Toxoplasma gondii Syntaxin 6 is required for vesicular transport between endosomal-like compartments and the Golgi Complex

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears thatToxoplasma has retained a plant-like secretory pathway

Multipath-assisted maximum-likelihood indoor positioning using UWB signals

Multipath-assisted indoor positioning (using ultrawideband signals) exploits the geometric information contained in deterministic multipath components. With the help of a-priori available floorplan information, robust localization can be achieved, even in absence of a line-of-sight connection between anchor and agent. In a recent work, the Cramér-Rao lower bound has been derived for the position estimation variance using a channel model which explicitly takes into account diffuse multipath as a stochastic noise process in addition to the deterministic multipath components. In this paper, we adapt this model for position estimation via a measurement likelihood function and evaluate the performance for real channel measurements. Performance results confirm the applicability of this approach. A position accuracy better than 2.5 cm has been obtained in 90% of the estimates using only one active anchor at a bandwidth of 2GHz and robustness against non-line-of-sight situations has been demonstrated

Host cell invasion by apicomplexan parasites: the junction conundrum

No abstract available

Two essential light chains regulate the MyoA lever arm to promote Toxoplasma gliding motility

Key to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca2+ in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca2+ or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites

Schaffung einer Datenbasis zur Erfassung der Mengen von in Deutschland wiederverwendeten Produkten : Zwischenbericht

Im Rahmen des Projektes werden mögliche Bewertungsmaßstäbe für die Erfolgsmessung von Abfallvermeidungsmaßnahmen vertiefend analysiert und bewertet sowie ein passendes Set an Indikatoren erarbeitet und ein geeignetes Daten-Erfassungskonzeptes entwickelt. Bezogen auf das Datenerfassungskonzept gibt es im Bereich Wiederverwendung (WV) erhebliche Datenlücken zu den tatsächlich wiederverwendeten Mengen gebrauchter Produkte. Der vorliegende Zwischenbericht enthält die Ergebnisse einer ersten Datenerhebung auf Basis einer Befragung der 400 Wiederverwendungseinrichtungen in Deutschland. Ergänzend wurde das Potential der aus Online-Angeboten gewinnbaren Gebrauchtprodukte zur WV recherchiert

Conditional U1 gene silencing in Toxoplasma gondii

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3’-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3’-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail

VCSEL-Based Optical Frequency Combs: Toward Efficient Single-Device Comb Generation

Optical frequency combs generators (OFCGs) have demonstrated to be extremely useful tools in a wide variety of applications. The current research trends look toward compact devices that are able to offer high phase correlation between optical lines, and in this sense, mode-locked laser diodes (MLLDs), with repetition frequencies in the few gigahertz (GHz) range, and especially microresonators, with repetition frequencies of hundreds of GHz, are the most promising devices fulfilling these requirements. Nevertheless, focusing in the few GHz frequency rate, MLLDs cannot provide continuous tunability and require special devices that are still far from offering reliability and repeatability for commercial use. In this letter, we demonstrate for the first time the generation of a flat OFCG based on a single commercial vertical cavity surface emitting laser under gain-switching regime with 20 optical lines (spaced by 4.2 GHz) in a 3-dB bandwidth, offering wide tunability range and very high phase correlation between optical modes. This OFCG does not need any external modulator and it is the most energy-efficient OFCG reported to date.Publicad

Liquid cooled micro-scale gradient system for magnetic resonance

Schaltbare magnetische Feldgradientspulen sind ein geeignetes Werkzeug für die Modulation der Kernspinpräzession in der gepulsten Kernspinresonanzspektroskopie und Bildgebung. Die Magnetresonanztomographie von mikroskopischen Proben benötigt starke, schnell schaltbare Magnetfeldgradienten, um diffusionsbedingte Artefakte zu unterdrücken, Suszeptibilitätseffekte abzuschwächen und um die Messzeit zu verkürzen. Verschiedene Techniken können eingesetzt werden, um eine hohe Gradientenintensität zu erreichen, wie zum Beispiel die Erhöhung der Stromstärke oder die Steigerung der Windungsdichte der Feldspule. Ein weiterer, geeigneter technischer Ansatz besteht darin, die Gradientenspulen näher an der Probe zu platzieren. Als Konsequenz wird aber die durch die Joule-Erwärmung verursachte Wärmeentwicklung zu einem zentralen Problem. In dieser Arbeit wird ein neuartiges Design, ein Mikroherstellungsprozess und eine Kernspin-Evaluierung eines Feldgradientenchips präsentiert. Die Gradientenspulen wurden besonders hoch miniaturisiert und durch den Einsatz von verbesserten und neuartigen Strukturierungsverfahren entwickelt. Zuerst wird ein Fertigungsverfahren zur Herstellung einer kompakten Hochfrequenzspule vorgestellt. Durch den Einsatz einer maskenlosen Rückseitenlithographie konnte die Prozesskomplexität reduziert werden. Dieses Verfahren wurde durch Tintenstrahldruck mit Nanopartikeln realisiert, wobei die gedruckten Strukturen selbst als lithographische Maske für die Herstellung einer galvanischen Form dienen. Somit werden die Seitenwände der galvanischen Form durch die gedruckte Seed-Schicht optimal selbst ausgerichtet. Dies ermöglichte eine anisotrope Galvanisierung, um eine höhere elektrische Leitfähigkeit der gedruckten Leiterbahnen zu erzielen. Aus den Erkenntnissen der ausgearbeiteten Herstellungsprozesse wurde ein optimiertes Spulendesign für ein-axiale sowie drei-axiale linearen Gradientenchips entwickelt. Die einachsige lineare $z$-Gradientenspule wurde mit der Stream-Function-Methode berechnet, wobei die Optimierung darauf abgestimmt wurde, eine minimale Verlustleistung zu erzielen. Die Gradientenspulen wurden auf zwei Doppellagen implementiert, die mittels Cu-Galvanik in Kombination mit fotodefinierbaren Trockenfilm-Laminaten aufgebracht wurden. Bei dem hier vorgestellten Herstellungsverfahren diente die erste Metallisierungschicht gleichzeitig dazu, Widerstands-Temperaturdetektoren zu integrieren. Um niederohmige Spulen zu realisieren wurde der Galvanisierungsprozess soweit angepasst, um eine hohe Schichtdicke zu erzielen. Die Chipstruktur beinhaltet ein aktives Kühlsystem, um dem Aufheizen der Spulen entgegenzuwirken. Thermographische Aufnahmen in Kombination mit den eingebetteten Temperatursensoren ermöglichen es, die Erhitzung der Spule zu analysieren, um die Strombelastbarkeit zu ermitteln. Die Gradientenspule wurde mit einer Hochfrequenz-Mikrospule in einer Flip-Chip-Konfiguration zusammengebaut, und mit diesem Aufbau wurde ein eindimensionales Kernspinexperiment durchgeführt. Es wurde eine Gradienteneffizienz von 3.15 $T\,m^{−1}\,A^{−1}$ bei einer Profillänge von 1.2 $mm$ erreicht

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii. Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite

Continuous wave sub-THz photonic generation with VCSEL-based optical frequency comb

A simple and energy-efficient photonic system to generate continuously tunable, low phase noise, sub-THz waves based on COTS components is presented. The optical scheme is based on the use of a commercial vertical cavity surface emitting laser under gain switching modulation that provides a very flat optical frequency comb generator (OFCG) with 23 modes in a 20 dB bandwidth. The laser only needs 15 dBm continuous wave radiofrequency input power and 9 mA of bias current to provide this OFCG. Two optical injection locking stages filter and amplify the two desired modes that are detected in a photodiode to produce the desired sub-THz signal at the frequency difference of these two selected modes. As an example, demonstrated is the generation of a very stable 88.2 GHz tone with lower linewidth than 10 Hz using a reference of 4.2 GHz to generate the OFCG.The work by Á.R. Criado has been supported by the Spanish Ministry of Science and Technology under the FPI Program, grant no. BES2010-030290Publicad