from acting in parallel and might reduce complexity, the material still poses higher cognitive load than written text vignettes or other traditionally used static material. In particular, the information they deliver is transient and can, therefore, easily be missed. Signaling can guide learners’ attention to central aspects of a video, thereby reducing cognitive load and enhancing learning outcomes. In the current project, pre-service teachers acquired scientific knowledge about learning strategies and their promotion in a computer-based learning environment. We explored the effect of different arrangements of signaling in classroom video-examples on conceptual knowledge and the reasoning-component of professional vision. Therefore, we conducted a set of two studies with 100 student teachers including two signal arrangements in order to investigate how signaling can help learning to reason about classroom videos. In addition, we varied if participants received information on the use of signals in advance (informed) or not (uninformed). We measured conceptual knowledge by asking participants what they knew about self-regulation strategies. Additionally, we assessed reasoning by asking participants to notice sequences in a video where teachers induced learning strategies, and to reason in what respect the observed behavior was useful to induce the strategy. Uninformed signaling did not affect the acquisition of conceptual knowledge and reasoning. Informed signaling led to significantly better conceptual knowledge than uninformed signaling. It is argued that the signal-induced extraneous load exceeded the load reduction due to the signal’s selection advantage in the uninformed conditions. In a third, exploratory study, nine participants were interviewed on the perception of different signals and indicated that spotlight and zoom-in signals foster processing of classroom videos

Positional dynamics and glycosomal recruitment of developmental regulators during trypanosome differentiation

African trypanosomes are parasites of sub-Saharan Africa responsible for both human and animal disease. The parasites are transmitted by tsetse flies, and completion of their life cycle involves progression through several development steps. The initiation of differentiation between blood and tsetse fly forms is signaled by a phosphatase cascade, ultimately trafficked into peroxisome-related organelles called glycosomes that are unique to this group of organisms. Glycosomes undergo substantial remodeling of their composition and function during the differentiation step, but how this is regulated is not understood. Here we identify a cytological site where the signaling molecules controlling differentiation converge before the dispersal of one of them into glycosomes. In combination, the study provides the first insight into the spatial coordination of signaling pathway components in trypanosomes as they undergo cell-type differentiation.Glycosomes are peroxisome-related organelles that compartmentalize the glycolytic enzymes in kinetoplastid parasites. These organelles are developmentally regulated in their number and composition, allowing metabolic adaptation to the parasite’s needs in the blood of mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates differentiation between parasite developmental forms, comprising a tyrosine phosphatase, Trypanosoma brucei PTP1 (TbPTP1), which dephosphorylates and inhibits a serine threonine phosphatase, TbPIP39, which promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to glycosomes during differentiation from bloodstream “stumpy” forms to procyclic forms. Detailed microscopy and live-cell imaging during the synchronous transition between life cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site closely associated with TbVAP, which defines the flagellar pocket endoplasmic reticulum. TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of stumpy-enriched mRNAs. This site provides a molecular node for the interaction between TbPTP1 and TbPIP39. Within 30 min of the initiation of differentiation, TbPIP39 relocates to glycosomes, whereas TbPTP1 disperses to the cytosol. Overall, the study identifies a “stumpy regulatory nexus” (STuRN) that coordinates the molecular components of life cycle signaling and glycosomal development during transmission of Trypanosoma brucei

The relationship between teleworking, traffic and air pollution

Traffic congestion is one of the foremost problems confronted by the urban and suburban tenants of today, which increases vehicle emissions and degrades air quality. Urban planners and policy makers have consequently been always investigating choices to alleviate traffic congestion and to enhance air quality. Teleworking is one option that has received significant consideration and has been studied in the recent past. The aim of the study is to explore the relationship between teleworking, air quality and traffic in Switzerland. The analysis relies on panel individual and household level data over the period 2002–2013. We examine five main air pollutants; the sulphur dioxide (SO 2 ), the ground-level ozone (O 3 ) the nitrogen dioxide (NO 2 ), the carbon monoxide (CO) and the particulate matter less than 10 μm (PM 10 ). Based on the fixed effects estimates, teleworking reduces traffic volume by 1.9 per cent. Furthermore, the reduction observed on air pollution is higher for NO 2 , CO and PM 10 ranging between 3.3 and 3.7 per cent, followed by O 3 at 2.3 per cent and SO 2 at 2.1 per cent. According to instrumental variable (IV) approach and the two stage least squares (2SLS) method, the effect is higher ranging between 2.6 and 4.1 per cent. The respective reduction on traffic becomes 2.7 per cent. Overall, the main concluding remark of the study is that teleworking can be a promising tool for urban planning and development, focusing at the traffic volume reduction, and the air quality improvement. We further discuss additional policy implications of teleworking and its beneficial effects for the society

Einzelmolekül-Fluoreszenzmikroskopie in lebenden TrypanosomaTrypanosoma bruceibrucei und Modellmembranen

Der eukaryotische Parasit Trypanosoma brucei hat komplexe Strategien entwickelt um der Immunantwort eines Wirtes zu entkommen und eine persistente Infektion innerhalb dessen aufrechtzuerhalten. Ein zentrales Element seiner Verteidigungsstrategie stützt sich auf die Schutzfunktion seines Proteinmantels auf der Zelloberfläche. Dieser Mantel besteht aus einer dichten Schicht aus identischen, Glykosylphosphatidylinositol (GPI)-verankerten variablen Oberflächenglykoproteinen (VSG). Der VSG Mantel verhindert die Erkennung der darunterliegenden, invarianten Epitope durch das Immunsystem. Obwohl es notwendig ist die Funktionsweise des VSG Mantels zu verstehen, vor allem um ihn als mögliches Angriffsziel gegen den Parasiten zu verwenden, sind seine biophysikalischen Eigenschaften bisher nur unzureichend verstanden. Dies ist vor allem der Tatsache geschuldet, dass die hohe Motilität der Parasiten mikroskopische Studien in lebenden Zellen bisher weitestgehend verhinderten. In der vorliegenden Arbeit wird nun hochmoderne Einzelmolekül-Fluoreszenzmikroskopie (EMFM) als Möglichkeit für mikroskopische Untersuchungen im Forschungsbereich der Trypanosomen vorgestellt. Die Arbeit umfasst Untersuchungen der VSG Dynamik unter definierten Bedingungen künstlicher Membransysteme. Es wurde zuerst der Einfluss der lateralen Proteindichte auf die VSG Diffusion untersucht. Experimente mittels Fluoreszenz- Wiederkehr nach irreversiblem Photobleichen und komplementäre Einzelmolekül- Verfolgungs Experimente offenbarten, dass ein molekularer Diffusionsschwellenwert existiert. Über diesem Schwellenwert wurde eine dichteabhänige Reduzierung des Diffusionskoeffizienten gemessen. Eine relative Quantifizierung der rekonstituierten VSGs verdeutlichte, dass der Oberflächenmantel der Trypanosomen sehr nahe an diesem Schwellenwert agiert. Der VSG Mantel ist optimiert um eine hohe Proteindichte bei gleichzeitiger hoher Mobilität der VSGs zu gewährleisten. Des Weiteren wurde der Einfluss der VSG N-Glykosylierung auf die Diffusion des Proteins quantitativ untersucht. Die Messungen ergaben, dass die N-Glykosylierung dazu beiträgt eine hohe Mobilität bei hohen Proteindichten aufrechtzuerhalten. Eine detaillierte Analyse von VSG Trajektorien offenbarte, dass zwei unterschiedliche Populationen frei diffundierender VSGs in der künstlichen Membran vorlagen. Kürzlich wurde entdeckt, dass VSGs zwei strukturell unterschiedliche Konformationen annehmen können. Die Messungen in der Arbeit stimmen mit diesen Beschreibungen überein. Die Ergebnisse der EMFM in künstlichen Membranen wurden durch VSG Einzelmolekül- Verfolgungs Experimente auf lebenden Zellen ergänzt. Es wurde eine hohe Mobilität und Dynamik einzelner VSGs gemessen, was die allgemein dynamische Natur des VSG Mantels verdeutlicht. Dies führte zu der Schlussfolgerung, dass der VSG Mantel auf lebenden Trypanosomen ein dichter und dennoch dynamischer Schutzmantel ist. Die Fähigkeit der VSGs ihre Konformation flexibel anzupassen, unterstützt das Erhalten der Fluidität bei variablen Dichten. Diese Eigenschaften des VSG Mantels sind elementar für die Aufrechterhaltung einer presistenden Infektion eines Wirtes. In dieser Arbeit werden des Weiteren verschiedene, auf Hydrogel basierende Einbettungsmethoden vorgestellt. Diese ermöglichten die Zellimmobilisierung und erlaubten EMFM in lebenden Trypanosomen. Die Hydrogele wiesen eine hohe Zytokompatibilität auf. Die Zellen überlebten in den Gelen für eine Stunde nach Beginn der Immobilisierung. Die Hydrogele erfüllten die Anforderungen der Superresolution Mikroskopie (SRM) da sie eine geringe Autofluoreszenz im Spektralbereich der verwendeten Fluorophore besaßen. Mittels SRM konnte nachgewiesen werden, dass die Hydrogele die Zellen effizient immobilisierten. Als erstes Anwendungsbeispiel der Methode wurde die Organisation der Plasmamembran in lebenden Trypanosomen untersucht. Die Untersuchung eines fluoreszenten Tracers in der inneren Membranschicht ergab, dass dessen Verteilung nicht homogen war. Es wurden spezifische Membrandomänen gefunden, in denen das Molekül entweder vermehrt oder vermindert auftrat. Dies führte zu der Schlussfolgerung, dass diese Verteilung durch eine Interaktion des Tracers mit Proteinen des zellulären Zytoskeletts zustande kam. Die in dieser Arbeit präsentierten Ergebnisse zeigen, dass EMFM erfolgreich für verschiedene biologische Untersuchungen im Forschungsfeld der Trypanosomen angewendet werden kann. Dies gilt zum Beispiel für die Untersuchung von der VSG Dynamik in künstlichen Membransystemen, aber auch für Studien in lebenden Zellen unter Verwendung der auf Hydrogelen basierenden Zelleinbettung.The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to escape the host immune response and maintain a persistent infection inside a host. One central feature of the parasite’s defense mechanism relies on the shielding function of their surface protein coat. This coat is composed of a dense arrangement of one type of glycosylphosphatidylinositol (GPI)-anchored variant surface glycoproteins (VSGs) which impair the identification of epitopes of invariant surface proteins by the immune system. In addition to the importance of understanding the function of the VSG coat and use it as a potential target to efficiently fight the parasite, it is also crucial to study its biophysical properties as it is not yet understood sufficiently. This is due to the fact that microscopic investigations on living trypanosomes are limited to a great extent by the intrinsic motility of the parasite. In the present study, state-of-the-art single-molecule fluorescence microscopy (SMFM) is introduced as a tool for biophysical investigations in the field of trypanosome research. The work encompasses studies of VSG dynamics under the defined conditions of an artificial supported lipid bilayer (SLB). First, the impact of the lateral protein density on VSG diffusion was systematically studied in SLBs. Ensemble fluorescence after photobleaching (FRAP) and complementary single-particle tracking experiments revealed that a molecular crowding threshold (MCT) exists, above which a density dependent decrease of the diffusion coefficient is measured. A relative quantification of reconstituted VSGs illustrated that the VSG coat of living trypanosomes operates very close to its MCT and is optimized for high density while maintaining fluidity. Second, the impact of VSG N-glycosylation on VSG diffusion was quantitatively investigated. N-glycosylation was shown to contribute to preserving protein mobility at high protein concentrations. Third, a detailed analysis of VSG trajectories revealed that two distinct populations of freely diffusing VSGs were present in a SLB, which is in agreement with the recent finding, that VSGs are able to adopt two main structurally distinct conformations. The results from SLBs were further complemented by single-particle tracking experiments of surface VSGs on living trypanosomes. A high mobility and free diffusion were measured on the cell surface, illustrating the overall dynamic nature of the VSG coat. It was concluded that the VSG coat on living trypanosomes is a protective structure that combines density and mobility, which is supported by the conformational flexibility of VSGs. These features are elementary for the persistence of a stable infection in the host. Different hydrogel embedding methods are presented, that facilitated SMFM in immobilized, living trypanosomes. The hydrogels were found to be highly cytocompatible for one hour after cross-linking. They exhibited low autofluorescence properties in the spectral range of the investigations, making them suitable for super-resolution microscopy (SRM). Exemplary SRM on living trypanosomes illustrated that the hydrogels efficiently immobilized the cells on the nanometer lever. Furthermore, the plasma membrane organization was studied in living trypanosomes. A statistical analysis of a tracer molecule inside the inner leaflet of the plasma membrane revealed that specific membrane domains exist, in which the tracer appeared accumulated or diluted. It was suggested that this distribution was caused by the interaction with proteins of the underlying cytoskeleton. In conclusion, SMFM has been successfully introduced as a tool in the field of trypanosome research. Measurements in model membranes facilitated systematic studies of VSG dynamics on the single-molecule level. The implementation of hydrogel immobilization allowed for the study of static structures and dynamic processes with high spatial and temporal resolution in living, embedded trypanosomes for the first time

Thymic changes after chorioamnionitis in fetal sheep

Regulatorische T-Lymphozyten differenzieren sich im fetalen Thymus unter dem Einfluss des Transkriptionsfaktors FoxP3. Sie sind für die Aufrechterhaltung des Gleichgewichts des Immunsystems wichtig. Es wurde untersucht ob eine Chorioamnionitis, induziert durch intraamniotische Endotoxingabe, die fetale Thymusentwicklung beeinflusst. Den Mutterschafen wurde fünf Tage, zwei Tage, einen Tag oder fünf Stunden vor der Sectio cesarea 10mg Endotoxin intraamniotisch verabreicht. Die Sectio cesarea wurde bei einem Gestationsalter von 123 Tagen durchgeführt. Der entnommene Thymus wurde gewogen, Nabelschnurblutlymphozyten und Plamakortisolwerte wurden bestimmt. Glukokortikoidrezeptoren, aktivierte Caspase-3-, Ki67-, PCNA-, NFkB- und FoxP3-positive Zellen wurden immunohistochemisch nachgewiesen. Das Thymusgewicht war im Verhältnis zum Körpergewicht der Lämmer nach intraamniotischer Endotoxingabe zu allen gemessenen Zeitpunkten verringert. Die zirkulierenden Lymphozyten im Nabelschnurblut nahmen einen Tag nach Endotoxingabe um 40% ab. Die Endotoxingabe führte zu einem vorübergehenden Anstieg der Plasmakortisolwerte, zu einer Verdoppelung NFkB positiver Zellen und zu einer Abnahme Foxp3 positiver Zellen in der Thymusrinde einen Tag nach Endotoxingabe. Die intraamniotische Verabreichung eines Endotoxins führte im Schafmodell zu Veränderungen im fetalen Thymus.Regulatory T-lymphocytes differentiate in the fetal thymus under the control of the transcription factor FoxP3. T-lymphocytes mediate homeostasis of the immune system. The objective was whether chorioamnionitis, caused by endotoxin,would modulate fetal thymus development. An intaamniotic injection of 10mg endotoxin was given to the sheep five days, two days, one day or five hours before delivery at 123 gestation days. Thymus weight, cord blood lymphocytes and plasma cortisol were measured. Glucocorticoid receptor-, activated caspase-3-, Ki67-, proliferating cell nuclear antigen-, nuclear factor kB-, and FoxP3-positive cells were immunohistochemically evaluated. Thymus-to-body weight ratios were reduced in all endotoxin groups. There was a decrease of circulation lymphoctes after intraamniotic endotoxin exposure by 40% after one day. Plasma cortisol concentration increased transiently, nuclear factor kB positive cells in thymic cortex doubled and FoxP3 positive cells were reduced one day after endotoxin exposure. Intraamniotic exposure to endotoxin induced thymic changes in fetal sheep