mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints.

Model-Based Classification for Digital PCR: Your Umbrella for Rain.

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples.

Flexible analysis of digital PCR experiments using generalized linear mixed models.

The use of digital PCR for quantification of nucleic acids is rapidly growing. A major drawback remains the lack of flexible data analysis tools. Published analysis approaches are either tailored to specific problem settings or fail to take into account sources of variability.

A new survivin tracer tracks, delocalizes and captures endogenous survivin at different subcellular locations and in distinct organelles.

Survivin, the smallest member of the inhibitor of apoptosis protein family, plays a central role during mitosis and exerts a cytoprotective function. Survivin is highly expressed in most cancer types and contributes to multiple facets of carcinogenesis. The molecular mechanisms underlying its highly diverse functions need to be extensively explored, which is crucial for rational design of future personalized therapeutics.

An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model.

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade.

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization.

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion.

A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction.

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration.

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain.

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive.

Down-regulation of myopodin expression reduces invasion and motility of PC-3 prostate cancer cells.

Enhanced motility of cancer cells by remodelling of the actin cytoskeleton is crucial in the process of cancer cell invasion and metastasis. Although several studies propose a tumor suppressor role for the actin bundling protein myopodin, it was also shown previously that overexpression of mouse myopodin promotes invasion in vitro. In the present study, the role of myopodin in human cancer cell motility and invasion was explored using RNA interference with siRNA duplexes designed to down-regulate all human myopodin isoforms currently identified.

The tandem PDZ protein Syntenin interacts with the aminoacyl tRNA synthetase complex in a lysyl-tRNA synthetase-dependent manner.

Syntenin-1 is a tandem PDZ protein that binds a diverse array of signaling molecules that are often associated with cell adhesion and intracellular trafficking. With the use of a MS-based functional proteomics approach, we identified several members of the aminoacyl-tRNA synthetase macromolecular (ARS) complex in a syntenin-1 pull down assay. Interaction of these proteins with syntenin-1 was confirmed by co-immunoprecipitation from cultured cells.

Multiple isoforms of the tumor suppressor myopodin are simultaneously transcribed in cancer cells.

Expression of myopodin, an actin associated protein, is frequently lost in invasive prostate cancers due to partial or complete deletion of the gene. Screening of public databases reveals that two human myopodin isoforms have been proposed. Remarkably both isoforms deviate profoundly from the human or mouse isoforms examined to date.

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin.

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus.