Multiplexed Droplet Digital PCR Assays for the Simultaneous Screening of Major Genetic Alterations in Tumors of the Central Nervous System.

The increased integration of molecular alterations to define tumor type or grade in central nervous system (CNS) tumor classification brings new challenges for the pathologist to make the best use of a precious limited tissue specimen for molecular studies. Within the different methods available to identify gene alterations, the droplet digital PCR (dPCR) constitutes a rapid, cost-effective, and very sensitive tool. In this study, we describe the development and validation of five multiplexed dPCR assays to detect major CNS biomarkers by using only small amounts of DNA extracted from formalin-fixed paraffin-embedded specimens.

[Development of molecular analysis by digital PCR for clinical practice: positioning, current applications and perspectives].

This review is the second part of the workshop on digital PCR (dPCR) proposed by the working group of the French society of clinical biology. The first part of the paper discusses the advantages and limitations of dPCR for the search of different molecular abnormalities such as point mutations, copy number variants, DNA methylation, RNA analysis and a more innovative application, the single-cell dPCR. This synthesis makes it possible to propose a positioning of the dPCR compared to the other available technologies in a medical laboratory.

BIABooster: Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/μL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA.

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/μL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces.

Coxsackievirus Adenovirus Receptor Loss Impairs Adult Neurogenesis, Synapse Content, and Hippocampus Plasticity.

Unlabelled: Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus.

HIV-1 Tat inhibits phagocytosis by preventing the recruitment of Cdc42 to the phagocytic cup.

Most macrophages remain uninfected in HIV-1-infected patients. Nevertheless, the phagocytic capacity of phagocytes from these patients is impaired, favouring the multiplication of opportunistic pathogens. The basis for this phagocytic defect is not known.

Disruption of the coxsackievirus and adenovirus receptor-homodimeric interaction triggers lipid microdomain- and dynamin-dependent endocytosis and lysosomal targeting.

The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalization is unclear as studies suggest that its intracellular domain is dispensable for some AdV infection. We previously showed that in motor neurons, AdV induced CAR internalization and co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries.

Analysis of the early steps of herpes simplex virus 1 capsid tegumentation.

Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an in vitro nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay.

Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a RNA interference screen.

Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle.

Herpesviruses exploit several host compartments for envelopment.

Enveloped viruses acquire their host-derived membrane at a variety of intracellular locations. Herpesviruses are complex entities that undergo several budding and fusion events during an infection. All members of this large family are believed to share a similar life cycle.

An adenovirus traffic update: from receptor engagement to the nuclear pore.

Adenoviruses have a bipolar nature: they are ubiquitous pathogens that occasionally cause life-threatening diseases or they can be engineered into powerful gene transfer vectors. The goal of this article is to summarize the most recent advances in adenovirus receptor engagement, internalization, endosomal maturation, endosomal escape and trafficking to the nuclear pore. A better understanding of this initial part of the adenovirus lifecycle may identify new mechanistic-based treatments for adenovirus-induced diseases and help in the engineering of more efficient vectors.

An endocytic CARriage tale: Adenoviruses internalization and trafficking in neurons.

In immune-competent hosts, adenoviruses (Ads) are mild pathogens that cause mainly infections of the respiratory and ocular tracks. The advent of Ad-based gene transfer vectors made the understanding of their interaction with the host cellular machinery an intensive field of research over the last decade. As studies focused primarily on epithelial-like cells, the mechanism of neuronal uptake of Ads was still missing.

A capsid-encoded PPxY-motif facilitates adenovirus entry.

Viruses use cellular machinery to enter and infect cells. In this study we address the cell entry mechanisms of nonenveloped adenoviruses (Ads). We show that protein VI, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport.

CAR-associated vesicular transport of an adenovirus in motor neuron axons.

Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons.

The cell adhesion molecule "CAR" and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution.

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species.

The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies.

Background: The human T-cell leukemia virus type I (HTLV-I) basic leucine-zipper factor (HBZ) has previously been shown to modulate transcriptional activity of Jun family members. The presence of a novel isoform of HBZ, termed HBZ-SP1, has recently been characterized in adult T-cell leukemia (ATL) cells and has been found to be associated with intense nuclear spots. In this study, we investigated the role of these nuclear bodies in the regulation of the transcriptional activity of JunB.