Schematic of binding and unbinding for a divalent antibody and tandem epitope.

A tandem epitope (green) with variable linker length is bound in two steps by a divalent antibody (blue). If the proximity-dependent rebinding rate (kb) is much larger than the monovalent unbinding rate (koff) then avidity is greatly increased.</p

Performance of homodimeric antibodies with tandem epitopes.

<p>(<b>A</b>) Binding of recombinant C11L34 antibody to a single or tandem GCN epitope placed within the PCDH15 sequence and expressed in RPMI 2650 cells. Binding was normalized to the signal of an anti-PCDH15 antibody. The binding to the tandem epitope was about 15x better, if the linker was 19 amino acids (green line) but not if the linker was shorter (magenta). (<b>B</b>) Immunoblots of cell extracts showing much stronger binding to the tandem epitope with longer linker. Lysates from HEK cells expressing PCDH15 with different tags were run on two identical gels; one was probed with the divalent C11L34 anti-GCN at a fixed concentration and one with anti-PCDH15 as a loading control. Full sized immunoblots are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s003" target="_blank">S3 Fig</a>. (<b>C</b>) Binding of recombinant anti-THAP antibody or biotinylated BTX to a single or tandem HAP epitope placed within the PCDH15 sequence, normalized to the anti-PCDH15 signal. Anti-THAP avidity for the THAP tag was about 7X better than to the single HAP tag. (<b>D</b>) Immunoblots of cell extracts showing much stronger binding to the tandem epitope than to the single HAP. Protocol as in (B). Lower panel shows the binding to the tagged PCDH15 by anti-THAP relative to anti-PCDH15. Full sized immunoblots are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s004" target="_blank">S4 Fig</a>. (<b>E</b>) Biacore surface plasmon resonance analysis of binding between the recombinant antibodies to HAP or THAP peptides. One antibody (anti-THAP; shown as BTX:BTX) contained symmetric BTX binding domains, while another (BTX:con) was asymmetric with one arm containing BTX and the other an irrelevant control binding domain. The THAP peptide showed higher affinity, but it was substantially higher only for the anti-THAP antibody, and only when the linker length was 10 rather than 14 amino acids. Representative traces are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s001" target="_blank">S1 Fig</a>. Measured rate constants and dissociation constants are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s005" target="_blank">S1 Table</a>. (<b>F</b>) Single-molecule pull-down assay. Cell lysates containing monomeric YFP tagged with THAP and 6xHis were applied at different dilutions to slides with immobilized anti-His or anti-THAP antibodies, or anti-HA as a negative control. Insets show molecules pulled down. Full immunoblots for panels B and D are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s004" target="_blank">S4</a> Figs.</p

Strategy for designing high-affinity antibodies.

<p>(<b>A</b>) Schematic of the scFv C11L34-Fc fusion binding to a tandem GCN epitope. The recombinant antibody has identical Fc regions and is homodimeric. (<b>B</b>) THAP and anti-THAP. Bungarotoxin (BTX) replaces the variable region. (<b>C</b>) Recombinant bi-specific antibody to EGFP, created by different mutation of each Fc region to force heterodimeric binding. Each half of the dimer incorporates a nanobody (GBP1 or GBP2) targeting different regions of EGFP.</p

Binding of heterodimeric GBP antibody to EGFP.

<p>(<b>A</b>) Biacore surface plasmon resonance assay for effective affinity. Three test antibodies contained a GBP6-Fc fusion with an irrelevant Fc control arm (crtl+GBP6), a GBP1-Fc with an irrelevant Fc (crtl+GBP1), and a heterodimeric GBP1+GBP6 Fc fusion (GBP1+GBP6). Different concentrations of recombinant EGFP, ranging from 0 to 10 nM, were injected over immobilized Fc fusion proteins. Representative traces are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s002" target="_blank">S2 Fig</a>; rate constants in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150125#pone.0150125.s005" target="_blank">S1 Table</a>. (<b>B</b>) CHO cells expressing EGFP assessed with EGFP fluorescence or anti-EGFP immunocytochemistry. CHO cells were transfected with EGFP fused to the transmembrane protein TMC1 as a carrier. Left panels show intrinsic EGFP fluorescence; right panels show label with either stock rabbit anti-GFP (top) or GBP1+6-Fc (bottom) with appropriate secondary antibodies. Imaging of the native EGFP fluorescence revealed bright EGFP-expressing cells, but also autofluorescence of non-transfected cells (top left). An anti-GFP antibody signal, adjusted for the same brightness, had less but not zero non-specific signal (top right). In comparison, the GBP1+6-Fc antibody, adjusted for the same brightness, had no detectable nonspecific label (bottom right). (<b>C</b>) Direct comparison of rabbit anti-GFP and GBP1+6-Fc. Cells expressing TMC1_EGFP were labeled with both rabbit anti-GFP and GBP1+6-Fc and with appropriate secondary antibodies of different colors, adjusted for equal brightness. Both labeled two strongly-expressing cells (upper part of field) and two weakly-expressing cells (middle, *), but the anti-GFP showed more nonspecific label—visible in the nuclei of the middle cells and in a non-expressing cell (bottom, #). (<b>D</b>) Single-molecule pull-down. Cell lysates containing tagged monomeric YFP were applied to slides coated with anti-HA (control), anti-GFP, or GBP1+6-Fc. The dashed box shows the optimal range for single-molecule pull-down analysis; GBP1+6-Fc is substantially better than anti-GFP in this range.</p

Additional file 1 of E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

Supplementary Material

Table_1_Core-Etched CC/SnO2 Nanotube Arrays as High-Performance Anodes for Lithium-Ion Batteries With Ionic Liquid Electrolyte.DOCX

Despite the design of nano-structured SnO2 anodes has attracted much attention because of its high theoretical capacity, good electron mobility, and low potential of lithium-ion intercalation, challenges remain due to their weak mechanical stability, complex processing and rapid capacity decay. The one-dimensional binder-free porous CC/SnO2 nanotube arrays are synthesized with a well-suited core etching method to meet the needs of steady operation of flexible devices under mechanical deformation. This porous, binder-free nanostructure has large contact area with the electrolyte and excellent electron transport performance. The electrochemical measurements demonstrate that these nanotube arrays have high energy density and high-rate capability. After 500 cycles at a current density of 200 mA g−1, their stable capacity remains at 595.7 mA h g−1.</p

Engineering Polymers via Understanding the Effect of Anchoring Groups for Highly Stable Liquid Metal Nanoparticles

Liquid metal nanoparticles (LMNPs) have recently attracted much attention as soft functional materials for various biorelated applications. Despite the fact that several reports demonstrate highly stable LMNPs in aqueous solutions or organic solvents, it is still challenging to stabilize LMNPs in biological media with complex ionic environments. LMNPs grafted with functional polymers (polymers/LMNPs) have been fabricated for maintaining their colloidal and chemical stability; however, to the best of our knowledge, no related work has been conducted to systematically investigate the effect of anchoring groups on the stability of LMNPs. Herein, various anchoring groups, including phosphonic acids, trithiolcarbonates, thiols, and carboxylic acids, are incorporated into brush polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization to graft LMNPs. Both the colloidal and chemical stability of such polymer/LMNP systems are then investigated in various biological media. Moreover, the influence of multidentate ligands is also investigated by incorporating different numbers of carboxylic or phosphonic acid into the brush polymers. We discover that increasing the number of anchoring groups enhances the colloidal stability of LMNPs, while polymers bearing phosphonic acids provide the optimum chemical stability for LMNPs due to surface passivation. Thus, polymers bearing multidentate phosphonic acids are desirable to decorate LMNPs to meet complex environments for biological studies

Additional file 4 of Prediction of pandemic risk for animal-origin coronavirus using a deep learning method

Additional file 4. The number of virus data with artificial negative data

Additional file 1 of Prediction of pandemic risk for animal-origin coronavirus using a deep learning method

Additional file 1. The number for the initial virus data

Additional file 3 of Prediction of pandemic risk for animal-origin coronavirus using a deep learning method

Additional file 3. The Entry ID for the selected negative virus to recombine initial positive virus