Hemagglutination assay.

<p>(a) Hemagglutination assay was performed as indicated in Material and methods using serial two-fold diluted samples of duplicate (R1, R2) TSP fractions extracted from leaves expressing H1-HFBI or untagged H1. The two bottom rows contain inactivated A/Texas/05/2009(H1N1) virus as a positive control or a GFP-HFBI extract as a negative control. (b) Hemagglutination assay using serial two-fold diluted samples of ATPS-purified H1-HFBI (triplicates, R1–R3). The two bottom rows contain bovine serum albumin as a negative control or inactivated A/Texas/05/2009(H1N1) virus as a positive control. The hemagglutination titer (HT) or the amount of hemagglutination units (HAU) was calculated according to the well with the highest dilution giving a complete hemagglutination. This test was also used to quantify inactivated virus concentration in terms of HAU for inhibition assay.</p

ELISA-based assessment of the immune response of H1-HFBI-immunized mice.

<p>(a) Ten mice were immunized with H1-HFBI as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115944#s4" target="_blank">Experimental procedures</a>. Anti-HA antibodies were assayed by ELISA in the pre-immune sera (open circle) and the sera collected after the 4^th (blue triangle) and 6^th (red square) boost. Plates were coated with 5 µg/ml of recombinant Influenza A/Texas/05/2009(H1N1) ectodomain expressed in mammalian cells (Sino Biologicals, 11085-V08H). HRP-conjugated anti-mouse secondary antibody was used for detection. HA titer was calculated as the highest dilution giving a signal higher than three times the signal coming from the negative control. (b) Box and whisker analysis of antibody titers obtained after endpoint ELISA titer analysis of the test groups. Each dot represents the antibody titer from an individual mouse. (p-values  = 0.18 (boost 4/boost 6), 1.8.10⁻⁴ (boost 4/pre-immune), 3.8.10⁻⁴ (boost 6/pre-immune))</p

Endoglycosidase H treatment of H1-HFBI.

<p>Time course incubation of a leaf TSP extract (5 µg) in the presence (+) or absence (−) of endoglycosidase H (0.2 U/ml). Samples were analyzed by Western blotting after 0, 15, or 60 min of incubation. The signal was detected with polyclonal anti-influenza A antibodies.</p

H1-HFBI is found as protein bodies in transgenic <i>N. tabacum</i> BY-2 suspension cells.

<p>Wild-type cells (a), cells expressing H1 (b) and cells expressing H1-HFBI (c–e) at the exponential phase (3 days after dilution) were submitted to <i>in situ</i> immunolocalization as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115944#s4" target="_blank">Experimental procedures</a> using an FITC-conjugated anti-influenza H1N1. Bars  = 25 µm (a, b, c) and 5 µm (d, e).</p

Neutralizing properties of antibodies induced by H1-HFBI.

<p>Sera from the ten vaccinated mice were serially 2-fold diluted and incubated with inactivated virus for 30 min, and then RBCs were added. Pre-immune sera were used as a negative control. Hemagglutination inhibition titers were determined. The mean for each test group was calculated, and bars represent SD.</p

Schematic representation of the pEAQ plasmids used to express H1-HFBI and H1.

<p>RB and LB: right and left T-DNA borders, En₂PMA4: <i>N. plumbaginifolia</i> (NpPMA4) promoter reinforced by two copies of the CaMV 35S enhancer (De Muynck et al., 2009); 5′ and 3′UTR: Cowpea Mosaic Virus Untranslated Regions (translational enhancers); SP: <i>A. thaliana</i> basic endochitinase signal peptide, HA: hemagglutinin ectodomain (residues 18 to 529), HFBI: hydrophobin I; KDEL: ER retention signal; pNos, NosT: nopaline synthase gene promoter or terminator; p35S, 35ST: Cauliflower Mosaic Virus 35S promoter or terminator; p19: suppressor of RNA silencing; nptII: <i>neomycin phosphotransferase II</i> gene.</p

Selective ammonium sulfate precipitation of ATPS-purified H1-HFBI.

<p>Solid ammonium sulfate corresponding to 5% or 10% saturation was added to ATPS-purified H1-HFBI samples (4 mg/ml), left for 1 h at 4°C, and centrifuged. The pellet (P) was dissolved in the same volume of PBS as the supernatant volume (SN).</p

Purification of H1-HFBI by ATPS.

<p>A TSP extract from H1-HFBI-expressing leaves was subjected to purification by ATPS as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115944#s4" target="_blank">Experimental procedures</a>. Samples (40 µl) of TSP, the upper phase discarded after the first phase separation, and the lower phase recovered after the second phase separation, were analyzed by SDS-PAGE. The identification of bands 1–5 by mass spectrometry is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115944#pone.0115944.s007" target="_blank">S1 Table</a>.</p

Accumulation of HA fused or not to HFBI.

<p>The H1 and H1-HFBI constructs were transiently expressed in <i>N. benthamiana</i> leaves. One construct was abaxially infiltrated on one half of the leaf and the other one on the other half. (a) Coomassie-blue stained gel of 30 µg TSP from four representative extracts. TSP from a plant infiltrated with an <i>A. tumefaciens</i> strain containing an empty vector was used as a negative control (C-). The bands corresponding to H1-HFBI and H1 are indicated by an arrow. (b) Western blot analysis of 5 µg TSP coming from the same extracts as in (a). The signal was detected with polyclonal anti-influenza A.</p

Isolation of High-Purity Cellulose Nanofibers from Wheat Straw through the Combined Environmentally Friendly Methods of Steam Explosion, Microwave-Assisted Hydrolysis, and Microfluidization

High-purity cellulose nanofibers were isolated from wheat straw through an environmentally friendly, multistep treatment process that combined steam explosion, microwave-assisted hydrolysis, and microfluidization. The cellulose content of the processed nanofibers increased from 44.81% to 94.23%, whereas the hemicellulose and lignin contents significantly decreased. Scanning electron microscopy revealed the effects of the isolation treatments on fiber morphology and width. Atomic force microscopy was used to observe the changes in the components, surface roughness, and crystallinity of the fibers. Transmission electron microscopy showed long, loose nanofiber bundles that were 10–40 nm wide with an average individual diameter of 5.42 nm. Fourier transform infrared spectroscopy showed that noncellulosic components were effectively removed. X-ray diffraction analysis revealed the improved crystallinity of the processed fibers, as well as the partial crystalline transformation of cellulose I to cellulose II. Thermogravimetric analysis and derivative thermogravimetric results showed the enhanced thermal properties of the nanofibers. The removal of hemicellulose and lignin increased the crystallinity of the fibers, thus enhancing the thermal properties of the processed fibers. Results indicated that the efficient, environmentally friendly, multistep treatment process yields nanofibers with potential advanced applications