Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity

Cyber Insurance

This volume covers a wide spectrum of issues relating to economic and political development enabled by information and communication technology (ICT)

Mechanical Properties of Polymer Cement-Fiber-Reinforced Concrete (PC-FRC): Comparison Based on Experimental Studies

peer reviewedThe paper presents results of the comparative tests on OC and PCC modified with a constant volume of steel, macro-, and micro-polypropylene fibers. The influence of these fibers on FRC and PC-FRC mechanical properties was evaluated by flexural and compressive strength tests, as well as bending strength and breaking load of a concrete pavement slab in accordance with EN 1339. Significant differences in results were observed between FRC and PC-FRC. Increase of flexural strength in PC-FRC beams made with steel and macro-polypropylene fibers in comparison with reference PCC samples has been shown. Breaking load of PC-FRC slabs was close to reference PCC ones. The enhancement of compressive strength on cubic samples of PC-FRC in relation to PCC was also observed. Further research with different polymer additives and fibers with other volume contents is considered

Vor-Ort-Nachweis bioterroristisch relevanter Agenzien

In Europa besteht eine abstrakte Gefährdungslage nicht nur für konventionell durchgeführte Anschläge mit Waffen oder Sprengstoffen, sondern auch für Anschläge, bei denen biologische Agenzien eingesetzt werden. Zur Gefahrenabwehr werden daher kontinuierlich schnelle und zuverlässige Nachweisverfahren entwickelt und erprobt. Für die Anwendung im stationären Labor wurde für bioterroristisch relevante Agenzien bereits ein umfassendes Spektrum an Nachweismethoden etabliert. Für eine Vor-Ort-Detektion aus Umweltproben werden darüber hinaus von den Einsatzkräften zunehmend vergleichbar verlässliche mobile Nachweissysteme zur ersten Lagebewertung gewünscht. Basierend auf den Funktionsprinzipien können generische, immunologische und nukleinsäurebasierte Vor-Ort-Detektionsverfahren unterschieden werden. Diese sollten einfach durchzuführen, schnell, sensitiv und spezifisch sein. Kommerziell erhältliche Vor-Ort-Detektionssysteme haben systembedingt häufig eine eingeschränkte Sensitivität und sind zumeist nicht von unabhängiger Seite validiert. Darüber hinaus stellt die Vielfalt an potenziell nachzuweisenden Agenzien in komplexen Umweltproben eine besondere Herausforderung dar. Daher ist detailliertes Wissen über Einsatzbereiche und Limitation der verwendeten Testsysteme zwingend erforderlich, um erhaltene Ergebnisse zielführend bewerten und Handlungsempfehlungen ableiten zu können. Ziel dieses Artikels ist es, einen Überblick über die Messprinzipien von Vor-Ort-Detektionssystemen für bioterroristisch relevante Viren, Bakterien und Toxine sowie Vor- und Nachteile der Testsysteme zu geben. Trotz vielversprechender Entwicklungen sind derzeit erhältliche Testsysteme zur Vor-Ort-Detektion noch beschränkt aussagekräftig. Deshalb sind Expertenlabore zur gesicherten Befundung von Umweltproben weiterhin einzubeziehen