Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways.

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery

Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery

Spectral line shape of resonant four-wave mixing induced by broad-bandwidth lasers

We present a theoretical and experimental study of the line shape of resonant four-wave mixing induced by broad-bandwidth laser radiation that revises the theory of Meacher, Smith, Ewart, and Cooper (MSEC) [Phys. Rev. A 46, 2718 (1992)]. We adopt the same method as MSEC but correct for an invalid integral used to average over the distribution of atomic velocities. The revised theory predicts a Voigt line shape composed of a homogeneous, Lorentzian component, defined by the collisional rate Γ, and an inhomogeneous, Doppler component, which is a squared Gaussian. The width of the inhomogeneous component is reduced by a factor of √2 compared to the simple Doppler width predicted by MSEC. In the limit of dominant Doppler broadening, the width of the homogeneous component is predicted to be 4Γ, whereas in the limit of dominant homogeneous broadening, the predicted width is 2Γ. An experimental measurement is reported of the line shape of the four-wave-mixing signal using a broad-bandwidth, "modeless", laser resonant with the Q1 (6) line of the A2 Σ - X2 Π(0,0) system of the hydroxyl radical. The measured widths of the Voigt components were found to be consistent with the predictions of the revised theory

RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests

Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids

A mathematical model of the metabolic and perfusion effects on cortical spreading depression

Cortical spreading depression (CSD) is a slow-moving ionic and metabolic disturbance that propagates in cortical brain tissue. In addition to massive cellular depolarization, CSD also involves significant changes in perfusion and metabolism -- aspects of CSD that had not been modeled and are important to traumatic brain injury, subarachnoid hemorrhage, stroke, and migraine. In this study, we develop a mathematical model for CSD where we focus on modeling the features essential to understanding the implications of neurovascular coupling during CSD. In our model, the sodium-potassium--ATPase, mainly responsible for ionic homeostasis and active during CSD, operates at a rate that is dependent on the supply of oxygen. The supply of oxygen is determined by modeling blood flow through a lumped vascular tree with an effective local vessel radius that is controlled by the extracellular potassium concentration. We show that during CSD, the metabolic demands of the cortex exceed the physiological limits placed on oxygen delivery, regardless of vascular constriction or dilation. However, vasoconstriction and vasodilation play important roles in the propagation of CSD and its recovery. Our model replicates the qualitative and quantitative behavior of CSD -- vasoconstriction, oxygen depletion, extracellular potassium elevation, prolonged depolarization -- found in experimental studies. We predict faster, longer duration CSD in vivo than in vitro due to the contribution of the vasculature. Our results also help explain some of the variability of CSD between species and even within the same animal. These results have clinical and translational implications, as they allow for more precise in vitro, in vivo, and in silico exploration of a phenomenon broadly relevant to neurological disease.Comment: 17 pages including 9 figures, accepted by PLoS On

Potential conservation of circadian clock proteins in the phylum Nematoda as revealed by bioinformatic searches

Although several circadian rhythms have been described in C. elegans, its molecular clock remains elusive. In this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (Mus musculus, Drosophila melanogaster, Neurospora crassa, Arabidopsis thaliana and Synechoccocus elongatus) in the proteomes of C. elegans and other members of the phylum Nematoda. With this approach we found that the Nematoda contain proteins most related to the core and accessory proteins of the insect and mammalian clocks, which provide new insights into the nematode clock and the evolution of the circadian system.Fil: Romanowski, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; ArgentinaFil: Garavaglia, Matías Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Goya, María Eugenia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Golombek, Diego Andres. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Localisation of Human Papillomavirus 16 E7 Oncoprotein Changes with Cell Confluence

E7 is one of the best studied proteins of human papillomavirus type 16, largely because of its oncogenic potential linked to cervical cancer. Yet the sub-cellular location of E7 remains confounding, even though it has been shown to be able to shuttle between the nucleus and the cytoplasm. Here we show with immunocytochemistry that E7 proteins are located in the nucleus and cytoplasm in sub-confluent cells, but becomes cytoplasmic in confluent cells. The change in E7's location is independent of time in culture, cell division, cell cycle phase or cellular differentiation. Levels of E7 are also increased in confluent cells as determined by Western blotting. Our investigations have also uncovered how different analytical techniques influence the observation of where E7 is localised, highlighting the importance of technical choice in such analysis. Understanding the localisation of E7 will help us to better comprehend the function of E7 on its target proteins

A Pearson-Dirichlet random walk

A constrained diffusive random walk of n steps and a random flight in Rd, which can be expressed in the same terms, were investigated independently in recent papers. The n steps of the walk are identically and independently distributed random vectors of exponential length and uniform orientation. Conditioned on the sum of their lengths being equal to a given value l, closed-form expressions for the distribution of the endpoint of the walk were obtained altogether for any n for d=1, 2, 4 . Uniform distributions of the endpoint inside a ball of radius l were evidenced for a walk of three steps in 2D and of two steps in 4D. The previous walk is generalized by considering step lengths which are distributed over the unit (n-1) simplex according to a Dirichlet distribution whose parameters are all equal to q, a given positive value. The walk and the flight above correspond to q=1. For any d >= 3, there exist, for integer and half-integer values of q, two families of Pearson-Dirichlet walks which share a common property. For any n, the d components of the endpoint are jointly distributed as are the d components of a vector uniformly distributed over the surface of a hypersphere of radius l in a space Rk whose dimension k is an affine function of n for a given d. Five additional walks, with a uniform distribution of the endpoint in the inside of a ball, are found from known finite integrals of products of powers and Bessel functions of the first kind. They include four different walks in R3 and two walks in R4. Pearson-Liouville random walks, obtained by distributing the total lengths of the previous Pearson-Dirichlet walks, are finally discussed.Comment: 33 pages 1 figure, the paper includes the content of a recently submitted work together with additional results and an extended section on Pearson-Liouville random walk