Physical characterization of icosahedral virus ultra structure, stability, and integrity using electrospray differential mobility analysis

We present a rapid and quantitative method to physically characterize the structure and stability of viruses. Electrospray differential mobility analysis (ES-DMA) is used to determine the size of capsomers (i.e., hexons) and complete capsids. We demonstrate how to convert the measured mobility size into the icosahedral dimensions of a virus, which for PR772 become 68.4 nm for vertex-to-vertex, 54.4 nm for facet-to-facet, and 58.2 nm for edge-to-edge lengths, in reasonable agreement with dimensions from transmission electron microscopy for other members of the family Tectiviridae (e.g., PRD1). These results indicate ES-DMA's mobility diameter most closely approximates the edge-to-edge length. Using PR772's edge length (36.0 nm) and the size of the major capsid hexon (≈8.4 nm) from ES-DMA with icosahedral geometry, PR772's T = 25 symmetry is confirmed and the number of proteins in the capsid shell is determined. We also demonstrate the use of ES-DMA to monitor the temporal disintegration of PR772, the thermal degradation of PP7, and the appearance of degradation products, essential to viral stability assays. These results lay groundwork essential for the use of ES-DMA for a variety of applications including monitoring of vaccine and gene therapy vector products, confirmation of viral inactivation, and theoretical studies of self-assembling macromolecular structures.     

Method of linearizing the 3d L3/L2 white line ratio as a function of magnetic moment

We have developed a parameterization method which linearizes the relationship between local magnetic moment and the 3d L3/L2 "white line" ratio as observed in electron energy loss spectroscopy or X-ray near edge absorption spectroscopy. We first establish that the parameterization linearizes an existing theoretical result for ratio versus moment. We then test our method on data sets for which a white line ratio has been previously published by other authors, who have studied a series of compounds using a consistent deconvolution procedure. Finally, we apply our linearization method to the observed ratios of a series of 3d transition metals, and to the Cr L edges for a Au(x)Cr(1 - x) alloy. In addition we obtain, for the first time, experimental results on the Au L3 and L2 edge white lines of this alloy system. These results are consistent with a model in which the large local moment in this system is not limited to Cr dopants, but extends into the gold matrix.     

The N-terminal extracellular segments of the chemokine receptors CCR1 and CCR3 are determinants for MIP-1alpha and eotaxin binding, respectively, but a second domain is essential for efficient receptor activation

CCR1 and CCR3 are seven-transmembrane domain G protein-coupled receptors specific for members of the CC chemokine subgroup of leukocyte chemoattractants. Both have been implicated in the inflammatory response, and CCR3, through its expression on eosinophils, basophils, and Th2 lymphocytes, may be especially important in allergic inflammation. CCR1 and CCR3 are 54% identical in amino acid sequence and share some ligands but not others. In particular, macrophage inflammatory protein 1alpha (MIP-1alpha) is a ligand for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1. To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1alpha and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1alpha nor eotaxin were effective agonists at either chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites determine chemokine selectivity, but others are needed for receptor activation. Agents that bind to the N-terminal segments of CCR1 and CCR3 may be useful in blocking receptor function.     

Differential generation of class I H-2D- versus H-2K-restricted cytotoxicity against a demyelinating virus following central nervous system infection

Despite the fact that both H-2K and D molecules are up-regulated in the central nervous system (CNS) following Theiler's murine encephalomyelitis virus (TMEV) infection, resistance in this virus model of multiple sclerosis maps exclusively to D. To address this paradox, we examined the ability of the K and D molecules to present viral antigens to cytotoxic T lymphocytes (CTL). Whereas no virus-specific CTL were detected in the CNS of susceptible B10.Q and B10.S mice 7 days post-infection, D-restricted CTL were identified readily in the CNS of resistant B10 animals. There was no evidence of K-restricted CTL in the CNS of B10 mice at day 7 post-infection. The presence of both K- and D-restricted virus-specific CTL in the spleen of immunized B10 mice demonstrates that the exclusive use of D molecules by CTL in the CNS of mice 7 days post-infection is not due to the inability of the K molecules to present viral peptides to lymphocytes. We conclude that the prominent role of the D locus in determining resistance or susceptibility to TMEV-induced demyelination is determined by factors governing the regulation of the immune response, and not by the presence or absence of CTL precursors capable of recognizing viral peptides presented by the K and D antigen-presenting molecules, or by differences in the ability of the K and D molecules to present viral peptides.     

Degenerate recognition of alloantigenic peptides on a positive-selecting class I molecule

The well-defined 2C T cell was used to investigate alloreactive degeneracy. A panel of class I molecules that are known ligands for the 2C TCR were sensitized with three known peptide ligands, p2Ca (LSPFPFDL), dEV-8 (EQYKFYSV), and SIYR-8 (SIYRYYGL). The peptide p2Ca was originally identified as the allopeptide seen in the Ld class I molecule by 2C T cells, 2C recognizes the dEV-8 peptide as the ligand in the Kbm3 class I molecule, and SIYR-8 was recently identified as a peptide ligand for 2C in the context of the Kb class I molecule. Strong recognition of all three Ag-presenting molecules occurred in the context of their respective allopeptides, but 2C recognized all three peptides to a measurable extent in the context of Kb. Molecular modeling of these Kb/peptide complexes revealed a high degree of similarity between dEV-8 and SIYR-8, but very little conformational similarity of either of these peptides with p2Ca. Furthermore, the structural changes in the mutant Kbm3 binding site resulted in generalized changes in the conformation of each of five bound peptides compared with those of the same peptides bound to Kb. The finding that degenerate recognition occurs on Kb, the restriction element responsible for selecting 2C T cells, suggests a unique relationship between a TCR and the Ag-presenting molecule that mediates its positive selection.     

The ARTEMIS under‐ice AUV docking system

The ARTEMIS docking system demonstrates autonomous docking capability applicable to robotic exploration of sub‐ice oceans and sub‐glacial lakes on planetary bodies, as well as here on Earth. In these applications, melted or drilled vertical access shafts restrict vehicle geometry as well as the in‐water infrastructure that may be deployed. The ability of the vehicle to return reliably and precisely to the access point is critical for data return, battery charging, and/or vehicle recovery. This paper presents the mechanical, sensor, and software components that make up the ARTEMIS docking system, as well as results from field deployment of the system to McMurdo Sound, Antarctica in the austral spring of 2015. The mechanical design of the system allows the vehicle to approach the dock from any direction and to pitch up after docking for recovery through a vertical access shaft. It uses only a small volume of in‐water equipment and may be deployed through a narrow vertical access shaft. The software of the system reduces position estimation error with a hierarchical combination of dead reckoning, acoustic aiding, and machine vision. The system provides critical operational robustness, enabling the vehicle to return autonomously and precisely to the access shaft and latch to the dock with no operator input.     

Self-formation of sub-60-nm half-pitch gratings with large areas through fracturing

Periodic micro- and nanostructures (gratings) have many significant applications in electronic, optical, magnetic, chemical and biological devices and materials. Traditional methods for fabricating gratings by writing with electrons, ions or a mechanical tip are limited to very small areas and suffer from extremely low throughput. Interference lithography can achieve relatively large fabrication areas, but has a low yield for small-period gratings. Photolithography, nanoimprint lithography, soft lithography and lithographically induced self-construction all require a prefabricated mask, and although electrohydrodynamic instabilities can self-produce periodic dots without a mask, gratings remain challenging. Here, we report a new low-cost maskless method to self-generate nano- and microgratings from an initially featureless polymer thin film sandwiched between two relatively rigid flat plates. By simply prising apart the plates, the film fractures into two complementary sets of nonsymmetrical gratings, one on each plate, of the same period. The grating period is always four times the thickness of the glassy film, regardless of its molecular weight and chemical composition. Periods from 120 nm to 200 microm have been demonstrated across areas as large as two square centimetres.     

Perforin-dependent neurologic injury in a viral model of multiple sclerosis

In this study we demonstrate perforin-mediated cytotoxic effector function is necessary for viral clearance and may directly contribute to the development of neurologic deficits after demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. We previously demonstrated major histocompatability complex (MHC) class I-deficient (beta2m-deficient) mice with an otherwise resistant genotype develop severe demyelination with minimal neurologic disease when chronically infected with TMEV. These studies implicate CD8(+) T cells as the pathogenic cell in the induction of neurologic disease after demyelination. To determine which effector mechanisms of CD8(+) T cells, granule exocytosis or Fas ligand expression, play a role in the development of demyelination and clinical disease, we infected perforin-deficient, lpr (Fas mutation), and gld (Fas ligand mutation) mice with TMEV. Perforin-deficient mice showed viral persistence in the CNS, chronic brain pathology, and demyelination in the spinal cord white matter. Perforin-deficient mice demonstrated severely impaired MHC class I-restricted cytotoxicity against viral epitopes, but normal MHC class II-restricted delayed-type hypersensitivity responses to virus antigen. Despite demyelination, virus-infected perforin-deficient mice showed only minimal neurologic deficits as indicated by clinical disease score, activity monitoring, and footprint analysis. Perforin- and MHC class II-deficient mice (with functional CD8(+) T cells and perforin molecules and an H-2(b) haplotype) had comparable demyelination and genotype, however, only the latter showed severe clinical disease. Gld and lpr mice demonstrated normal TMEV-specific cytotoxicity and maintained resistance to TMEV-induced demyelinating disease. These studies implicate perforin release by CD8(+) T cells as a potential mechanism by which neurologic deficits are induced after demyelination.