Elasticity Theory and Shape Transitions of Viral Shells

Recently, continuum elasticity theory has been applied to explain the shape transition of icosahedral viral capsids - single-protein-thick crystalline shells - from spherical to buckled/faceted as their radius increases through a critical value determined by the competition between stretching and bending energies of a closed 2D elastic network. In the present work we generalize this approach to capsids with non-icosahedral symmetries, e.g., spherocylindrical and conical shells. One key new physical ingredient is the role played by nonzero spontaneous curvature. Another is associated with the special way in which the energy of the twelve topologically-required five-fold sites depends on the background local curvature of the shell in which they are embedded. Systematic evaluation of these contributions leads to a shape phase diagram in which transitions are observed from icosahedral to spherocylindrical capsids as a function of the ratio of stretching to bending energies and of the spontaneous curvature of the 2D protein network. We find that the transition from icosahedral to spherocylindrical symmetry is continuous or weakly first-order near the onset of buckling, leading to extensive shape degeneracy. These results are discussed in the context of experimentally observed variations in the shapes of a variety of viral capsids.Comment: 53 pages, 17 figure

Mobility and survival of Salmonella Typhimurium and human adenovirus from spiked sewage sludge applied to soil columns

Aims: This study investigated the survival and transport of sewage sludge-borne pathogenic organisms in soils. Methods and Results: Undisturbed soil cores were treated with Salmonella enterica ssp. enterica serovar Typhimurium-lux (STM-lux) and human adenovirus (HAdV)-spiked sewage sludge. Following an artificial rainfall event, these pathogens were analysed in the leachate and soil sampled from different depths (0-5 cm, 5-10 cm and 10-20 cm) after 24 h, 1 and 2 months. Significantly more STM-lux and HAdV leached through the soil cores when sewage sludge was present. Significantly more STM-lux were found at all soil depths, at all time periods in the sewage sludge treatments, compared to the controls. The rate of decline of STM-lux in the controls was more rapid than in the sewage sludge treatments. Survival and transport of HAdV were minimal. Conclusions: The presence of sewage sludge can significantly influence the transport and survival of bacterial pathogens in soils, probably because of the presence of organic matter. Environmental contamination by virus is unlikely because of strong soil adsorption. Significance and Impact of the Study: This study suggests that groundwater contamination from vertical movement of pathogens is a potential risk and that it highlights the importance of the treatment requirements for biosolids prior to their application to land

Electron microscopy as an emerging analytical tool for characterizing vaccines

Characterization of nanoparticles and biologics is a critical step in the development of important new pharmaceutical products and biosimilars. Biologics pose unique characterization challenges that require an interdisciplinary approach in which several orthogonal methods are used to provide a complete picture. The physical characteristics of a biological product include properties such as the size, shape, morphology and aggregation state of the particles. These properties are often dependent on the specific environment of the particles and thus ideally must be assessed under conditions that reflect the final formulation of the pharmaceutical. Electron microscopy (EM) and in particular cryo-electron microscopy (cryoEM), has a unique advantage in that it provides a direct means of observing the individual particles in a sample, preserved in their natural hydrated state (cryoEM), simultaneously providing information on homogeneity, size distribution, titer, morphology, preservation state, flexibility, and aggregation state. For particles with a regular size and shape, particle averaging methods can provide 3D structural information, complementing X-ray crystallography analysis. We will demonstrate the use of EM as an analytical and structural characterization tool by presenting a number of case studies as highlights. Specifically, we will discuss the characterization of Human Papilloma Virus (HPV) VLPs in GARDASIL®, including the structure of the VLPs alone, on adjuvants, and when interacting with neutralizing antibodies [1]. We will also show how TEM was used as a non-intrusive tool to understand the structure and function of Hepatitis B surface antigen (rHBsAg) VLPs, the active component in the HBV vaccine [2]. We will furthermore demonstrate how TEM can be used to provide supporting information for characterization of a biosimilar drug delivery nanoparticle, a recombinant tuberculosis vaccine antigen, interacting with a lipid-based adjuvant [3], and a bi-specific, tetravalent immunoglobulin G-like molecule [4]. References: [1] Zhao Q, et al. 2013. Characterization of virus-like particles in GARDASIL(R) by cryo transmission electron microscopy. Hum Vaccin Immunother.10:1-6. [2] Mulder AM, et al. 2012. Toolbox for non-intrusive structural and functional analysis of recombinant VLP based vaccines: a case study with hepatitis B vaccine. PLoS One 7:e33235. [3] Fox CB, et al. 2014. Cryogenic transmission electron microscopy of recombinant tuberculosis vaccine antigen with anionic liposomes reveals formation of flattened liposomes. Int J Nanomedicine 9:1367-77. [4] Correia I, et al. 2013.The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen. MAbs. 5:364-72

Fourier transform ion cyclotron resonance MS reveals the presence of a water molecule in an enzyme transition-state analogue complex

The structures of several powerful inhibitors of hydrolytic enzymes resemble that of the altered substrate in the transition state, except that a hydrogen atom replaces one substituent (typically the leaving group). To test the hypothesis that a water molecule might be present in the gap resulting from this replacement, we examined a transition-state analogue complex formed by Escherichia coli cytidine deaminase by Fourier transform ion cyclotron resonance MS in electrospray mode. Upon nebularization from aqueous solution under conditions (pH 5.6) where the enzyme is active, cytidine deaminase remains dimeric in the vapor phase. In the presence of inhibitor, the enzyme's exact mass can be used to infer the presence at each active site of zinc, 5-fluoro-3,4-dihydrouridine, and a single water molecule

Conceptual design for a receiving station for the nondestructive assay of PuO/sub 2/ at the fuels and materials examination facility

We propose a conceptual design for a receiving station for input accountability measurements on PuO/sub 2/ received at the Fuels and Materials Examination Facility at the Hanford Engineering Development Laboratory. Nondestructive assay techniques are proposed, including neutron coincidence counting, calorimetry, and isotopic determination by gamma-ray spectroscopy, in a versatile data acquisition system to perform input accountability measurements with precisions better than 1% at throughputs of up to 2 M.T./yr of PuO/sub 2/

Structural Evidence of T Cell Xeno-reactivity in the Absence of Molecular Mimicry

The T cell receptor (TCR), from a xeno-reactive murine cytotoxic T lymphocyte clone AHIII12.2, recognizes murine H-2Db complexed with peptide p1027 (FAPGVFPYM), as well as human HLA-A2.1 complexed with peptide p1049 (ALWGFFPVL). A commonly proposed model (the molecular mimicry model) used to explain TCR cross-reactivity suggests that the molecular surfaces of the recognized complexes are similar in shape, charge, or both, in spite of the primary sequence differences. To examine the mechanism of xeno-reactivity of AHIII12.2, we have determined the crystal structures of A2/p1049 and Db/p1027 to 2.5 Å and 2.8 Å resolution, respectively. The crystal structures show that the TCR footprint regions of the two class I complexes are significantly different in shape and charge. We propose that rather than simple molecular mimicry, unpredictable arrays of common and differential contacts on the two class I complexes are used for their recognition by the same TCR

Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry

With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis