Acidulant Effect on Greening, Reducing Capacity, and Tryptophan Fluorescence of Sunflower Butter Cookie Dough During Refrigerated Storage

BACKGROUND: Sunflower seed derived butter can be a source of protein and phenolic antioxidants in refrigerated dough. Chlorogenic quinone-amino acid induced greening can however occur at alkaline pH, which could result in less bioavailable conjugated phenol-amino acids. Acidulants were tested as potential anti-greening ingredients in refrigerated chemically leavened cookie dough. Effect of refrigerated storage time, leavening agents and acidulants on tryptophan fluorescence (»ex=280nm, »em=300-500), color (hunter LAB), reducing capacity (DPPH and Folin-Ciocalteu reagent reducing capacity/FCRC), and hydroxycinnamic acids were measured. RESULTS: The pH range of acidified doughs was 4.83–6.98 compared to 7.65- 9.18 in nonacidified leavened doughs after 24 days. Greening was higher in baking soda dough control (a*=-0.54) than baking powder dough control (a*=2.98) after 24 days, attributed to higher pH (9.18) of the former compared to pH 7.14 in the later. Tryptophan fluorescence intensity in baking soda dough decreased in the order: control \u3e glucono-delta lactone H citric acid after 24 days. The DPPH and FCRC of acidified doughs were greater than corresponding control doughs. CONCLUSION: The use of acidulants would prevent greening in sunflower dough without lowering its phenolic concentration, making use of sunflower butter in refrigerated dough for baked goods feasible

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein

A novel receptor-induced activation site in the Nipah virus attachment glycoprotein (G) involved in triggering the fusion glycoprotein (F)

Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (beta6S4/beta1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein

Hexameric Assembly of NiV-F glycoprotein trimers.

<p>The six NiV-F copies are colored in blue, green, pink, yellow, grey and purple, respectively. The C-terminal helical bundles of the NiV-F trimers are colored in red. Six NiV-F trimers assemble into a hexagonal ring around a three-fold crystallographic symmetry axis. The hexameric assembly of NiV-F is presented in cartoon views from the top (<b>A</b>), bottom (<b>B</b>) and side (<b>C</b>). The inset in panel C shows a hydrophobic patch of the hexameric interface between two neighboring NiV-F trimers (interface 1). Specifically, the cathepsin-L cleavage site-containing loop of one F trimer inserts into a hydrophobic pocket of the adjacent F trimer. Residue R109 is embedded into a pocket defined by P52, L53, Y248, T250, L256, F282, P283 and I284. The surrounding residues V108, A111, Q393 and G398 also contribute to the interaction. In the two interface inserts, the blue, green, pink, and purple text marks residues in the monomers of the corresponding colors.</p

Structure the NiV-F trimer.

<p>The NiV-F trimer has a tree-like overall shape with the three copies of the F glycoprotein (colored in blue, green and violet) twined around a central axis (Axis-T). The fusion Peptide (FP) is colored in red. The NiV-F trimer is shown viewed from side, and from the top. Glycosylation moieties and disulfide bonds are shown as sticks. The inset shows a close-up view of the FP, which is located at the N-terminus of the F₁ subunit, and docks into a groove formed by the F₂ subunit of a neighboring F molecule within the trimer. Furthermore, the C-terminus of F₂ and N-terminus of FP fold into a β-hairpin conformation, forming a continuous β-sheet (strands S1-S6) with the F₁ subunit, which fixes the position of FP and stabilizes the pre-fusion state. The N-terminal FP residue, L110, is shown as a red sphere.</p

A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion

The antiviral activity of LJ001 is dependent on its ability to generate singlet oxygen (¹O₂).

<p>(<b>A</b>) LJ001, or control LJ025, was added to a solution of DMA and kept under light. After 6 h, DMA conversion was detected by ¹H-NMR (DMA∶oxiDMA = 3.1 ppm:2.1 ppm (methyl peak)). Reactions were performed in CDCl₃ using 1 equivalent of each reagent. CDCl₃ was saturated with oxygen by bubbling O₂ through the solvent for 30 min and the reaction was kept under O₂ gas atmosphere, except for Ar where oxygen was exchanged with argon by freeze/thaw method. Data represents the mean ± SD of duplicate experiments. (<b>B</b>) HIV-1_IIIB, Herpes Simplex Virus-1 (HSV) or Newcastle disease virus (NDV) were incubated with 0.25 µM of LJ001 in the presence of 50 µM α-tocopherol or DMA, or 100 mM NaN₃. Infectivity was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003297#s4" target="_blank">Materials and Methods</a>, and results presented as infection relative to untreated virus (100%). HIV: mean ± SD of duplicate measurements, representative of three independent experiments. HSV and NDV: results representative of three independent experiments. #: NaN₃ was toxic to TZM-Bl cells used to assay HIV entry. (<b>C</b>) HSV was incubated with 5, 50 or 500 nM of LJ001 and exposed to white light for 2, 5, 10, 20, 40 or 80 min. Infectivity was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003297#s4" target="_blank">Materials and Methods</a>, and results presented as infection relative to untreated virus (100%) at a given time, to account for loss of infectivity over time, and as a function of time of light exposure. Data are representative of two independent experiments. (<b>D</b>) HIV-1_IIIB, HSV or NDV were treated in the dark with 1 µM of LJ001, and subsequently either exposed to a white light source or left in the dark, for 10 min, before infection of cells in the dark. Relative infectivity was determined as in (<b>B</b>). LJ001-treated viruses exposed to light had >99% reduction in infectivity. Data represents the mean ± SD of two independent experiments.</p