No abstract

SummaryThe liver stage of the Plasmodium parasite remains one of the most promising targets for intervention against malaria as it is clinically silent, precedes the symptomatic blood stage and represents a bottleneck in the parasite life cycle. However, many aspects of the development of the parasite during this stage are far from understood. During the liver stage, the parasite undergoes extensive replication, forming tens of thousands of infectious merozoites from each invading sporozoite. This implies a very efficient and accurate process of cytokinesis and thus also of organelle development and segregation. We have generated for the first time Plasmodium berghei double-fluorescent parasite lines, allowing visualization of the apicoplast, mitochondria and nuclei in live liver stage parasites. Using these we have seen that in parallel with nuclear division, the apicoplast and mitochondrion become two extensively branched and intertwining structures. The organelles then undergo impressive morphological and positional changes prior to cell division. To form merozoites, the parasite undergoes cytokinesis and the complex process of organelle development and segregation into the forming daughter merozoites could be analysed in detail using the newly generated transgenic parasites.

We demonstrate super-resolution imaging of proteins and nucleic acids that were densely labelled with fluorophores using the concept of “click chemistry”.

Background: Novel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today. The higher spatial resolution of super-resolution microscopy techniques demands for precise correction of drift, spectral and spatial offset of images recorded at different axial planes.

DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules.

Gene InhIbItIondirectly to cRGD peptide or to internalizing RGD (iRGD), which is a variant RGD peptide that triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin. The cRGD-and iRGD-conjugated KRAS Adaptors were tested for efficacy against subcutaneous MiaPaca-2 xenografts, and tumor growth was inhibited to equal or greater extent as with the original cRGD-dendrimer system. iRGD may be of particular benefit for pancreatic adenocarcinomas, which have a densely fibrotic stroma that impedes drug delivery. Enhanced delivery of small-and large-molecule therapeutics into primary pancreatic adenocarcinomas in KPC mice has been achieved previously by conjugating or coinjecting iRGD peptide.We have shown that U1 Adaptors can successfully target human KRAS both in vitro and in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS in pancreatic cancer.