Analysis and visualization of H7 influenza using genomic,evolutionary and geographic information in a modular web service

We have reported previously on use of a web‐based application, Supramap ( http://supramap.org ) for the study of biogeographic, genotypic, and phenotypic evolution. Using Supramap we have developed maps of the spread of drug‐resistant influenza and host shifts in H1N1 and H5N1 influenza and coronaviruses such as SARS. Here we report on another zoonotic pathogen, H7 influenza, and provide an update on the implementation of Supramap as a web service. We find that the emergence of pathogenic strains of H7 is labile with many transitions from high to low pathogenicity, and from low to high pathogenicity. We use Supramap to put these events in a temporal and geospatial context. We identify several lineages of H7 influenza with biomarkers of high pathogenicity in regions that have not been reported in the scientific literature. The original implementation of Supramap was built with tightly coupled client and server software. Now we have decoupled the components to provide a modular web service for POY ( http://poyws.org ) that can be consumed by a data provider to create a novel application. To demonstrate the web service, we have produced an application, Geogenes ( http://geogenes.org ). Unlike in Supramap, in which the user is required to create and upload data files, in Geogenes the user works from a graphical interface to query an underlying dataset. Geogenes demonstrates how the web service can provide underlying processing for any sequence and metadata database. © The Willi Hennig Society 2012.     

A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Characterization of products formed during the autoxidation of beta-carotene

The anticarcinogenic action of carotenoids such as beta-carotene has been frequently ascribed to their antioxidant properties. However, very little is actually known about the nature of the antioxidant reaction or the products that are formed. beta-Carotene was exposed to either spontaneous autoxidation conditions or to radical-initiated autoxidation conditions. The products were separated by reverse-phase HPLC, and individual peaks were characterized with an on-line diode array detector. Carbonyl products were isolated and characterized by several procedures, including borohydride reduction to the corresponding alcohols, derivatization with O-ethyl-hydroxylamine to the corresponding O-ethyl-oximes of the carbonyls, and analysis by GC-MS. Under the conditions of the experiments, the formation of a homologous series of carbonyl products was demonstrated, including beta-apo-13-carotenone, retinal, beta-apo-14'-carotenal, beta-apo-12'-carotenal, and beta-apo-10'-carotenal. Several very hydrophobic compounds were formed, which have not been previously identified. In addition, the products of NaOCl-treatment of beta-carotene were analyzed, and shown to be significantly different from the autoxidation products. This type of product analysis should be useful in determining the nature of the oxidants reacting with beta-carotene in vivo.     

Light waveguiding in bioinspired peptide nanostructures

Basic optical properties of bioinspired peptide nanostructures are deeply modified by thermally mediated refolding of peptide secondary structure from α‐helical to β‐sheet. This conformational transition is followed by the appearance in the β‐sheet structures of a wideband optical absorption and fluorescence in the visible region. We demonstrate that a new biophotonic effect of optical waveguiding recently observed in peptide/protein nanoensembles is a structure‐sensitive bimodal phenomenon. In the primary α‐helical structure input, light propagates via optical transmission window demonstrating conventional passive waveguiding, based on classical optics. In the β‐sheet structure, fluorescent (active) light waveguiding is revealed. The latter can be attributed to completely different physical mechanism of exciton‐polariton propagation, characterized by high effective refractive index, and can be observed in nanoscale fibers below diffraction limit. It has been shown that peptide material requirements for passive and active waveguiding are dissimilar. Original biocompatibility and biodegradability indicate high potential future applications of these bioinspired waveguiding materials in precise photobiomedicine towards advanced highly selective bioimaging, photon diagnostics, and optogenetics.     

Decreased aortic early atherosclerosis and associated risk factors in hypercholesterolemic hamsters fed a high- or mid-oleic acid oil compared to a high-linoleic acid oil

Currently, diets higher in polyunsaturated fat are believed to lower blood cholesterol concentrations, and thus reduce atherosclerosis, greater than diets containing high amounts of saturated or possibly even monounsaturated fat. The present study was designed to investigate the effect of diets containing mid- or high-linoleic oil versus the typical high-linoleic sunflower oil on LDL oxidation and the development of early atherosclerosis in a hypercholesterolemic hamster model. Animals were fed a hypercholesterolemic diet containing 10% mid-oleic sunflower oil, high-oleic olive oil, or high-linoleic sunflower oil (wt/wt) plus 0.4% cholesterol (wt/wt) for 10 weeks. After 10 weeks of dietary treatment, only the animals fed the mid-oleic sunflower oil had significant reductions in plasma LDL-C levels (-17%) compared to the high-linoleic sunflower oil group. The high-oleic olive oil-fed hamsters had significantly higher plasma triglyceride levels (+41%) compared to the high-linoleic sunflower oil-fed hamsters. The tocopherol levels in plasma LDL were significantly higher in hamsters fed the mid-oleic sunflower oil (+77%) compared to hamsters fed either the high-linoleic sunflower or high-oleic olive oil. Measurements of LDL oxidation parameters, indicated that hamsters fed the mid-oleic sunflower oil and high-oleic olive oil diets had significantly longer lag phase (+66% and +145%, respectively) and significantly lower propagation rates (-26% and -44%, respectively) and conjugated dienes formed (-17% and -25%, respectively) compared to the hamsters fed the high-linoleic sunflower oil. Relative to the high-linoleic sunflower oil, aortic cholesterol ester was reduced by -14% and -34% in the mid-oleic sunflower oil and high-oleic olive oil groups, respectively, with the latter reaching statistical significance. Although there were no significant associations between plasma lipids and lipoprotein cholesterol with aortic total cholesterol and cholesterol esters for any of the groups, the lag phase of conjugated diene formation was inversely associated with both aortic total and esterified cholesterol in the high-oleic olive oil-fed hamsters (r = -0.69, P < 0.05). The present study suggests that mid-oleic sunflower oil reduces risk factors such as lipoprotein cholesterol and oxidative stress associated with early atherosclerosis greater than the typical high-linoleic sunflower oil in hypercholesterolemic hamsters. The high-oleic olive oil not only significantly reduced oxidative stress but also reduced aortic cholesterol ester, a hallmark of early aortic atherosclerosis greater than the typical high-linoleic sunflower oil.     

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.