Publications by authors named "Hak Sung Kim"

192 Publications

Epitopes of Protein Binders Are Related to the Structural Flexibility of a Target Protein Surface.

J Chem Inf Model 2021 Apr 8. Epub 2021 Apr 8.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.

Protein binders including antibodies are known not to bind to random sites of target proteins, and their functional effectiveness mainly depends on the binding region, called the epitope. For the development of protein binders with desired functions, it is thus critical to understand which surface region protein binders prefer (or do not prefer) to bind. The current methods for epitope prediction focus on static indicators such as structural geometry or amino acid propensity, whereas protein binding events are in fact a consequence of dynamic interactions. Here, we demonstrate that the preference for a binding site by protein binders is strongly related to the structural flexibility of a target protein surface. Molecular dynamics simulations on unbound forms of antigen structures revealed that the antigen surface in direct contact with antibodies is less flexible than the rest of the surface. This tendency was shown to be similar in other non-antibody protein binders such as affibody, DARPin, monobody, and repebody. We also found that the relatedness of epitopes to the structural flexibility of a target protein surface is dependent on the secondary structure elements of paratopes. Monobody and repebody, whose binding sites are composed of β-strands, distinctively prefer to bind to a relatively more rigid region of a target protein. These observations enabled us to develop a simple epitope prediction method which shows a comparable performance to the commonly used ones.
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http://dx.doi.org/10.1021/acs.jcim.0c01397DOI Listing
April 2021

Computationally-guided design and affinity improvement of a protein binder targeting a specific site on HER2.

Comput Struct Biotechnol J 2021 27;19:1325-1334. Epub 2021 Feb 27.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea.

A protein binder with a desired epitope and binding affinity is critical to the development of therapeutic agents. Here we present computationally-guided design and affinity improvement of a protein binder recognizing a specific site on domain IV of human epidermal growth factor receptor 2 (HER2). As a model, a protein scaffold composed of Leucine-rich repeat (LRR) modules was used. We designed protein binders which appear to bind a target site on domain IV using a computational method. Top 10 designs were expressed and tested with binding assays, and a lead with a low micro-molar binding affinity was selected. Binding affinity of the selected lead was further increased by two-orders of magnitude through mutual feedback between computational and experimental methods. The utility and potential of our approach was demonstrated by determining the binding interface of the developed protein binder through its crystal structure in complex with the HER2 domain IV.
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http://dx.doi.org/10.1016/j.csbj.2021.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941009PMC
February 2021

Synergistic anticancer effect of docosahexaenoic acid and isoliquiritigenin on human colorectal cancer cells through ROS-mediated regulation of the JNK and cytochrome c release.

Mol Biol Rep 2021 Feb 27;48(2):1171-1180. Epub 2021 Jan 27.

Department of Internal Medicine, Digestive Disease Research Institute, Wonkwang University School of Medicine, Iksan, Jeonbuk, 54538, Republic of Korea.

A large body of research has demonstrated a synergistic anticancer effect between docosahexaenoic acid (DHA) and standard chemotherapy regimens against colorectal cancer (CRC). In this study, we investigated the chemotherapeutic potential of cotreatment with DHA and isoliquiritigenin (ISL) against CRC HCT-116 cells. Apoptosis was confirmed by Annexin V/PI staining and expression of apoptosis-associated proteins. The synergistic effect of DHA and ISL combination on apoptosis was detected using combination index approaches. Flow cytometry was carried out using fluorescent probes to measure the production of reactive oxygen species (ROS). DHA and ISL in combination synergistically enhanced the decrease in cell viability versus the compounds used alone. Moreover, we demonstrated that the synergistic anti-CRC activity of cotreatment with these two compounds was achieved by inducing the apoptosis caspase-dependently mediated through augmented ROS generation followed by increased Fas ligand mRNA expression and cytochrome c release. Our data also demonstrated that cotreating with DHA and ISL strongly upregulated the phosphorylation of ERK and JNK, which are functionally associated with ROS induced by the two compounds in combination. Interestingly, further study revealed that inhibiting ERK phosphorylation strongly enhanced Fas ligand mRNA expression and the combination of the two compounds induced stronger cytotoxicity, whereas inhibiting JNK phosphorylation significantly reduced the apoptotic signals mediated by cotreatment with these two compounds. Excessive ROS-induced JNK activation and cytochrome c release from mitochondria played a key role in the synergistic anticancer activity of CRC cells by cotreating with DHA and ISL.
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http://dx.doi.org/10.1007/s11033-021-06159-6DOI Listing
February 2021

Biomechanical evaluation of the stability of extra-articular distal radius fractures fixed with volar locking plates according to the length of the distal locking screw.

Comput Methods Biomech Biomed Engin 2020 Dec 21:1-11. Epub 2020 Dec 21.

Department of Orthopedic Surgery, Hanyang University Guri hospital, Guri, Korea.

Surgeons usually used short screws to avoid extensor tendon problems during volar locking plate fixation in distal radius fracture. However, the stability according to the length of distal locking screws have not been fully understood. We investigated this issue through finite element analysis and compression test using synthetic radius. Our results demonstrated that the bi-cortical full-length fixation does not contribute to the stiffness increase in the axial compression direction, and a reduction in length of up to more than 50% length can still provide similar stability to full-length screws. Our data can support that surgeon should undersize the distal screw.
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http://dx.doi.org/10.1080/10255842.2020.1861254DOI Listing
December 2020

Structure-guided rational design of the substrate specificity and catalytic activity of an enzyme.

Methods Enzymol 2020 4;643:181-202. Epub 2020 May 4.

Department of Biological sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea. Electronic address:

The quest for an enzyme with desired property is high for biocatalyic production of valuable products in industrial biotechnology. Synthetic biology and metabolic engineering also increasingly require an enzyme with unusual property in terms of substrate spectrum and catalytic activity for the construction of novel circuits and pathways. Structure-guided enzyme engineering has demonstrated a prominent utility and potential in generating such an enzyme, even though some limitations still remain. In this chapter, we present some issues regarding the implementation of the structural information to enzyme engineering, and exemplify the structure-guided rational approach to the design of an enzyme with desired functionality such as substrate specificity and catalytic efficiency.
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http://dx.doi.org/10.1016/bs.mie.2020.04.050DOI Listing
May 2020

Computer-guided binding mode identification and affinity improvement of an LRR protein binder without structure determination.

PLoS Comput Biol 2020 08 31;16(8):e1008150. Epub 2020 Aug 31.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea.

Precise binding mode identification and subsequent affinity improvement without structure determination remain a challenge in the development of therapeutic proteins. However, relevant experimental techniques are generally quite costly, and purely computational methods have been unreliable. Here, we show that integrated computational and experimental epitope localization followed by full-atom energy minimization can yield an accurate complex model structure which ultimately enables effective affinity improvement and redesign of binding specificity. As proof-of-concept, we used a leucine-rich repeat (LRR) protein binder, called a repebody (Rb), that specifically recognizes human IgG1 (hIgG1). We performed computationally-guided identification of the Rb:hIgG1 binding mode and leveraged the resulting model to reengineer the Rb so as to significantly increase its binding affinity for hIgG1 as well as redesign its specificity toward multiple IgGs from other species. Experimental structure determination verified that our Rb:hIgG1 model closely matched the co-crystal structure. Using a benchmark of other LRR protein complexes, we further demonstrated that the present approach may be broadly applicable to proteins undergoing relatively small conformational changes upon target binding.
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http://dx.doi.org/10.1371/journal.pcbi.1008150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485979PMC
August 2020

Site-Specific Lipidation of a Small-Sized Protein Binder Enhances the Antitumor Activity through Extended Blood Half-Life.

ACS Omega 2020 Aug 30;5(31):19778-19784. Epub 2020 Jul 30.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

Protein and peptide therapeutics tend to have a short blood circulation time mainly caused by rapid clearance in kidney, leading to a low therapeutic efficacy. Here, we demonstrate that the antitumor activity of a small-sized protein binder can be significantly enhanced by prolonged blood half-life through site-specific lipidation. An unnatural amino acid was genetically incorporated into a specific site with the highest accessibility in a human interleukin-6 (IL-6)-targeting protein binder with a size of 30.8 kDa, followed by conjugation with palmitic acid using cooper-free click chemistry. The resulting protein binder was shown to have a binding capacity for serum albumin, maintaining a comparable binding affinity for human IL-6 to the native protein binder. The terminal half-life of the lipidated protein binder was estimated to be 10.7 h, whereas the native one had a half-life of 20 min, resulting in a significantly enhanced tumor suppression effect. The present approach can be generally applied to small-sized therapeutic proteins for the elongation of circulation time and increase of bioavailability in blood, consequently enhancing their therapeutic efficacy.
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http://dx.doi.org/10.1021/acsomega.0c02555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424708PMC
August 2020

Functionalized gold nanoparticles with zinc finger-fused proteins as a colorimetric immunoassay platform.

Anal Chim Acta 2020 Aug 18;1126:154-162. Epub 2020 Jun 18.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea. Electronic address:

The quest for highly sensitive and specific detection of disease biomarkers is high, despite many advances in analysis system. Here, we present a sensitive immunoassay platform using DNA-tethered gold nanoparticles and DNA-binding zinc fingers (ZFs). Monomeric alkaline phosphatase (mAP) and human TNF-α were employed as a signal generator and a disease biomarker, respectively. Gold nanoparticles (AuNPs) were first grafted with double-stranded DNAs having specific sequences for two different types of ZFs (QNK and zif268). The alkaline phosphatase and TNF-α-specific protein binder were genetically fused to each of two different types of ZFs, respectively, followed by conjugation with the DNA-tethered AuNPs in a sequence-specific manner. The use of the functionalized AuNPs as a signal generator in a colorimetric immunoassay of TNF-α led to LOD of 120 pg/ml, showing about 161-fold higher sensitivity than a protein binder-fused mAP. The present immunoassay platform could be applied to other analytes by simply replacing a targeting moiety, allowing a versatile and reproducible colorimetric immunoassay.
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http://dx.doi.org/10.1016/j.aca.2020.06.019DOI Listing
August 2020

The surface plasmonic welding of silver nanowires via intense pulsed light irradiation combined with NIR for flexible transparent conductive films.

Nanoscale 2020 Sep 19;12(34):17725-17737. Epub 2020 Jun 19.

Department of Mechanical Engineering Hanyang University, 17 Haendang-Dong, Seongdong-Gu, Seoul 133-791, South Korea. and Institute of Nano Science and Technology, Hanyang University, 17 Haendang-Dong, Seongdong-Gu, Seoul, 133-791, South Korea.

In this work, surface plasmonic welding of silver nanowires (AgNWs) by intense pulse light (IPL) combined with NIR was investigated. AgNWs were coated on a flexible PET (polyethylene terephthalate) substrate using a bar-coater. The coated AgNW films were welded at room temperature and under ambient conditions by white IPL from a xenon lamp, assisted with light from a UV-C (ultraviolet C) and NIR (near infra-red) lamp using an in-house multi-wavelength IPL welding system. In order to investigate the welding mechanism, in situ monitoring with a Wheatstone bridge electrical circuit was performed. The sheet resistance changes of AgNW films during the welding process were monitored under various IPL conditions (e.g. light energy and on-time) with and without UV-C and NIR light irradiation. The microstructure of the welded AgNW film and the interface between the AgNW film and the PET substrate were observed using a scanning electron microscope (SEM) and transmission electron microscope (TEM). COMSOL multi-physics simulations were conducted and compared with the in situ monitoring results to discuss the in-depth mechanism of the IPL welding of AgNWs and its dependence on the wavelength of light. From this study, the optimal IPL welding conditions and appropriate wavelength were suggested, and the optimized IPL welding process could produce AgNW film with a lower sheet resistance (45.2 Ω sq) and high transparency (96.65%) without damaging the PET substrate.
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http://dx.doi.org/10.1039/c9nr10819jDOI Listing
September 2020

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms.

Nat Commun 2020 05 8;11(1):2319. Epub 2020 May 8.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École polytechnique fédérale de Lausanne (EPFL, 1015, Lausanne, Switzerland.

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.
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http://dx.doi.org/10.1038/s41467-020-16128-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210950PMC
May 2020

Targeted delivery of a human Bcl-2-specific protein binder effectively induces apoptosis of cancer cells.

Biochem Biophys Res Commun 2020 05 28;526(2):447-452. Epub 2020 Mar 28.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea. Electronic address:

Bcl-2 family proteins are critical switches to control cell death and survival, and Bcl-2 is a key regulator in pro-survival signaling, causing various diseases including cancers. Bcl-2 has drawn a considerable attention as a potential target for developing a pro-apoptotic agent for cancers. We here present the development of a specific protein binder against human Bcl-2 and its cytosolic delivery to effectively induce apoptosis of cancer cells. The protein binder composed of leucine-rich repeat modules was selected for human Bcl-2, and its binding affinity was increased up to 60 nM through a modular evolution-based approach. The protein binder was efficiently delivered into cancer cells by an intracellular delivery system using a translocation domain from a bacterial exotoxin, resulting in a strong suppression of anti-apoptotic signaling in cancer cells. Our results demonstrate that the human Bcl-2-specific protein binder can act as a potent therapeutic agent for cancers.
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http://dx.doi.org/10.1016/j.bbrc.2020.03.113DOI Listing
May 2020

A regulatory SH2 domain-targeting protein binder effectively inhibits the activity of Bruton's tyrosine kinase and its drug-resistant variants.

Biochem Biophys Res Commun 2020 05 16;526(1):8-13. Epub 2020 Mar 16.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea. Electronic address:

Human Bruton's tyrosine kinase (hBtk) plays a key role in growth and metabolism of B cells, but its dysfunctions cause various B-cell malignancies. Inhibitors targeting the ATP-binding pocket of hBtk have been developed, but they have several drawbacks such as adverse side effects and occurrence of drug-resistant mutations. Here, we present a protein binder which specifically binds to an allosteric regulatory SH2 domain of hBtk. The protein binder effectively inhibited the hBtk activity, indicating a critical role of the SH2 domain in allosteric regulation of the hBtk activity. Cytosolic delivery of the protein binder led to a significant inhibition on the BCR-mediated signaling and viability of B lymphoma cells. The utility of our approach was demonstrated by effective inhibition of drug-resistant hBtk variants by the protein binder. Based on the computationally predicted binding mode, the protein binder is likely to inhibit the hBtk activity by disrupting the interaction between the SH2 domain and kinase domain. The present approach can be used for developing therapeutic agents with improved efficacy for B-cell lymphoma.
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http://dx.doi.org/10.1016/j.bbrc.2020.03.006DOI Listing
May 2020

Effective inhibition of C3a-mediated pro-inflammatory response by a human C3a-specific protein binder.

Biotechnol Bioeng 2020 06 4;117(6):1904-1908. Epub 2020 Mar 4.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.

Complement component 3a (C3a) plays a crucial role in the immune response and host defense, but it is also involved in pro-inflammatory responses, causing many inflammatory disorders. Blockade of C3a has been regarded as a potent therapeutic strategy for inflammatory diseases. Here, we present the development of a human C3a (hC3a)-specific protein binder, which effectively inhibits pro-inflammatory responses. The protein binder, which is composed of leucine-rich repeat modules, was selected against hC3a through phage display, and its binding affinity was matured up to 600 pM by further expanding the binding interface in a module-by-module manner. The developed protein binder was shown to have more than 10-fold higher specificity to hC3a compared with human C5a, exhibiting a remarkable suppression effect on pro-inflammatory response in monocyte, by blocking the interaction between hC3a and its receptor. The hC3a-specific protein binder is likely to have a therapeutic potential for C3a-mediated inflammatory diseases.
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http://dx.doi.org/10.1002/bit.27309DOI Listing
June 2020

Modular protein-DNA hybrid nanostructures as a drug delivery platform.

Nanoscale 2020 Feb;12(8):4975-4981

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea.

With the increasing number of identified intracellular drug targets, cytosolic drug delivery has gained much attention. Despite advances in synthetic drug carriers, however, construction of homogeneous and biocompatible nanostructures in a controllable manner still remains a challenge in a translational medicine. Herein, we present the modular design and assembly of functional DNA nanostructures through sequence-specific interactions between zinc-finger proteins (ZnFs) and DNA as a cytosolic drug delivery platform. Three kinds of DNA-binding ZnF domains were genetically fused to various proteins with different biological roles, including targeting moiety, molecular probe, and therapeutic cargo. The engineered ZnFs were employed as distinct functional modules, and incorporated into a designed ZnF-binding sequence of a Y-shaped DNA origami (Y-DNA). The resulting functional Y-DNA nanostructures (FYDN) showed self-assembled superstructures with homogeneous morphology, strong resistance to exonuclease activity and multi-modality. We demonstrated the general utility of our approach by showing efficient cytosolic delivery of PTEN tumour suppressor protein to rescue unregulated kinase signaling in cancer cells with negligible nonspecific cytotoxicity.
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http://dx.doi.org/10.1039/c9nr08519jDOI Listing
February 2020

Prolonged half-life of small-sized therapeutic protein using serum albumin-specific protein binder.

J Control Release 2019 12 22;315:31-39. Epub 2019 Oct 22.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea. Electronic address:

Many small-sized proteins and peptides, such as cytokines and hormones, are clinically used for the treatment of a variety of diseases. However, their short half-life in blood owing to fast renal clearance usually results in a low therapeutic efficacy and frequent dosing. Here we present the development of a human serum albumin (HSA)-specific protein binder with a binding affinity of 4.3nM through a phage display selection and modular evolution approach to extend the blood half-life of a small-sized therapeutic protein. As a proof-of-concept, the protein binder composed of LRR (Leucine-rich repeat) modules was genetically fused to the N-terminus of Glucagon-like Peptide-1 (GLP-1). The fused GLP-1 was shown to have a significantly improved pharmacokinetic property: The terminal half-life of the fused GLP-1 increased to approximately 10h, and the area under the curve was 5-times higher than that of the control. The utility and potential of our approach was demonstrated by the efficient control of the blood glucose level in type-2 diabetes mouse models using the HSA-specific protein binder-fused GLP-1 over a prolonged time period. The present approach can be effectively used in enhancing the efficacy of small-sized therapeutic proteins and peptides through an enhanced blood circulation time.
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http://dx.doi.org/10.1016/j.jconrel.2019.09.017DOI Listing
December 2019

A small-sized protein binder specific for human PD-1 effectively suppresses the tumour growth in tumour mouse model.

J Drug Target 2020 04 26;28(4):419-427. Epub 2019 Sep 26.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.

Immune checkpoint inhibitors have drawn a consider attention as an effective cancer immunotherapy, and several monoclonal antibodies targeting the immune checkpoint receptors, such as human programmed cell death-1 (hPD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are clinically used for treatment of various cancers. Here we present the development of a small-sized protein binder which specifically binds to hPD-1. The protein binder, which is composed of leucine-rich repeat (LRR) modules, was selected against hPD-1 through phage display, and its binding affinity was maturated up to 17 nM by modular evolution approach. The protein binder was shown to be highly specific for hPD-1, effectively inhibiting the interaction between hPD-1 and its ligand, hPD-L1. The protein binder restored T-cell function , and exhibited a strong anti-tumour activity in tumour mouse model, indicating that it acts as an effective checkpoint blockade. Based on the results, the developed protein binder specific for hPD-1 is likely to find a potential use in cancer immunotherapy.
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http://dx.doi.org/10.1080/1061186X.2019.1669042DOI Listing
April 2020

B-SIDER: Computational Algorithm for the Design of Complementary β-Sheet Sequences.

J Chem Inf Model 2019 10 25;59(10):4504-4511. Epub 2019 Sep 25.

Department of Biological Sciences , Korea Advanced Institute of Science and Technology (KAIST) , Daejeon 34141 , Republic of Korea.

The β-sheet is an element of protein secondary structure, and intra-/intermolecular β-sheet interactions play pivotal roles in biological regulatory processes including scaffolding, transporting, and oligomerization. In nature, a β-sheet formation is tightly regulated because dysregulated β-stacking often leads to severe diseases such as Alzheimer's, Parkinson's, systemic amyloidosis, or diabetes. Thus, the identification of intrinsic β-sheet-forming propensities can provide valuable insight into protein designs for the development of novel therapeutics. However, structure-based design methods may not be generally applicable to such amyloidogenic peptides mainly owing to high structural plasticity and complexity. Therefore, an alternative design strategy based on complementary sequence information is of significant importance. Herein, we developed a database search method called β-Stacking Interaction DEsign for Reciprocity (B-SIDER) for the design of complementary β-strands. This method makes use of the structural database information and generates target-specific score matrices. The discriminatory power of the B-SIDER score function was tested on representative amyloidogenic peptide substructures against a sequence-based score matrix (PASTA 2.0) and two popular ab initio protein design score functions (Rosetta and FoldX). B-SIDER is able to distinguish wild-type amyloidogenic β-strands as favored interactions in a more consistent manner than other methods. B-SIDER was prospectively applied to the design of complementary β-strands for a splitGFP scaffold. Three variants were identified to have stronger interactions than the original sequence selected through a directed evolution, emitting higher fluorescence intensities. Our results indicate that B-SIDER can be applicable to the design of other β-strands, assisting in the development of therapeutics against disease-related amyloidogenic peptides.
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http://dx.doi.org/10.1021/acs.jcim.9b00548DOI Listing
October 2019

Erratum to: Nondestructive Estimation of Lean Meat Yield of South Korean Pig Carcasses Using Machine Vision Technique.

Food Sci Anim Resour 2019 Jun 30;39(3):520. Epub 2019 Jun 30.

Department of Biosystems Machinery Engineering, College of Agricultural and Life Science, Chungnam National University, Daejeon 34134, Korea.

[This corrects the article DOI: 10.5851/kosfa.2018.e44.].
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http://dx.doi.org/10.5851/kosfa.2019.e47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612793PMC
June 2019

Peptide Aptamer of Complementarity-determining Region to Detect Avian Influenza Virus.

J Biomed Nanotechnol 2019 Jun;15(6):1185-1200

Despite significant progress in the development of diagnostic methods for influenza, avian influenza (AI) infection continues to represent a substantial threat to human health. Among the subtypes of AI, H5 influenza is highly infectious to animals and humans; however, there are no reliable H5 subtype-specific diagnostic systems owing to a scarcity of H5 subtype-specific detection elements. In this study, a new peptide aptamer (P1:KASGYTFTSF) was developed to recognize the H5 viral subtype using an bioinformatics approach for predicting complementarity-determining regions (CDRs), and the aptamer was evaluated by immunoassays. The three-dimensional structure of influenza hemagglutinin (HA) and the peptide were used in a molecular docking study, and the peptide was compared to the epitope-derived peptide aptamer (H5-P2:KPNGAINF). Interactions between the peptides and the virus were then assessed by fluorescence-linked sandwich immunosorbent assay (FLISA), immunofluorescence staining assay (IFA), and rapid fluorescent immunochromatographic assay (FICT). P1 and H5-P2 both significantly detected H5N3 at 15.6 HAU/mL ( 0.05), and P1 detected the virus more effectively ( 0.05), consistent with the docking result. An optical image of the peptide recognizing an H5N3-infected cell was acquired by IFA, and was consistent with the antibody-linked IFA result. FICT employing the peptide showed the ability for H5 subtype-specific diagnosis, with 2-fold higher performance than that of a conventional, antibody-linked rapid test. This work shows the potential of a CDR-predicted peptide aptamer as a probe for immunological assays that can specifically recognize AI virus.
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http://dx.doi.org/10.1166/jbn.2019.2772DOI Listing
June 2019

Intense Pulsed Light Annealing Process of Indium-Gallium-Zinc-Oxide Semiconductors via Flash White Light Combined with Deep-UV and Near-Infrared Drying for High-Performance Thin-Film Transistors.

ACS Appl Mater Interfaces 2019 Apr 27;11(14):13380-13388. Epub 2019 Mar 27.

Department of Mechanical Engineering , Hanyang University , Haengdang-dong, Seongdong-gu, Seoul 133-791 , Republic of Korea.

In this study, an intense pulsed light (IPL) process for annealing an indium-gallium-zinc-oxide (IGZO) semiconductor was conducted via flash white light combined with near-infrared (NIR) and deep-ultraviolet (DUV) drying to form a thin-film transistor (TFT). The IGZO thin-film semiconductor was fabricated using a solution-based process on a doped-silicon wafer covered with silicon dioxide. In order to optimize the IPL irradiation condition for the annealing process, the flash white light irradiation energy was varied from 70 to 130 J/cm. Drying by NIR and DUV irradiation was employed and optimized to improve the performance of the TFT during IPL annealing. A TFT with a bottom-gate and top-contact structure was formed by depositing an aluminum electrode on the source and drain on the IPL-annealed IGZO. The electrical transfer characteristic of the TFT was measured using a parameter analyzer. The field effect mobility of the saturation regime and on/off current ratio were evaluated. Changes of the metal-oxide bonds in the IGZO thin film were analyzed using X-ray photoelectron spectroscopy to verify the effect of NIR and DUV drying and IPL annealing. Also, the distributions of the carrier concentration on the IPL-annealed IGZO were measured through a hall-effect system to deeply investigate the transition of the electrical characteristic of the TFT. From the results, it was found that the bond between oxygen and the gallium compound was activated via DUV irradiation. The NIR- and DUV-assisted IPL-annealed IGZO-based TFT showed highly enhanced electrical performance with a 7.7 cm/V·s mobility and a 3 × 10 on/off ratio.
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http://dx.doi.org/10.1021/acsami.8b22458DOI Listing
April 2019

Ultra-High-Speed Intense Pulsed-Light Irradiation Technique for High-Performance Zinc Oxynitride Thin-Film Transistors.

ACS Appl Mater Interfaces 2019 Jan 16;11(4):4152-4158. Epub 2019 Jan 16.

Division of Materials Science and Engineering , Hanyang University , 222, Wangsimni-ro , Seongdong-gu, Seoul 04763 , Republic of Korea.

In this study, we investigated the effects of intense pulsed light (IPL) on the electrical performance properties of zinc oxynitride (ZnON) thin films and thin-film transistors (TFTs) with different irradiation energies. Using the IPL process on the oxide/oxynitride semiconductors has various advantages, such as an ultrashort process time (∼100 ms) and high electrical performance without any additional thermal processes. As the irradiation energy of IPL increased from 30 to 50 J/cm, the carrier concentration of ZnON thin films decreased from 5.07 × 10 to 9.96 × 10 cm and the electrical performance of TFTs changed significantly, which is optimized at an energy of 40 J/cm (field effect mobility of 48.4 cm V s). The properties of TFTs, such as mobility, subthreshold swing, and hysteresis, and the stability of the device under negative bias degraded as the irradiation energy increased. This degradation contributed to the change in nitrogen-related bonding states, such as nonstoichiometric Zn N and N-N bonding, rather than that of oxygen-related bonding states and the atomic composition of ZnON thin films. Optimization of the IPL process in our results makes it possible to produce high-performance ZnON TFTs very fast without any additional thermal treatment, which indicates that highly productive TFT fabrication can be achieved via this process.
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http://dx.doi.org/10.1021/acsami.8b20291DOI Listing
January 2019

Nondestructive Estimation of Lean Meat Yield of South Korean Pig Carcasses Using Machine Vision Technique.

Korean J Food Sci Anim Resour 2018 Oct 31;38(5):1109-1119. Epub 2018 Oct 31.

Department of Biosystems Machinery Engineering, College of Agricultural and Life Science, Chungnam National University, Daejeon 34134, Korea.

In this paper, we report the development of a nondestructive prediction model for lean meat percentage (LMP) in Korean pig carcasses and in the major cuts using a machine vision technique. A popular vision system in the meat industry, the VCS2000 was installed in a modern Korean slaughterhouse, and the images of half carcasses were captured using three cameras from 175 selected pork carcasses (86 castrated males and 89 females). The imaged carcasses were divided into calibration (n=135) and validation (n=39) sets and a multilinear regression (MLR) analysis was utilized to develop the prediction equation from the calibration set. The efficiency of the prediction equation was then evaluated by an independent validation set. We found that the prediction equation-developed to estimate LMP in whole carcasses based on six variables-was characterized by a coefficient of determination ( ) value of 0.77 (root-mean square error [RMSEV] of 2.12%). In addition, the predicted LMP values for the major cuts: ham, belly, and shoulder exhibited values≥0.8 (0.73 for loin parts) with low RMSEV values. However, lower accuracy ( =0.67) was achieved for tenderloin cuts. These results indicate that the LMP in Korean pig carcasses and major cuts can be predicted successfully using the VCS2000-based prediction equation developed here. The ultimate advantages of this technique are compatibility and speed, as the VCS2000 imaging system can be installed in any slaughterhouse with minor modifications to facilitate the on-line and real-time prediction of LMP in pig carcasses.
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http://dx.doi.org/10.5851/kosfa.2018.e44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238032PMC
October 2018

Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery.

Small 2018 12 6;14(52):e1802618. Epub 2018 Nov 6.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea.

With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model.
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http://dx.doi.org/10.1002/smll.201802618DOI Listing
December 2018

A new chalcone derivative, 3-phenyl-1-(2,4,6-tris(methoxymethoxy)phenyl)prop-2-yn-1-one), inhibits phorbol ester-induced metastatic activity of colorectal cancer cells through upregulation of heme oxygenase-1.

Eur J Pharmacol 2018 Dec 12;841:1-9. Epub 2018 Oct 12.

Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Jeonbuk 54538, Republic of Korea. Electronic address:

Chalcone (1,3-diphenyl-2-propen-1-one) derivatives exert anti-cancer activity by targeting key molecules that can lead to carcinogenesis. We synthesized the chalcone derivative 3-phenyl-1-(2,4,6-tris(methoxymethoxy)phenyl)prop-2-yn-1-one (KB-34) and previously reported its anti-inflammatory activity in macrophages. In this study, we examined the anti-metastatic activity of KB-34 against human colorectal cancer (CRC) cells and elucidated its underlying molecular mechanisms. KB-34 treatment significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced migration, as well as the invasion and proliferation of CRC cells (HT-29 and SW620). TPA-induced activation of NF-κB was also markedly suppressed by KB-34 in HT-29 cells. KB-34 suppressed the expression of matrix metalloproteinase-7 (MMP-7) at both the mRNA and protein levels in TPA-stimulated CRC cells (HT-29 and SW620). We also demonstrated that induced heme oxygenase-1 (HO-1) expression in CRC cells (HT-29 and SW620) and HO-1 is required for KB-34-mediated suppression of the expression of MMP-7 in TPA-stimulated HT-29 cells. Additionally, the cyclin-dependent kinase inhibitor p21 was significantly induced by treatment with KB-34 in CRC cells (HT-29 and SW620). Knockdown of HO-1 prevented the induction of p21 expression by KB-34 in HT-29 cells. Furthermore, we also demonstrated that 5-fluorouracil (5-FU) together with KB-34 produced a significantly greater inhibition of growth and stimulation of apoptosis of HT-29 cells than did 5-FU alone. In conclusion, KB-34 inhibits the TPA-stimulated metastatic potential of HT-29 cells by induction of HO-1 and may be a promising anti-cancer agent in chemotherapeutic strategies for CRC.
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http://dx.doi.org/10.1016/j.ejphar.2018.10.011DOI Listing
December 2018

Rational engineering of the shikimate and related pathways in Corynebacterium glutamicum for 4-hydroxybenzoate production.

J Biotechnol 2018 Sep 19;282:92-100. Epub 2018 Jul 19.

Major in Food Science & Biotechnology, School of Food Biotechnology & Nutrition, Kyungsung University, 309, Suyeong-ro, Nam-gu, Busan, 48434, Republic of Korea. Electronic address:

4-Hydroxybenzoate (4HBA) is a valuable platform intermediate for the production of commodity and fine chemicals, including protocatechuate, cis,cis-muconic acid, adipic acid, terephthalic acid, phenol, vanillin, and 4-hydroxybenzyl alcohol glycoside (gastrodin). Here we describe rational engineering of the shikimate and related pathways in Corynebacterium glutamicum ATCC13032 for over-producing 4HBA. As an approach to increase the carbon flux to 4HBA, we first introduced a mutated chorismate-pyruvate lyase (CPL) and feedback-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthases encoded by ubiC and aroF/aroG, respectively, from Escherichia coli along with blockage of carbon flux to the biosynthetic pathways for aromatic amino acids and the catabolic pathway for 4HBA by deletion of the genes trpE (encoding anthranilate synthase I), csm (chorismate mutase), and pobA (4HBA hydroxylase). In particular, CPL less sensitive to product inhibition was incorporated into the microorganism to enhance the conversion of chorismate to 4HBA. The subsequent steps involved expression of aroE (shikimate kinase) and aroCKB in the shikimate pathway and deletion of qsuABD coding for enzymes involved in the quinate/shikimate degradation pathway. Finally, to reduce accumulation of pathway intermediates, shikimate and 3-dehydroshikimate, shikimate-resistant AroK from Methanocaldococcus jannaschii was introduced. The resulting strain was shown to produce 19.0 g/L (137.6 mM) of 4HBA with a molar yield of 9.65% after 65 h in a fed-batch fermentation. The engineered strain can also be effectively applied for the production of other products derived from the shikimate pathway.
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http://dx.doi.org/10.1016/j.jbiotec.2018.07.016DOI Listing
September 2018

Repeat Module-Based Rational Design of a Photoswitchable Protein for Light-Driven Control of Biological Processes.

Angew Chem Int Ed Engl 2018 08 23;57(34):10859-10863. Epub 2018 Jul 23.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.

Light-driven control of biological processes using photoswitchable proteins allows high spatiotemporal interrogation or manipulation of such processes, assisting in understanding their functions. Despite considerable advances, however, the wide spread use of optical control has been hampered by a limited repertoire of photoswitchable proteins and a lack of generalized design strategy. Herein, we present a repeat module-based rational design of a photoswitchable protein composed of LRR (Leucine-rich repeat) modules using azobenzene as a photochromic ligand. Our design approach involves the rational selection of a C pair between two nearby modules within a convex region and subsequent cross-linking with a photochromic ligand. We demonstrate the general utility and potential of our strategy by showing the design of three target-specific photoswitchable proteins and a light-driven modulation of the cell signaling. With an abundance of LRR proteins in nature, our approach can expand the repertoire of photoswitchable proteins for light-driven control of biological processes.
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http://dx.doi.org/10.1002/anie.201803993DOI Listing
August 2018

Selective Wavelength Plasmonic Flash Light Welding of Silver Nanowires for Transparent Electrodes with High Conductivity.

ACS Appl Mater Interfaces 2018 Jul 5;10(28):24099-24107. Epub 2018 Jul 5.

Department of Mechanical Engineering , Hanyang University , 17 Haengdang-Dong , Seongdong-Gu, Seoul 133-791 , South Korea.

In this work, silver nanowires (AgNWs) printed on a polyethylene terephthalate substrate using a bar coater were welded via selective wavelength plasmonic flash light irradiation. To achieve high electrical conductivity and transparent characteristics, the wavelength of the flash white light was selectively chosen and irradiated by using high-pass, low-pass, and band-pass filters. The flash white light irradiation conditions such as on-time, off-time, and number of pulses were also optimized. The wavelength range (400-500 nm) corresponding to the plasmonic wavelength of the AgNW could efficiently weld the AgNW films and enhance its conductivity. To carry out in-depth study of the welding phenomena with respect to wavelength, a multiphysics COMSOL simulation was conducted. The welded AgNW films under selective plasmonic flash light welding conditions showed the lowest sheet resistance (51.275 Ω/sq) and noteworthy transmittance (95.3%). Finally, the AgNW film, which was welded by selective wavelength plasmonic flash light with optical filters, was successfully used to make a large area transparent heat film and dye-sensitized solar cells showing superior performances.
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http://dx.doi.org/10.1021/acsami.8b03917DOI Listing
July 2018

A dimeric form of a small-sized protein binder exhibits enhanced anti-tumor activity through prolonged blood circulation.

J Control Release 2018 06 23;279:282-291. Epub 2018 Apr 23.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea. Electronic address:

Small-sized non-antibody scaffolds have attracted considerable interest as alternatives to immunoglobulin antibodies. However, their short half-life is considered a drawback in the development of therapeutic agents. Here we demonstrate that a homo-dimeric form of a repebody enhances the anti-tumor activity than a monomeric form through prolonged blood circulation. Spytag and spycatcher were genetically fused to the C-terminus of a respective human IL-6-specific repebody, and the resulting two repebody constructs were mixed at an equimolar ratio to produce a homo-dimeric form through interaction between spytag and spycatcher. The homo-dimeric repebody was detected as a single band in the SDS-PAGE analysis with an expected molecular size (78 kDa), showing high stability and homogeneity. The dimeric repebody was shown to simultaneously accommodate two hIL-6 molecules, and its binding affinity for hIL-6 was estimated to be comparable to a monomeric repebody. The serum concentration of the dimeric repebody was observed to be about 5.5 times higher than a monomeric repebody, consequently leading to considerably higher tumor suppression effect in human tumor xenograft mice. The present approach can be effectively used for prolonging the blood half-life of small-sized protein binders, resulting in enhanced therapeutic efficacy.
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http://dx.doi.org/10.1016/j.jconrel.2018.04.039DOI Listing
June 2018

Electrostatically assembled dendrimer complex with a high-affinity protein binder for targeted gene delivery.

Int J Pharm 2018 Jun 11;544(1):39-45. Epub 2018 Apr 11.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea. Electronic address:

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas.
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http://dx.doi.org/10.1016/j.ijpharm.2018.04.015DOI Listing
June 2018

Large area growth of MoTe films as high performance counter electrodes for dye-sensitized solar cells.

Sci Rep 2018 01 8;8(1):29. Epub 2018 Jan 8.

Graphene Research Institute, Sejong University, Seoul, 143-747, Republic of Korea.

A cost effective and efficient alternative counter electrode (CE) to replace commercially existing platinum (Pt)-based CEs for dye-sensitized solar cells (DSSCs) is necessary to make DSSCs competitive. Herein, we report the large-area growth of molybdenum telluride (MoTe) thin films by sputtering-chemical vapor deposition (CVD) on conductive glass substrates for Pt-free CEs of DSSCs. Cyclic voltammetry (CV), Tafel curve analysis, and electrochemical impedance spectroscopy (EIS) results showed that the as-synthesized MoTe exhibited good electrocatalytic properties and a low charge transfer resistance at the electrolyte-electrode interface. The optimized MoTe CE revealed a high power conversion efficiency of 7.25% under a simulated solar illumination of 100 mW cm (AM 1.5), which was comparable to the 8.15% observed for a DSSC with a Pt CE. The low cost and good electrocatalytic properties of MoTe thin films make them as an alternative CE for DSSCs.
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http://dx.doi.org/10.1038/s41598-017-18067-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758823PMC
January 2018