3526 ANLNLWTDYIRW[2.97436] 1 Phage display (common panning) 3 PMID:32454443 Human colon cancer cell line HT29 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding ability of the selected phage clones was evaluated on colon cancer cells (SW480, HT29, HCT116, LoVo, and DLD1) as well as on a normal cell line (CCD-18co) by cell phage ELISA. Absorbance was measured at 450 nm (OD450) using a plate reader. The binding affinities (relative OD values) showed more than two- to five-fold higher selectivity for colorectal cells than the negative controls. The binding affinity (relative fold) was reproduced from the Supplementary Figure 1 and shown. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under three rounds of biopannning for the human colon cancer cells HT29. The phage clone with the highest optical density (450 nm) compared to the negative control cells was selected. This peptide(ANLNLWTDYIRW) probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. 3527 ANLNLWTDYIRW[2.07692] 1 Phage display (common panning) 3 PMID:32454443 Human colon cancer cell line LoVo Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding ability of the selected phage clones was evaluated on colon cancer cells (SW480, HT29, HCT116, LoVo, and DLD1) as well as on a normal cell line (CCD-18co) by cell phage ELISA. Absorbance was measured at 450 nm (OD450) using a plate reader. The binding affinities (relative OD values) showed more than two- to five-fold higher selectivity for colorectal cells than the negative controls. The binding affinity (relative fold) was reproduced from the Supplementary Figure 1 and shown. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under three rounds of biopannning for the human colon cancer cells LoVo. The phage clone with the highest optical density (450 nm) compared to the negative control cells was selected. This peptide(ANLNLWTDYIRW) probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. 3528 CGGQDLKSC CSNLTSP*C CPSSSREKC 3 Phage display (common panning) 3 PMID:32243979 Cystic fibrosis mucus model Not determined. Not determined. Not determined. CX7C T7 phage display library (CX7C) For the first round of selection, an initial phage amount of 4.2e9 phage plaque forming units (pfu) was added on top of the mucus layer in the donor compartment and incubated for 1 h at room temperature (25 °C). At 5, 10, 15, 30, and 45 min, 10 μL aliquots of the eluates were taken for further quantification. After 1 h, the entire eluate was collected from the basolateral side and titered using standard double layer plaque assay to quantify phage concentration. The eluted phage library was amplified in BL21 E. coli as described before, which was then quantified by plaque assay prior to the next round of selection. Two subsequent rounds of iterative selection were performed with initial amounts of 5.2e6 pfu and 4.2e9 pfu, respectively. We used combinatorial peptide-presenting phage libraries and next-generation sequencing (NGS) to identify hydrophilic, net-neutral charged peptide coatings that enable penetration through human cystic fibrosis (CF) mucus ex vivo with ~600-fold better penetration than control, improve uptake into lung epithelial cells compared to uncoated or PEGylated-nanoparticles, and exhibit enhanced uniform distribution and retention in the mouse lung airways. These peptide coatings address multiple delivery barriers and effectively serve as excellent alternatives to standard PEG surface chemistries to achieve mucus penetration and address some of the challenges encountered using these chemistries. This biomolecule-based strategy can address multiple delivery barriers and hold promise to advance efficacy of therapeutics for diseases like CF. 3529 EGGDSRH[0.137913±0.000046] RLRAIFS[0.134717±0.000040] VSRVAPC[0.129523±0.000046] WLEVYLG[0.138723±0.000046] 4 Phage display (common panning) 3 PMID:31344770 Pancreatic cancer cell line MIA PaCa-2 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence- activated cell sorter (FACS) were performed to examine the specific binding. Absorbance was determined at 450 nm. OD450 was reproduced from Fig. 1 in the reference. M5(WLEVYLG) bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. The noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes. 3530 SGVYKVAYDWQH(5)[NA] SWFSDWDLELHA(5)[56.40442] SQDIRTWNGTRS(3)[NA] ETWLRWSEKYPT(2)[66.06515] 4 Phage display (common panning) 3 PMID:32892011 N-propyl paraben (PP) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The binding affinity and selectivity test of the selected peptide that has binding affinity to target was carried out using Dynabeads® M-270 Amine. Data (ppm) shown were reproduced from Figure 1b in the reference. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs,Beverly, MA, USA) were used for affinity selection of specific peptides under three rounds of biopannning for the n-propyl paraben (PP)(≥99.0%) . We successfully screened two peptides specific to PP, namely PP3; the results showed that the PP concentration reduction in PP3 was the highest at 39%, and the specificity was measured by the capture rate between target and control. 3531 VHWDFRQWWQPS(7)[NA] DSQFNKYSIATV(3)[62.18905] SGVYKVAYDWQH(2)[NA] SQDIRTWNGTRS(1)[NA] 4 Phage display (common panning) 3 PMID:32892011 Methyl paraben (MP) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The binding affinity and selectivity test of the selected peptide that has binding affinity to target was carried out using Dynabeads® M-270 Amine. Data (ppm) shown were reproduced from Figure 1a in the reference. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs,Beverly, MA, USA) were used for affinity selection of specific peptides under three rounds of biopannning for the Methyl paraben (MP) (purity: ≥ 99.0%) . We successfully screened two peptides specific to MP namely, MP4; the results showed that the MP concentration reduction in MP4 was the highest at 44%, and the specificity was measured by the capture rate between target and control. 3532 VHWDRQWWQPS(12) SGVYKVAYDWQH(1) SQDIRTWNGTRS(1) STPIFAEATARS(1) 4 Phage display (common panning) 3 PMID:32892011 Petri dish (polystyrene, PS) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The Ph.D.-12 Phage Display Peptide Library (New England BioLabs,Beverly, MA, USA) were used for affinity selection of specific peptides under three rounds of biopannning for the petri dish. 3533 VHWDFRQWWQPS[0.40 ± 0.03] WHITPWAWWRPM[0.32 ± 0.02] LMTTAGTAFSLA[0.27 ± 0.03] AHKSKLHQHVMF[0.56 ± 0.06] KPHPKVPLEHWR[0.30 ± 0.03] DWSSWVYRDPQP[0.19 ± 0.04] 6 Phage display (common panning) 5 PMID:32166528 Trans-2-nonenal Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Absorbance was measured at 405–415 nm. The OD405 was reproduced from Figure 2 in the reference and shown. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs,Beverly, MA, USA) were used for affinity selection of specific peptides under three rounds of biopannning for the trans-2-nonenal. The phage with sequence of AHKSKLHQHVMFGGG (called as P4) in the end of tail, has shown the highest response. To explore a role of the peptide selected in sequence analysis and ELISA assay, the peptide was connected to magnetic beads. The peptide-coated beads were treated within trans-2-nonenal: treatment of P4 peptide shows significant decrease of trans-2-nonenal compared to negative peptide. Based on our results, it is suggested that the peptide, which is selected by phage display, could be used for the removal of trans-2-nonenal and odor associated with aging. 3534 VHWDFRQWWQPS WHITPWAWWRPM 2 Phage display (common panning) 5 PMID:32166528 Petri dish (polystyrene, PS) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) 3535 LMTTAGTAFSLA 2 Phage display (common panning) 5 PMID:32166528 Glass dish Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) 3536 QSHDQNNYNQRS(33/69)[0.86 ± 0.62] DSFNGRYEHVPH(12/69)[0.3 ± 0.13] QTNILTRIHRNR(11/69)[0.95 ± 0.41] KPMRRHRSLHRR(7/69)[3.6 ± 1.3] RKPPRNTMQIRI(2/69)[6.3 ± 2.1] RPRTMPMPTSMR(2/69)[2.8 ± 0.95] RTPLRPIRSRKP(1/69)[8.4 ± 3.8] RINRTRRIRPTP(1/69)[8.39 ± 3.7] 8 Phage display (competitive panning) 5 PMID:32619663 Anthrax toxin receptor 1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance was measured using a SpectroMax M2 Multi-Mode Microplate Reader (Molecular Devices) at 450 nm. The value (OD450) shown in Table S2 in the reference was obtained in a binding saturation curve fitting from three independent experiments. The Ph.D.-12 Phage Display Peptide Library (New England BioLabs) were used for affinity selection of specific peptides under five rounds of biopannning for the purified recombinant ATR/TEM8 VWA (rATR/TEM8 VWA). In the first round, 0.1% Tween-20 was used, and In subsequent biopanning (from the 2nd through 5th rounds), the concentration of detergent (Tween-20) or salt (NaCl) in the wash buffer was increased, creating progressively more harsh conditions as follows: 0.3% (2nd round) and 0.5% (3rd to 5th round) Tween-20, and 500 mM NaCl (4th and 5th round). After the final round of biopanning, the protein-bound phages were eluted by the addition of a 10 molar excess of rATR/TEM8 VWA. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides(QSHDQNNYNQRS,DSFNGRYEHVPH) binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-D-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptidebased approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment. 3537 MISTSRK9(40)[0.37345] QKRNTIT(2)[0.20761] 2 Phage display (subtractive panning) 3 PMID:31092638 Patients' sera pool containing Alternaria specific IgE (s-IgE) Anti-human IgE mAbs Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA Absorbance was measured at 450 nm (OD450). OD450 was reproduced from Fig.3a in the reference. Phages from the library of phage (Ph.D.-7 phage Display Peptide Library Kit; New England BioLabs) were screened by non-Alternaria s-IgE (plate A) for three-round selections (each time for a new plate A). The nonbinding phages were harvested and then screened by Alternaria s-IgE (plate B) three times (each time for a new plate B). The bound phages (targeted phages) were washed off from plate B. Third, the targeted phages in a monoclonal colony were picked and amplified respectively, and then the amplified phages were purified by polyethylene glycol/NaCl-precipitation and titrated. Two mimotopes (MISTSRK and QKRNTIT) presented high binding ability with the sera of the Alternaria-allergic patients and mice and, therefore, were selected for immunotherapy in the mouse model. Compared with irrelevant phage control, model, and natural extract immunotherapy group, mimotope immunotherapy group significantly reduced serum IgE levels, inflammatory cells infiltration in the lung tissue, and IL-4 levels in bronchoalveolar lavage fluid, whereas serum IgG1 and IFN-γ levels in bronchoalveolar lavage fluid were increased. The results indicate that B cell mimotopes of Alternaria alleviates allergic response in a mouse model and have potential as novel therapeutic agents for IgE-mediated Alternaria-allergic diseases. 3538 GYFDVVLGGFGP(3)[0.49593] SLHTGATNLYLH(1)[0.62228] FIRPNDWGFGPW(1)[0.54192] HVLNSTVWNTRI(1)[0.57257] HSAQASITIKMA(1)[0.631180±0.001074] SYFDALERMLPG(1)[0.72191] DX(1,4)GFGP 6 Phage display (competitive panning) 4 PMID:31452334 Anti-fibronectin-binding protein A (FnBPA) N-terminal A domain (FnBPA-A) polyclonal antibody Fibronectin-binding protein A Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding ability of the selected six mimotope peptides synthesized in vitro reacted with anti-FnBPA-A antibodies was measured using a microplate reader (Model 450; BioRad Laboratories) at 450nm. OD450 was reproduced from Fig.4 in the reference. To obtain phages binding to anti-rFnBPA-A antibodies, a random Ph.D.-12 phage display peptide library (New England Biolabs) was screened with purified anti-FnBPA-A antibodies according to the manufacturer's instructions. For each round of biopanning, phages (1.5e12 PFU/mL diluted with pure normal rabbit IgG) were applied to a 96-well plate precoated with anti-rFnBPA-A antibodies (10 μg/well). Eight anti-rFnBPA-A antibody-binding phage clones were selected for sequencing, and six different 12-mer peptides were displayed by these phages. Although these displayed peptides shared no more than three consecutive amino acid residues identical to the sequence of FnBPA-A, they could be recognized by the FnBPA-A-specific antibodies in vitro and could induce specific antibodies against FnBPA-A in vivo, suggesting that these displayed peptides were mimotopes of FnBPA-A. Finally, the protective efficiencies of these mimotopes were investigated by mouse vaccination and challenge experiments. Compared with that of control group mice, the relative percent survival of mice immunized with phage clones displaying a mimotope was 13.33% (C2: SLHTGATNLYLH or C15: HVLNSTVWNTRI), 0% (C8: GYFDVVLGGFGP), 6.67% (C10: FIRPNDWGFGPW), 26.67% (C19: HSAQASITIKMA or 1:2 mixture of C23: SYFDALERMLPG and C19), 53.33% (C23), 33.33% (1:1 mixture of C23 and C19), and 66.67% (2:1 mixture of C23 and C19). Overall, five peptides mimicking FnBPA-A protein epitopes were obtained, and a partially protective immunity against S. aureus infection could be stimulated by these mimotope peptides in mice. 3539 PSPHRQRQHILR(25/50) QTIRIIIRRSRT(6/60) SLHMRHKRKPRR(4/50) SSRSMQRTLIIS(2/50) IRSIRMRRILIL(2/50) KTSMRPLILIHI(2/50) 6 Phage display (subtractive panning) 4 PMID:32417652 Brain tumor initiating cells (BTICs) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) A BTIC-specific phage library was generated by subtracting against U87MG cells, U251 N cells, and human normal fetal astrocytes to deplete the library of background phage and common binders, and a mixture of patient-derived BTICs were used for selection of candidate phage. A combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Six unique 12 amino acid (12mer) peptide sequences were identified more than once, and four were retained (PSPHRQRQHILR,QTIRIIIRRSRT,SLHMRHKRKPRR,SSRSMQRTLIIS) based on back screening of the synthetically made corresponding peptides against a non-invasive tumor cell line used for subtraction. 3540 SVSVGMKPSPRP(21/100) GISLSSYLQSTQ(20/100) EHMALTYPFRPP(13/100) HWAPSMYDYVSW(7/100) RTVPDYTAHVRT(5/100) SGHQLLLNKMPN(4/100) TNSIWYTAPYMF(3/100) GMSLSRQMLWSL(2/100) HLFPQSNYGGHS(2/100) CIQLANPPRLXG(2/100) 10 Phage display (subtractive panning) 4 PMID:32417652 Invasive U87MG glioblastoma cell line Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The phage library was first incubated with U87T cells to deplete the library of background phage. The supernatant was then incubated with U87R cells. 3541 VQHNTKYSVVIR(14)[7.04026] VQHNTKYSVVIR(10)[NA] HGTAPALKILK(2)[NA] HGTAPALKILK(1)[2.15974] LHHWNARSALAN(1)[NA] SSIEPAYYLHAT(1)[NA] EALNQWHIFLGR(1)[NA] HYKTPNLYKGWH(1)[NA] PSIFKHPKHLA(1)[NA] 9 Phage display (subtractive panning) 3 PMID:32040673 SUS316L stainless steel Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Binding ability of SBP-A and SBP-B on SUS316L surface was determined based on the amount of each biotinylated (Bio)-peptide binding on the SUS316L disk surface, as assessed by the relative SA-HRP activity measured by ABTS assay. Data shown were reproduced from Fig.2a in the reference. To remove phage particles non-specifically binding to the polystyrene dish, 5E+10 pfu of the phage library in phosphate buffered saline (PBS) (pH 7.4) containing 0.05% (w/v) Tween 20 (PBST) was added into a polystyrene dish. After 1 h incubation at room temperature with gentle agitation, the solution containing unbound phage was recovered and used as the primary library. After three rounds of phage panning procedure, 12 mer peptide (SBP-A; VQHNTKYSVVIR) was identified as SUS316L-binding peptide. The SBP-A peptide formed a stable bond to a SUS316L modified surface and was not toxic to HUVECs. The SBP-A was then used for anti-ICAM antibody modification on SUS316L to construct a vascular endothelial cell-selective surface. The constructed surface dominantly immobilized vascular endothelial cells to smooth muscle cells, demonstrating that the SBP-A enabled simple immobilization of biomolecules without disturbing their active biological function. 3542 VVSPDMNLLLTN(5/16)[0.912840±0.000310] VFSSMVHVLNTH(3/16)[0.140007±0.000179] SGVYKVAYDWQH(1/16)[0.235673±0.002483] GLHTSATNLYLH(1/16)[1.061523±0.002488] CYAGHDLYVAAD(1/16)[NA] SLSWLTKMQMEM(1/16)[NA] 6 Phage display (common panning) 3 PMID:31993846 Escherichia coli O157:H7 cells Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The color development was recorded using a microplate reader (Thermo Fisher, MultiskanTM GO) by monitoring absorbance at 405 nm. A405 shown was reproduced from Figure 2. Briefly, 10 μL phage library (~e13 pfu/mL) were added into 1 mL E. coli O157:H7 cell suspensions (OD600 = 0.5) and incubated for 1 h at room temperature with gentle agitation. Bacteria with bound phages were precipitated by spinning for 5 min at 16,000×g, and separated from unbound phages in solution by a series of 10 washing and centrifugation steps (16,000×g, 5 min) with 1 mL TBST buffer (50 mM Tris–HCl, 0.05% (v/v) Tween 20) each time. After washing, bound phages with E. coli O157:H7 were suspended in 200 μL elution buffer (0.2 M glycine–HCl, pH 2.2) with gentle shaking at room temperature for 10 min. The eluted phages were neutralized with 150 μL, 1 M Tris–HCl (pH 9.1). A 12-mer peptide (with the sequence of GLHTSATNLYLH) with high binding affinity toward Escherichia coli O157:H7 was discovered by biopanning of phage-displayed peptide library. The peptide modified with glycine residues (G3) and one cysteine (C) residue at C-terminal, could self-assemble on gold electrodes, enabling electrochemical impedance spectroscopy (EIS) analysis for quantitative detection of E. coli O157:H7. This method showed a low detection limit of 20 CFU/mL and a liner range from 2e2 to 2e6 CFU/mL. 3543 SGVYKVAYDWQH(6/16)[0.235673±0.002483] GLHTSATNLYLH(3/16)[1.061523±0.002488] VVSPDMNLLLTN(1/16)[0.140007±0.000179] GSAPLLTVDTSK(1/16)[NA] 4 Phage display (common panning) 4 PMID:31993846 Escherichia coli O157:H7 cells Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The color development was recorded using a microplate reader (Thermo Fisher, MultiskanTM GO) by monitoring absorbance at 405 nm. A405 shown was reproduced from Figure 2. Briefly, 10 μL phage library (~e13 pfu/mL) were added into 1 mL E. coli O157:H7 cell suspensions (OD600 = 0.5) and incubated for 1 h at room temperature with gentle agitation. Bacteria with bound phages were precipitated by spinning for 5 min at 16,000×g, and separated from unbound phages in solution by a series of 10 washing and centrifugation steps (16,000×g, 5 min) with 1 mL TBST buffer (50 mM Tris–HCl, 0.05% (v/v) Tween 20) each time. After washing, bound phages with E. coli O157:H7 were suspended in 200 μL elution buffer (0.2 M glycine–HCl, pH 2.2) with gentle shaking at room temperature for 10 min. The eluted phages were neutralized with 150 μL, 1 M Tris–HCl (pH 9.1). A 12-mer peptide (with the sequence of GLHTSATNLYLH) with high binding affinity toward Escherichia coli O157:H7 was discovered by biopanning of phage-displayed peptide library. The peptide modified with glycine residues (G3) and one cysteine (C) residue at C-terminal, could self-assemble on gold electrodes, enabling electrochemical impedance spectroscopy (EIS) analysis for quantitative detection of E. coli O157:H7. This method showed a low detection limit of 20 CFU/mL and a liner range from 2e2 to 2e6 CFU/mL. 3544 MHPNAGHGSLMR(8/19)[3.193117±0.007169] SGVYKVAYDWQH(8/19)[2.932297±0.006400] TGENHVADDRKN(1/19)[2.093027±0.008066] GLDGYRHPRDAW(1/19)[1.885507±0.010138] GLVDEGDHDGER(1/19)[2.386910±0.008071] 5 Phage display (subtractive panning) 3-4 PMID:31270859 Matrix metalloproteinase-9, MMP-9 Integrin beta-5 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Binding of the phages on MMP-9 and BSA was examined with phage ELISA. The absorbance was measured on a Multiskan ELISA reader at 450nm. The binding ratio was calculated and reproduced from Figure 3 in the reference. For this process, we added 1e11 pfu phages from the M13 phage display peptide library (Ph.D.‐12) (New England Biolabs, Herts, UK) to 2 μg of active human recombinant MMP‐9 in the first round and incubated this mixture for 2 hours in 200 μL of tris‐buffered saline with Tween 20 (TBST) buffer (0.1% Tween 20). After that, we added 50 μL of Ni magnetic beads (His60 Ni Magnetic Beads, Takara Bio Inc, Kusatsu, Shiga Prefecture, Japan) to the mixture and incubated that for another 2 hours. The supernatant containing unbound phage was removed via magnetic separation. By elution magnetic beads 10 times with 1 mL of TBST buffer (0.1% Tween 20), we removed feeble or nonspecifically bound phage. In the end, according to the guideline of the manufacturer the bound phage particles were recovered via infecting Escherichia coli ER2738 host. We also preincubated one portion of the first round amplified phage with the beads in the lack of target to eliminate nickel magnetic beads-specific phage particles. By applying magnetic fishing, the unbound phage fraction was incubated with recombinant human MMP‐9 and specifically bound phages were separated. Interestingly, in silico molecular docking showed strong interactions between the peptide threedimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence (MHPNAGHGSLMR) was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2- driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. 3545 WHWRLWDVPDNP(22)[2.081931±0.454971] SFVNLWTPRYSL(5)[0.777530±0.359939] TLFSKPYPNSSR(1)[0.907007±0.005364] TPMHYPATPSPH(1)[1.105431±0.004797] QFGPVFTWLNHA(1)[4.510443±0.010535] TITNAPIKDLTP(1)[0.680020±0.007786] LTPHKHHKHLHA(1)[0.93332] DPHGSLFPRTHP(1)[1.945993±0.003895] TQYPIDGDIFRR(1)[0.999870±0.007093] HLTWIPSVVRNS(1)[0.278147±0.003574] WHWAWYSPTARM(1)[2.754893±0.008042] 11 Phage display (subtractive panning) 3 PMID:32737386 Non-structural protein 1, NS1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Optical density (OD) was measured at 450 nm (measurement wavelength) and 620 nm (reference wavelength) using a Synergy H1 multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA). The OD450 was reproduced from Figures 2a and 2b in the reference and shown. The phage-displayed peptide library ( e11 pfu) was incubated in the BSA-coated wells at RT for 1 h to deplete BSAbinding phages. Unbound phages from the BSA-coated wells were transferred to the DENV NS1-coated wells and incubated at RT for 1 h. The DENV NS1-coated wells were then thoroughly washed to remove unbound phages. DENV NS1-binding phages were eluted from the wells by incubating with glycine elution buffer (0.2 M glycine–HCl pH 2.2 and 1 mg/ml BSA) at RT for 20 min, followed by neutralization with 1 M Tris–HCl pH 9.1. The eluted phages were amplified in E. coli ER2738 cultures and titrated according to the manufacturer’s protocol with minor modifications. The amplified phages ( e11 pfu) from the first round were used for the next round of biopanning, and a total of 3 rounds of biopanning were performed. The number of phages obtained from each round was titrated to evaluate the efficiency of phage recovery. We performed biopanning assays using a phage-displayed peptide library and identified 11 different sequences of 12-mer peptides binding to DENV NS1. In silico analyses of peptide-protein interactions revealed 4 peptides most likely to bind to DENV NS1 at specific positions and their association was analysed by surface plasmon resonance. Treatment of Huh7 cells with these 4 peptides conjugated with N-terminal fluorescent tag and C-terminal cell penetrating tag at varying time-of-addition post-DENV infection could inhibit the production of DENV-2 in a time- and dose-dependent manner. The inhibitory effects of the peptides were also observed in other virus serotypes (DENV-1 and DENV-4), but not in DENV-3. These findings indicate the potential application of peptides targeting DENV NS1 as antiviral agents against DENV infection. 3546 NVFTVSP(6)[0.816657±0.001570, 12.09] NSLSISY(3)[0.371417±0.001570, NA] SQQGKLN(2)[0.525270±0.002944, NA] ELTPLTL(2)[0.182410±0.003243, NA] QLAVAPS(2)[0.255427±0.001570, NA] DRLSHTR(2)[0.252820±0.002944, NA] QTLNVKP(2)[0.215853±0.001570, NA] VVTPKTA(2)[0.220397±0.001570, NA] TTQVLEA(2)[0.208373±0.000981, NA] NLLDSLH(1)[0.208033±0.001570, NA] WSLSELH(1)[0.319490±0.002720, NA] 11 Phage display (common panning) 3 PMID:31805343 Fibroblast growth factor 9, FGF-9 FGFR-1, FGFR-2, FGFR-3 and FGFR-4 Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The binding activity of the 11 positive phage clones to FGF9 was verified by ELISA assay. The absorbance was measured at 450 nm. The OD450 value (in the first column) was reproduced from Figure 1A and shown. In addition, to confirm the interaction between P4 peptide and FGF9, the surface plasmon resonance (SPR) on the immobilized FGF9 was applied to determine the affinity constants. The binding kinetics of the peptides to FGF9 was detected by OpenSPR™ (Nicoya Lifesciences, Waterloo, Canada). The Kd (μM) value was shown in the second column of the affinity values. A sterile polystyrene petri dish was coated with 10 μg/ml recombinant human FGF9 or PBS at 4 °C in 0.1 M NaHCO3 overnight, and blocked with bovine serum albumin (BSA) at 5 mg/ml in 0.1 M NaHCO3 for 2 h at room temperature. After the dish was washed five times (1 min each) with 0.05 % PBST (PBS containing 0.05 % Tween-20), the diluted original Ph.D.-7 library (2.0e11) was added and incubated for 2 h at room temperature. The unbound phages were removed through washing ten times (1 min each) with 0.05 % PBST prior to elution of the bound phages with continuous shaking in 1 ml of 0.1 M glycine-HCl for 10 min at room temperature and subsequent neutralization with 100 μl of 1 M Tris-HCl. The eluate was amplified and purified for the next round of screening. Two additional rounds of selection were conducted under more rigorous conditions. In short, dishes were coated with less amount of FGF9 (5 μg/ml and 2.5 μg/ml for the second and third round, respectively), incubated with the previous eluate for shorter time (1.5 h and 1 h for the second and third rounds, respectively), and washed with higher concentration of PBST for a longer time (0.1 % PBST for 10 × 2 min and 0.3 % PBST for 10 × 3 min for the second and third round, respectively). After three successive rounds of strict selection, the phage clones obtained by infection of E. coli ER2738, individual selection and amplification were exposed to Enzyme-linked immunosorbent assay (ELISA). Sequence comparison showed that P4 (NVFTVSP) shared high homology with the conserved motif in the immunoglobulin-like (Ig-like) domain II∼III (D2-D3) linker of the FGF9 high-affinity receptor (FGFR3c). The interaction between P4 and FGF9 was confirmed by the surface plasmon resonance (SPR) assay. Functional analysis indicated that P4 counteracted FGF9-induced aggressive phenotype, including cell proliferation, migration, and invasion in vitro, as well as suppressed tumor growth in vivovia down-regulation of the MAPKs and Akt cascades. More importantly, we found that FGF9 served as an underlying mechanism of the chemoresistance in GC and BC cells, and P4 could increase the sensitivity to the chemical agent via antagonizing the suppression effects of FGF9 on cell apoptosis. Taken together, our study identified a novel binding peptide for FGF9, which may serve as a potential therapeutic agent for malignant tumors featured by abnormally up-regulation of FGF9. 3547 LPHWHPHSHLQP(42.9%) RQRPKDHFFSRP(14.3%) SPVMPFSPYSTW(14.3%) VPWYKHPRHPHL(14.3%) FHKHKNPGSPII(7.1%) FHRHHSPPPSII(7.1%) 6 Phage display (common panning) 5 PMID:31289973 Silica, Si Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The phages binding on the silica wafer were eluted by incubating with 200 μL of glycine–HCl solution (0.2 M, pH 2.2) at room temperature for 10 min. After neutralization by adding 30 μL of 1 M Tris–HCl (pH 9.1), eluted phages were amplified in E. coli ER2738, and better affinity-binding phages were obtained after the first round of biopanning. After that, the amplified phages were incubated with the silica wafer to perform the above biopanning round by round. A total of five rounds of biopanning were conducted and the silica affinity-binding phages were enriched. The optimal 12-mer SAP sequence that specifically and intensively binding to silica through affinity interaction was obtained by DNA sequencing of the SAP selected from round-5 biopanning phages. The optimal screened SAP (LPHWHPHSHLQP) was selected from a M13 phage display peptide library and fused to the C-terminal of DAAO to obtain fused DAAOs with one, two and three SAP tags, respectively. The activity of DAAO-SAP-MPS was superior comparing with DAAO-2SAP-MPS and DAAO-3SAP-MPS; meanwhile DAAO-SAP-MPS shows 36% higher activity than that of DAAO-MPS. Fusion with one SAP improved the thermal stability with a 10% activity increase for immobilized DAAO-SAP-MPS compared to that of DAAO-MPS at 50 °C for 3 h. Moreover, the activity recovery of immobilized DAAO-SAP-MPS was 25% higher in operation stability assessment after six-batch conversions of cephalosporin to glutaryl-7-amino cephalosporanic acid than that of DAAO-MPS. 3548 EIAYPARYANTY IPWTQHMAMSPM QNKLWDTPSNPW TALGHQPLMRNT SGGMPTARMSHQ APWHNSWSEERT ESGLWYSIDMKP YLDEFAWYRFTH WPRPYYGDWFQT YPPPDSHSERVE SMQGKAYGGTVM WPRPYYGEGFQT HPLTWNLRSSPA ADWYHWRSHSSS VVSPDMNLLLTN TSLDGRISYHNR VHWDFRQWWQPS SQWETSQMIQKM LPLTRGYVGDQY FSPHADWVVVSG GFAVGARDSLMF 21 Phage display (subtractive panning) 3 PMID:30789148 Sera from 30 Systemic lupus erythematosus (SLE) patients Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Serum pools were obtained from 30 HCs and 30 SLE patients. Microtitre wells were coated overnight at 4°C with 150 μl sera from healthy controls diluted with NaHCO3. Plates were blocked with 3% non-fat milk for 2 h at 37°C, and subsequently washed six times with phosphate-buffered saline with Tween 20 (0.05% Tween 20). A 100-μl diluted random 12-peptide phage display library with a titre of 2.0e11 pfu/ml was added to the coated plates. After incubation for 1 h at room temperature, unbound phages were collected and 100μl was added per well to SLE serumcoated plates. Following incubation for 1 h at room temperature, bound phages were eluted with 100 μl 0.2 mol/l glycine–HCl (pH 2.2) and neutralised with 1 mol/l Tris-HCl (pH 9.1). Eluted phages were amplified in a host strain and purified by precipitation for about 4 h using one-sixth volume of polyethylene glycol/NaCl. Another two rounds of SLE-serum affinity selection were carried out using the same process. After a negative selection step with serum from healthy controls (HCs), a phage library of 12 peptides was used for three rounds of screening with sera from 30 SLE patients. After four rounds of biopanning, 21 positive peptides were sequenced. We produced 37-feature arrays containing 16 recombinant citrullinated peptides. The microarrays were tested with an independent validation set of serum samples from 50 HCs, 60 SLE patients, and 60 rheumatoid arthritis (RA) patients. 3549 SQSGPTIFYNLA(13)[2.203160±0.009388] AESRRPFFEPFM(2)[2.833720±0.009388] FNPSFAKMGNSL(1) SALTSAVSGSAA(1) 4 Phage display (common panning) 3 PMID:32808238 Anti-epstein-barr virus (EBV) multiple sclerosis(MS) brain IgG antibodies (MS 95–2) Epstein-barr virus (EBV) Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Purified MS brain 95–2 IgG in 0.1 M carbonate buffer (50 μl, 200 μg/ml) was coated onto wells of ELISA plates overnight at 4 °C. The wells were then blocked with 3% BSA for 2 h followed by incubation with purified phage 3–2 and 3–3 (5 × 109/well) for 1 h. After washing with 0.05% Tween-20/Tris buffered saline (TBST), the wells were incubated with a 1:500 dilution of mouse anti-M13 IgG-HRP (New England BioLabs) antibody for 1 h, followed by incubation with peroxidase substrate ABTS (Vector Laboratories, Burlingame, CA) for 20 min. The optical absorbance was measured at 415 nm with a Microplate Manager (BioRad, Hercules, CA). All samples were tested in duplicate and the ELISA was repeated at least one additional time. OD415 were reproduced from Figure 3A and shown. Wells were blocked with 3% BSA in Tris-buffed saline (TBS) for 2 h, washed 6 times with 0.1% Tween20–TBS (TBST), and the phage peptide library (1.5e11 phage in 100 μl of TBST) was added to the wells and incubated for 2 h at room temperature. Wells were washed 20–30 times with TBST, and bound phage were eluted with 100 μl of 0.2 M glycine, pH 2.2/0.1% BSA for 10 min. The phage-containing solution was immediately neutralized with 15 μl of 1 M Tris, pH 9.5, and eluted phage were titered and reamplified. Amplified phage (e11 pfu) from the first round of panning was applied to additional rounds of panning using the same conditions as in the first pan, except that the concentration of Tween20 in the washing buffer was increased to 0.5%. After the third round of panning, affinity-selected phage were titered. Single plaques were amplified and the peptides were sequenced. We screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. We identified two phage peptides that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays. 3550 TPDCTRWWCPLT YPICTDTLCRLS RQRTPPTRTIRS RPRTRLHTHRNR TQTTKSISTNRI 5 Phage display (common panning) 5 PMID:31812636 Recombinant envelope (E) protein E-TrX Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA After five rounds of bio-panning, phages showing high binding activity to the recombinant E protein based on an indirect enzyme-linked immunosorbent assay (ELISA) were selected (data not shown). The Ph.D.-12™ Phage Display Peptide Library (New England Bio labs, USA) was exposed to plates coated with purified recombinant protein E-Trx. Unbound phages were washed away, and specifically bound phages were eluted and amplified for a second set of screening. After five rounds of bio-panning, phages showing high binding activity to the recombinant E protein based on an indirect enzyme-linked immunosorbent assay (ELISA) were selected then sequenced. P1(TPDCTRWWCPLT) inhibits JEV infection in BHK-21 cells with 50% inhibitory capacity at a concentration of 35.9 μM. The time-of-addition assay indicates that JEV replication is significantly inhibited during pre-infection and co-infection of P1 with JEV while post-infection treatments with P1 have very little impact on JEV proliferation, showing that P1 inhibits JEV infection at early stages and indicating the potential prophylactic effect of P1.