Full Inactivation of Human Influenza Virus by High Hydrostatic Pressure Preserves Virus Structure and Membrane Fusion While Conferring Protection to Mice against Infection

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.     

Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper‐preserved archaeological bone using size exclusion chromatography

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Assessing Nonsuperiority,Noninferiority, or Equivalence When Comparing Two Regression Models Over a Restricted Covariate Region

Summary In many scientific problems the purpose of the comparison of two regression models, which describe the relationship between a same response variable and several same covariates for two different groups, is to demonstrate that one model is no higher than the other by a negligible amount, or to demonstrate that the models have only negligible differences and so they can be regarded as describing practically the same relationship between the response variable and the covariates. In this article, methods based on one‐sided pointwise confidence bands are proposed for assessing the nonsuperiority of one model to the other and for assessing the equivalence of two regression models. Examples from QT/QTc study and from drug stability study are used to illustrate the methods.     

Dispersal potential of the invasive green alga Codium fragile ssp. fragile

Since its introduction in 1989, the invasive green alga Codium fragile ssp. fragile (formerly Codium fragile ssp. tomentosoides) has spread rapidly in Atlantic Canada. Although its spread has likely been facilitated by human transport, C. fragile possesses diverse modes of natural dispersal. In addition to parthenogenetically developing swarmer cells, it can propagate through the release of vegetative buds, thallus fragments, and entire, dislodged thalli. We examined the natural dispersal potential of these propagules using a combination of field and laboratory experiments. Vegetative buds were most abundant on thalli in late August, coinciding with increasing late summer wave activity. At two locations within two field sites, we examined the effects of tidal state, wave action and topography on the dispersal of fragments and intact thalli. Over a 4-hour period, retention rate was generally lower and dispersal distance of retained propagules higher: during flood than ebb tide; at the more wave-exposed locations; and for fragments than thalli. Dispersal direction corresponded with topography or flood tide currents at certain locations, but was often bi-directional or random, suggesting an added role of wave action. Over periods of weeks, the retention rate of marked detrital thalli was negatively related to the magnitude of wave activity. In the laboratory, settling rate was negatively related to propagule length, and large buds had higher critical shear erosional velocities than either small buds or fragments. Our results indicate that large propagules of C. fragile, such as intact adult thalli, have a generally low dispersal potential (meters), but may be transported longer distances (kilometers) during storms or when positively buoyant. Fragments and buds are regularly transported 10's of meters at a time at average flow velocities. However, they also become resuspended in average flows, and probably disperse longer distances over multiple resuspension events, or when positively buoyant. Once settled, the smallest propagules may be less easily resuspended or transported along the bottom as they become entrapped by small-scale topographic features or turf algae. The wide variety of propagules produced by C. fragile and the variable distances over which different propagule types may be transported give C. fragile the advantage of both short- and long-distance dispersal, and have likely played a role in the invasive success of this alga.     

Two strings to the systems biology bow: co-extracting the metabolome and proteome of yeast

Experimental samples are valuable and can represent a significant investment in time and resources. It is highly desirable at times to obtain as much information as possible from a single sample. This is especially relevant for systems biology approaches in which several ‘omics platforms are studied simultaneously. Unfortunately, each platform has a particular extraction methodology which increases sample number and sample volume requirements when multiple ‘omics are analyzed. We evaluated the integration of a yeast extraction method; specifically we explored whether fractions from a single metabolite extraction could be apportioned to multiple downstream ‘omics analytical platforms. In addition, we examined how variations to a chloroform/methanol yeast metabolite extraction regime influence metabolite recoveries. We show that protein suitable for proteomic analysis can be recovered from a metabolite extraction and that recovery of lipids, while reproducible, are not wholly quantitative. Higher quenching solution temperatures (?30 °C) can be used without significant leakage of intracellular metabolites when lower fermentation temperatures (20 °C) are employed. However, extended residence time in quenching solution, in combination with vigorous washing of quenched cell pellets, leads to extensive leakage of intracellular metabolites. Finally, there is minimal difference in metabolite amounts obtained when metabolite extractions are performed at 4 °C compared to extractions at ?20 °C. The evaluated extraction method delivers material suitable for metabolomic and proteomic analyses from the same sample preparation.     

Re-Directing an Alkylating Agent to Mitochondria Alters Drug Target and Cell Death Mechanism

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.     

Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047–2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.