Development of a High-throughput Platform for the Determination of Antiviral Therapeutics

JC polyomavirus (JCPyV) persists in up to 90% of the global human population. In healthy individuals, the virus resides within the kidneys resulting in a low-level infection. However, in severely immunocompromised individuals, the virus can migrate to the central nervous system (CNS), causing the demyelinating disease progressive multifocal leukoencephalopathy (PML). Currently, this debilitating disease has no clinical therapeutic options and is almost universally fatal. Specifics of the JCPyV infectious cycle, as well as the limitations of traditional laboratory techniques, have previously hindered the search for antiviral agents with the potential to prevent or treat JCPyV infection. To this end, a new high-throughput, in vitro method to measure JCPyV infectivity, the In-cell Western (ICW) assay, has been adapted to allow for rapid, consistent, and impartial analysis of the antiviral properties of large libraries of drugs and other small compounds. Utilizing this ICW platform, a large-scale drug screen was performed using the National Institute for Health (NIH) Clinical Collection, a library of over 700 drugs and small compounds, to identify drugs and compounds that reduce JCPyV infectivity. Through analysis and characterization of these compounds, heretofore unknown therapeutic agents against JCPyV have been identified, including drugs that target cell surface receptors and biochemical pathways involved in calcium and MAP kinase signaling. These compounds are the focus of further characterization to identify the cell-based mechanism by which they inhibit JCPyV infection. Findings from this study provide new information that significantly advances the field in the development of antiviral compounds to treat or prevent PML

The response function of a sphere in a viscoelastic two-fluid medium

In order to address basic questions of importance to microrheology, we study the dynamics of a rigid sphere embedded in a model viscoelastic medium consisting of an elastic network permeated by a viscous fluid. We calculate the complete response of a single bead in this medium to an external force and compare the result to the commonly-accepted, generalized Stokes-Einstein relation (GSER). We find that our response function is well approximated by the GSER only within a particular frequency range determined by the material parameters of both the bead and the network. We then discuss the relevance of this result to recent experiments. Finally we discuss the approximations made in our solution of the response function by comparing our results to the exact solution for the response function of a bead in a viscous (Newtonian) fluid.Comment: 12 pages, 2 figure

High-Throughput Characterization of Viral and Cellular Protein Expression Patterns During JC Polyomavirus Infection

JC polyomavirus (JCPyV) is a ubiquitous human pathogen and the causative agent of a fatal demyelinating disease in severely immunocompromised individuals. Due to the lack of successful pharmacological interventions, the study of JCPyV infection strategies in a rapid and highly sensitive manner is critical for the characterization of potential antiviral therapeutics. Conventional methodologies for studying viral infectivity often utilize the detection of viral proteins through immunofluorescence microscopy-based techniques. While these methodologies are well established in the field, they require significant time investments and lack a high-throughput modality. Scanning imager-based detection methods like the In-cell Western (ICW)TM have been previously utilized to overcome these challenges incurred by traditional microscopy-based infectivity assays. This automated technique provides not only rapid detection of viral infection status, but can also be optimized to detect changes in host-cell protein expression during JCPyV challenge. Compared to traditional manual determinations of infectivity through microscopy-based techniques, the ICW provides an expeditious and robust determination of JCPyV infection. The optimization of the ICW for the detection of viral and cellular proteins during JCPyV infection provides significant time and cost savings by diminishing sample preparation time and increasing resource utilization. While the ICW cannot provide single-cell analysis information and is limited in the detection of quantitation of low-expressing proteins, this assay provides a high-throughput system to study JCPyV, previously unavailable to the field. Thus, the high-throughput nature and dynamic experimental range of the ICW can be applied to the study of JCPyV infection

Probing structural relaxation in complex fluids by critical fluctuations

Complex fluids, such as polymer solutions and blends, colloids and gels, are of growing interest in fundamental and applied soft-condensed-matter science. A common feature of all such systems is the presence of a mesoscopic structural length scale intermediate between atomic and macroscopic scales. This mesoscopic structure of complex fluids is often fragile and sensitive to external perturbations. Complex fluids are frequently viscoelastic (showing a combination of viscous and elastic behaviour) with their dynamic response depending on the time and length scales. Recently, non-invasive methods to infer the rheological response of complex fluids have gained popularity through the technique of microrheology, where the diffusion of probe spheres in a viscoelastic fluid is monitored with the aid of light scattering or microscopy. Here we propose an alternative to traditional microrheology that does not require doping of probe particles in the fluid (which can sometimes drastically alter the molecular environment). Instead, our proposed method makes use of the phenomenon of "avoided crossing" between modes associated with the structural relaxation and critical fluctuations that are spontaneously generated in the system.Comment: 4 pages, 4 figure

Double radio peak and non-thermal collimated ejecta in RS Ophiuchi following the 2006 outburst

We report Multi-Element Radio-Linked Interferometer Network, Very Large Array, One-Centimetre Radio Array, Very Long Baseline Array (VLBA), Effelsberg and Giant Metrewave Radio Telescope observations beginning 4.5 days after the discovery of RS Ophiuchi undergoing its 2006 recurrent nova outburst. Observations over the first 9 weeks are included, enabling us to follow spectral development throughout the three phases of the remnant development. We see dramatic brightening on days 4 to 7 at 6 GHz and an accompanying increase in other bands, particularly 1.46 GHz, consistent with transition from the initial ‘free expansion’ phase to the adiabatic expansion phase. This is complete by day 13 when the flux density at 5 GHz is apparently declining from an unexpectedly early maximum (compared with expectations from observations of the 1985 outburst). The flux density recovered to a second peak by approximately day 40, consistent with behaviour observed in 1985. At all times the spectral index is consistent with mixed non-thermal and thermal emission. The spectral indices are consistent with a non-thermal component at lower frequencies on all dates, and the spectral index changes show that the two components are clearly variable. The estimated extent of the emission at 22 GHz on day 59 is consistent with the extended east and west features seen at 1.7 GHz with the VLBA on day 63 being entirely non-thermal. We suggest a two-component model, consisting of a decelerating shell seen in mixed thermal and non-thermal emission plus faster bipolar ejecta generating the non-thermal emission, as seen in contemporaneous VLBA observations. Our estimated ejecta mass of 4 ± 2 × 10−7 M⊙ is consistent with a white dwarf (WD) mass of 1.4 M⊙. It may be that this ejecta mass estimate is a lower limit, in which case a lower WD mass would be consistent with the data

Intracellular microrheology of motile Amoeba proteus

The motility of motile Amoeba proteus was examined using the technique of passive particle tracking microrheology, with the aid of newly-developed particle tracking software, a fast digital camera and an optical microscope. We tracked large numbers of endogeneous particles in the amoebae, which displayed subdiffusive motion at short time scales, corresponding to thermal motion in a viscoelastic medium, and superdiffusive motion at long time scales due to the convection of the cytoplasm. Subdiffusive motion was characterised by a rheological scaling exponent of 3/4 in the cortex, indicative of the semiflexible dynamics of the actin fibres. We observed shear-thinning in the flowing endoplasm, where exponents increased with increasing flow rate; i.e. the endoplasm became more fluid-like. The rheology of the cortex is found to be isotropic, reflecting an isotropic actin gel. A clear difference was seen between cortical and endoplasmic layers in terms of both viscoelasticity and flow velocity, where the profile of the latter is close to a Poiseuille flow for a Newtonian fluid

Diffusing-wave spectroscopy of nonergodic media

We introduce an elegant method which allows the application of diffusing-wave spectroscopy (DWS) to nonergodic, solid-like samples. The method is based on the idea that light transmitted through a sandwich of two turbid cells can be considered ergodic even though only the second cell is ergodic. If absorption and/or leakage of light take place at the interface between the cells, we establish a so-called "multiplication rule", which relates the intensity autocorrelation function of light transmitted through the double-cell sandwich to the autocorrelation functions of individual cells by a simple multiplication. To test the proposed method, we perform a series of DWS experiments using colloidal gels as model nonergodic media. Our experimental data are consistent with the theoretical predictions, allowing quantitative characterization of nonergodic media and demonstrating the validity of the proposed technique.Comment: RevTeX, 12 pages, 6 figures. Accepted for publication in Phys. Rev.

Mapping far-IR emission from the central kiloparsec of NGC 1097

Using photometry of NGC 1097 from the Herschel PACS (Photodetector Array Camera and Spectrometer) instrument, we study the resolved properties of thermal dust continuum emission from a circumnuclear starburst ring with a radius ~ 900 pc. These observations are the first to resolve the structure of a circumnuclear ring at wavelengths that probe the peak (i.e. lambda ~ 100 micron) of the dust spectral energy distribution. The ring dominates the far-infrared (far-IR) emission from the galaxy - the high angular resolution of PACS allows us to isolate the ring's contribution and we find it is responsible for 75, 60 and 55% of the total flux of NGC 1097 at 70, 100 and 160 micron, respectively. We compare the far-IR structure of the ring to what is seen at other wavelengths and identify a sequence of far-IR bright knots that correspond to those seen in radio and mid-IR images. The mid- and far-IR band ratios in the ring vary by less than +/- 20% azimuthally, indicating modest variation in the radiation field heating the dust on ~ 600 pc scales. We explore various explanations for the azimuthal uniformity in the far-IR colors of the ring including a lack of well-defined age gradients in the young stellar cluster population, a dominant contribution to the far-IR emission from dust heated by older (> 10 Myr) stars and/or a quick smoothing of local enhancements in dust temperature due to the short orbital period of the ring. Finally, we improve previous limits on the far-IR flux from the inner ~ 600 pc of NGC 1097 by an order of magnitude, providing a better estimate of the total bolometric emission arising from the active galactic nucleus and its associated central starburst.Comment: Accepted for publication in the A&A Herschel Special Editio

A Study of Heating and Cooling of the ISM in NGC 1097 with Herschel-PACS and Spitzer-IRS

NGC 1097 is a nearby Seyfert 1 galaxy with a bright circumnuclear starburst ring, a strong large-scale bar and an active nucleus. We present a detailed study of the spatial variation of the far infrared (FIR) [CII]158um and [OI]63um lines and mid-infrared H2 emission lines as tracers of gas cooling, and of the polycyclic aromatic hydrocarbon (PAH) bands as tracers of the photoelectric heating, using Herschel-PACS, and Spitzer-IRS infrared spectral maps. We focus on the nucleus and the ring, and two star forming regions (Enuc N and Enuc S). We estimated a photoelectric gas heating efficiency ([CII]158um+[OI]63um)/PAH in the ring about 50% lower than in Enuc N and S. The average 11.3/7.7um PAH ratio is also lower in the ring, which may suggest a larger fraction of ionized PAHs, but no clear correlation with [CII]158{\mu}m/PAH(5.5 - 14um) is found. PAHs in the ring are responsible for a factor of two more [CII]158um and [OI]63um emission per unit mass than PAHs in the Enuc S. SED modeling indicates that at most 25% of the FIR power in the ring and Enuc S can come from high intensity photodissociation regions (PDRs), in which case G0 ~ 10^2.3 and nH ~ 10^3.5 cm^-3 in the ring. For these values of G0 and nH PDR models cannot reproduce the observed H2 emission. Much of the the H2 emission in the starburst ring could come from warm regions in the diffuse ISM that are heated by turbulent dissipation or shocks.Comment: 17 pages, 14 figures, 5 tables; accepted for publication in Ap

The Physics of the Colloidal Glass Transition

As one increases the concentration of a colloidal suspension, the system exhibits a dramatic increase in viscosity. Structurally, the system resembles a liquid, yet motions within the suspension are slow enough that it can be considered essentially frozen. This kinetic arrest is the colloidal glass transition. For several decades, colloids have served as a valuable model system for understanding the glass transition in molecular systems. The spatial and temporal scales involved allow these systems to be studied by a wide variety of experimental techniques. The focus of this review is the current state of understanding of the colloidal glass transition. A brief introduction is given to important experimental techniques used to study the glass transition in colloids. We describe features of colloidal systems near and in glassy states, including tremendous increases in viscosity and relaxation times, dynamical heterogeneity, and ageing, among others. We also compare and contrast the glass transition in colloids to that in molecular liquids. Other glassy systems are briefly discussed, as well as recently developed synthesis techniques that will keep these systems rich with interesting physics for years to come.Comment: 56 pages, 18 figures, Revie