Additional file 1: of De novo profile generation based on sequence context specificity with the long short-term memory network

Figure S1. Learning curves of the LSTM, Figure S2. ROC curves of similarity search for the target (HHBlits) and predictors, Figure S3. Comparison of profile generation time with simulation data, Figure S4. ROC curves of the similarity search for each iterative method, Table S1. Comparison of pAUC values for SCOP classes for SCOP20 test datasets. (PDF 857 kb

Box plots of differences in the index of familiarity (left) and rank differences (right) between opponents based on whether victims used vocalizations.

<p>Box edges represent the upper and lower quartiles, thick lines within the boxes represent medians, and open circles represent outliers (no vocalizations: <i>n</i> = 24; vocalizations: <i>n</i> = 48).</p

Development of recoverable adsorbents for Cr(VI) ions by grafting of a dimethylamino group-containing monomer on polyethylene substrate and subsequent quaternization

A polymeric adsorbent for removal of hexavalent chromium (Cr(VI)) ions was developed by the photografting of 2-(dimethylamino)ethyl methacrylate (DMAEMA) to a polyethylene (PE) mesh and subsequent quaternization with iodoalkanes of different alkyl chain lengths. The grafting of DMAEMA and subsequent quaternization were verified by the FT-IR and XPS measurements. The Cr(VI) ion adsorption capacity of the DMAEMA-grafted PE meshes had the maximum value at the grafted amount of 2.6 mmol/g in a 0.20 mM K2Cr2O7 solution at pH 3.0 and 30°C. The adsorption behaviour obeyed the pseudo-second order kinetic model and well expressed by Langmuir isotherm. These results suggest that the Cr(VI) ion adsorption occurs through the electrostatic interaction mainly between protonated dimethylamino groups and hydrochromate (HCrO4-) ions. The adsorption capacity of the quaternized PE-g-PDMAEMA meshes increased with an increase in the degree of quaternization and/or the alkyl chain length of the iodoalkanes used and the maximum adsorption ratio was obtained at the degree of quaternization of 54.2% for the iodoheptane-quaternized PE-g-PDMAEMA (PE-g-QC7PDMAEMA) mesh. This value was about 1.86 times higher than that of the PE-g-PDAMEMA mesh. Cr(VI) ions were successfully desorbed from the PE-g-PDMAEMA and PE-g-QC7PDMAEMA meshes in eluents such as NaOH, NaCl, and NH4Cl. In 0.50 M NaCl, 0.10 M NH4Cl, and 0.50 mM NaOH, the adsorption and desorption process was repeatedly performed without any considerable decrease and the desorption behaviour obeyed the pseudo-second order kinetic model. These results emphasise that the PE-g-PDMAEMA meshes and their quaternized products can be applied as an adsorbent for Cr(VI) ions.</p

Nuclear export activity of NP-NES3 mutant proteins.

<p>HeLa cells growing on cover glass were transfected with pCAGGS encoding wild-type NP-NES3 or its mutants (Φ1, Φ2, Φ3, or Φ4) for 48 h. The cells were then permeabilized with 50 µg/ml digitonin for 5 min on ice. The cytoplasmic components, including NP, were removed by washing (only NP in the nucleus remained). The nuclear export activity of NP was allowed to proceed in the presence of fresh total HeLa cell lysate at 30°C for 1 h (left column). Negative controls were incubated in the absence of total cell lysate (right panel). The cells were then stained with an anti-NP MAb followed by anti-mouse Alexa Flour 488 and Hoechst 333342, and observed under a confocal laser-scanning microscope. The white arrow head indicates nuclear export activity, whereas the yellow arrow head indicates a failure of nuclear export activity.</p

Summary of viral replication kinetics, nuclear localization, nuclear export capacity, and CRM1 binding of the NP-NES3 consensus sequence mutants.

a<p>% virus titer ± SD compared with WT at the 46 h of replication kinetic assay from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g004" target="_blank">Fig. 4C</a>.</p>b<p>No viral rescue by reverse genetics.</p>c<p>% cell count ± SD with nuclear localization of NP from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g005" target="_blank">Fig. 5B</a>.</p>d<p>−, ±, + indicate not occur, partially occur, occur of nuclear export capacity, respectively derived from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g006" target="_blank">Fig. 6</a>.</p>e<p>% intensity of the pull-downed NP band compared with WT from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g007" target="_blank">Fig. 7C</a>.</p

List of NES consensus sequence mutants.

a<p>Hydrophobic (Φ) residues, e.g., leucine, isoleucine, valine, and methionine, in the NES consensus sequence are underlined.</p

Summary of viral replication kinetics and nuclear localization of NES consensus sequence mutants.

a<p>% virus titer ± SD compared with WT at the 46 h of replication kinetic assay from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g001" target="_blank">Fig. 1C</a>.</p>b<p>No viral rescue by reverse genetics.</p>c<p>–, ±, + indicate no change, partial change, great change of NES mutant protein localization compared with WT from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g002" target="_blank">Fig. 2</a>.</p

Construction and expression of NP-NES3 consensus sequence mutants.

<p>(A) NP-NES harboring mutations in individual or all Φ residues (leucine was replaced by alanine by site-directed mutagenesis). (B) Expression of wild-type NP and NP-NES3 mutants (Φ1, Φ2, Φ3, and Φ4) was compared by Western blotting with an anti-WSN Ab and an anti-actin MAb.</p

Intracellular localization of NP-NES3 mutant proteins.

<p>(A) HeLa cells were grown on cover glass and transfected with pCAGGS encoding wild-type NP-NES3 or its mutants (Φ1, Φ2, Φ3, or Φ4) for 48 h before immunofluorescence staining with an anti-NP MAb followed by anti-mouse Alexa Fluor 488 and Hoechst 333342. The cells were then observed under a confocal laser-scanning microscope. The white and yellow arrow heads indicate predominant localization of NP in the cytoplasm (cytoplasmic staining > nuclear staining) and nucleus (nuclear staining > cytoplasmic staining), respectively. (B) Nuclear localization of NP wild-type and NP-NES3 mutants from A. Data are presented as the percentage (± SD) of total cell count with predominant nuclear or cytoplasmic staining of NP from five separate fields.</p

Binding of NP-NES3 mutant proteins to CRM1.

<p>(A) FLAG-tagged NP-NES3 wild-type and mutant (Φ1, Φ2, Φ3, and Φ4) proteins were prepared by transfecting the relevant pCAGGS plasmids into HEK-293T cells. The cells were then lysed. The proteins were captured by anti-FLAG agarose beads and eluted with a FLAG-peptide. CRM1-HA agarose beads were prepared by transfecting CRM1-HA/pCAGGS into HEK-293T cells. The cells were then lysed and the proteins were captured on anti-HA agarose beads. The purified proteins and beads were then run in 10% SDS-PAGE gels and stained with Coomassie Brilliant Blue. (B) NP/CRM1 binding was demonstrated by incubating equal amounts of CRM1-HA agarose beads or anti-HA agarose beads alone with purified NP proteins at 4°C for 3 h. After pull-down and washing, the beads were boiled with 4×SDS sample buffer and subjected to 10% SDS-PAGE and Western blot analysis with an anti-WSN Ab and an anti-CRM1 MAb. Input NP at 30% was included. (C) Equal amounts of purified wild-type NP-NES3-FLAG or mutant (Φ1, Φ2, Φ3, and Φ4) protein were co-incubated with CRM1-HA agarose beads at 4°C for 3 h, pulled-down, washed, boiled with 4×SDS sample buffer, and then subjected to Western blot analysis with an anti-WSN Ab and an anti-CRM1 MAb. NP/CRM1 binding affinity was compared by measuring the intensity of the NP band normalized against the CRM1 band. Each 30% input NP protein and remained NP protein after binding with the CRM1-HA agarose beads are also shown.</p