Dynamics of HIV-1 Assembly and Release

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics

The ScaleX campaign: scale-crossing land-surface and boundary layer processes in the TERENO-preAlpine observatory

Augmenting long-term ecosystem-atmosphere observations with multidisciplinary intensive campaigns aims at closing gaps in spatial and temporal scales of observation for energy- and biogeochemical cycling, and at stimulating collaborative research. ScaleX is a collaborative measurement campaign, co-located with a long-term environmental observatory of the German TERENO (TERrestrial ENvironmental Observatories) network in mountainous terrain of the Bavarian Prealps, Germany. The aims of both TERENO and ScaleX include the measurement and modeling of land-surface atmosphere interactions of energy, water, and greenhouse gases. ScaleX is motivated by the recognition that long-term intensive observational research over years or decades must be based on well-proven, mostly automated measurement systems, concentrated on a small number of locations

Genotyping of Circulating Free DNA Enables Monitoring of Tumor Dynamics in Synovial Sarcomas

Background: Synovial sarcoma (SS) is a malignant soft tissue tumor of mesenchymal origin that frequently occurs in young adults. Translocation of the SYT gene on chromosome 18 to the SSX genes on chromosome X leads to the formation of oncogenic fusion genes, which lead to initiation and proliferation of tumor cells. The detection and quantification of circulating tumor DNA (ctDNA) can serve as a non-invasive method for diagnostics of local or distant tumor recurrence, which could improve survival rates due to early detection. Methods: We developed a subtype-specific targeted next-generation sequencing (NGS) approach specifically targeting SS t(X;18)(p11;q11), which fuses SS18 (SYT) in chromosome 18 to SSX1 or SSX2 in chromosome x, and recurrent point mutations. In addition, patient-specific panels were designed from tumor exome sequencing. Both approaches were used to quantify ctDNA in patients’ plasma. Results: The subtype-specific assay allowed detection of somatic mutations from 25/25 tumors with a mean of 1.68 targetable mutations. The minimal limit of detection was determined at a variant allele frequency of 0.05%. Analysis of 29 plasma samples from 15 tumor patients identified breakpoint ctDNA in 6 patients (sensitivity: 40%, specificity 100%). The addition of more mutations further increased assay sensitivity. Quantification of ctDNA in plasma samples (n = 11) from one patient collected over 3 years, with a patient-specific panel based on tumor exome sequencing, correlated with the clinical course, response to treatment and tumor volume. Conclusions: Targeted NGS allows for highly sensitive tumor profiling and non-invasive detection of ctDNA in SS patients, enabling non-invasive monitoring of tumor dynamics

The challenge of lipid rafts

The Singer-Nicholson model of membranes postulated a uniform lipid bilayer randomly studded with floating proteins. However, it became clear almost immediately that membranes were not uniform and that clusters of lipids in a more ordered state existed within the generally disorder lipid milieu of the membrane. These clusters of ordered lipids are now referred to as lipid rafts. This review summarizes current thinking on the nature of lipid rafts focusing on the role of proteomics and lipidomics in understanding the structure of these domains. It also outlines the contribution of single-molecule methods in defining the forces that drive the formation and dynamics of these membrane domains