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. When compared to HRM-sequencing, NGS-sequencing, RNA-sequencing, or simplex digital PCR assays used as "gold standard" methods, these multiplexed dPCR assays displayed 100% specificity and sensitivity for the simultaneous detection of: 1/BRAF V600E mutation and KIAA1549:BRAF fusion; 2/FGFR1 N546K and K656E mutations and FGFR1 duplication; 3/H3F3A K27M and G34R/V mutations; 4/IDH1 R132X and IDH2 R172X mutations; and 5/TERT promoter mutations C228T and C250T. In light of the increased integration of molecular alteration, we believe that such strategies might help laboratories to optimize their screening strategies for routine diagnosis of pediatric and adult CNS tumors.

[Development of molecular analysis by digital PCR for clinical practice: positioning, current applications and perspectives]. / Développement des analyses moléculaires par PCR digitale pour la pratique clinique : positionnement, applications actuelles et 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. In a second part, the main current applications of the dPCR will be addressed including the oncology of solid tumors and liquid biopsies, oncohematology and the follow-up of hemopathies treatments by hematopoietic stem cell transplantation. We will also detail non-invasive prenatal diagnosis and diagnosis of mosaic genetic disease, using the example of McCune-Albright syndrome. Several French specialists in the field who have implemented these techniques in their laboratory have written these different examples of applications jointly. In summary, this manuscript offers an up-to-date view of the positioning of dPCR in relation to other existing technologies in order to best meet the expectations of precision medicine.

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. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ∼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.

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. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimer's disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains. SIGNIFICANCE STATEMENT: This study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.

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. HIV-1 Tat protein is efficiently secreted by infected cells. Secreted Tat can enter uninfected cells and reach their cytosol. Here we found that extracellular Tat, at the subnanomolar concentration present in the sera of HIV-1-infected patients, inhibits the phagocytosis of Mycobacterium avium or opsonized Toxoplasma gondii by human primary macrophages. This inhibition results from a defect in mannose- and Fcγ-receptor-mediated phagocytosis, respectively. Inhibition relies on the interaction of Tat with phosphatidylinositol (4,5)bisphosphate that interferes with the recruitment of Cdc42 to the phagocytic cup, thereby preventing Cdc42 activation and pseudopod elongation. Tat also inhibits FcγR-mediated phagocytosis in neutrophils and monocytes. This study provides a molecular basis for the phagocytic defects observed in uninfected phagocytes following HIV-1 infection.

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. Here, we characterized the mechanisms of CAR endocytosis in neurons and neuronal cells. We found that CAR internalization was lipid microdomain-, actin-, and dynamin-dependent, and subsequently followed by CAR degradation in lysosomes. Moreover, ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalization cascade involving sequences in its intracellular tail.

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. As anticipated, in vitro cytoplasmic capsids did not harbor UL34, UL31, or viral glycoproteins but contained US3. In agreement with previous findings, both nuclear and in vitro capsids were positive for ICP0 and ICP4. Unexpectedly, nuclear C capsids and cytoplasmic capsids produced in vitro without any cytosolic viral proteins also scored positive for UL36 and UL37. Immunoelectron microscopy confirmed that these tegument proteins were closely associated with nuclear capsids. When cytosolic viral proteins were present in the in vitro assay, no additional tegument proteins were detected on the capsids. As previously reported, the tegument was sensitive to high-salt extraction but, surprisingly, was stabilized by exogenous proteins. Finally, some tegument proteins seemed partially lost during egress, while others possibly were added at multiple steps or modified along the way. Overall, an emerging picture hints at the early coating of capsids with up to 5 tegument proteins at the nuclear stage, the shedding of some viral proteins during nuclear egress, and the acquisition of others tegument proteins during reenvelopment.

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. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception) indicating that the modulation was specific for HSV-1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions.

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. However, they seemingly differ in terms of acquisition of their mature envelope. Herpes simplex virus is often believed to bud into an existing intracellular compartment, while the related cytomegalovirus may acquire its final envelope from a novel virus-induced assembly compartment. This review focuses on recent advances in the characterization of cellular compartment(s) potentially contributing to herpes virion final envelopment. It also examines the common points between seemingly distinct envelopment pathways and highlights the dynamic nature of intracellular compartments in the context of herpesvirus infections.

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. Using a combination of biochemical and cell biology approaches, we characterized the axonal trafficking pathway used by the canine adenovirus serotype 2 (CAV-2) to reach the neuronal soma. We showed that CAV-2 and CAR (coxsackievirus and adenovirus receptor) are entering a vesicular pathway coupled to the axonal transport machinery. The lumen of the multivalent Rab7 (+) vesicles that transport CAV-2 and CAR is, surprisingly, pH neutral. Among other issues, our study opens numerous questions concerning the neuronal function of CAR.

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. We found that a PPxY motif within protein VI recruits Nedd4 E3 ubiquitin ligases to bind and ubiquitylate protein VI. We further show that this PPxY motif is involved in rapid, microtubule-dependent intracellular movement of protein VI. Ads with a mutated PPxY motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. Likewise, depletion of Nedd4 ligases attenuates nuclear accumulation of incoming Ad particles and infection. Our data provide the first evidence that virus-encoded PPxY motifs are required during virus entry, which may be of significance for several other pathogens.

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. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

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. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad-erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.

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. RESULTS: Using fluorescence microscopy, we found that the HBZ-SP1 protein localizes to intense dots corresponding to HBZ-NBs and to nucleoli. We analyzed the relative mobility of the EGFP-HBZ-SP1 fusion protein using fluorescence recovery after photobleaching (FRAP) analysis and found that the deletion of the ZIP domain perturbs the association of the HBZ-SP1 protein to the HBZ-NBs. These data suggested that HBZ needs cellular partners, including bZIP factors, to form HBZ-NBs. Indeed, by cotransfection experiments in COS cells, we have found that the bZIP factor JunB is able to target delocalized form of HBZ (deleted in its nuclear localization subdomains) into the HBZ-NBs. We also show that the viral protein is able to entail a redistribution of JunB into the HBZ-NBs. Moreover, by transfecting HeLa cells (known to express high level of JunB) with a vector expressing HBZ-SP1, the sequestration of JunB to the HBZ-NBs inhibited its transcriptional activity. Lastly, we analyzed the nuclear distribution of HBZ-SP1 in the presence of JunD, a Jun family member known to be activated by HBZ. In this case, no NBs were detected and the HBZ-SP1 protein was diffusely distributed throughout the nucleoplasm. CONCLUSION: Our results suggest that HBZ-mediated sequestration of JunB to the HBZ-NBs may be causing the repression of JunB activity in vivo.