17 results on '"Eunkeu Oh"'
Search Results
2. Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems
- Author
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Meghna Thakur, Joyce C. Breger, Kimihiro Susumu, Eunkeu Oh, Joseph R. Spangler, Igor L. Medintz, Scott A. Walper, and Gregory A. Ellis
- Subjects
Medicine ,Science - Abstract
Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.
- Published
- 2022
3. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.
- Author
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Ying Fu, Juliana da Fonseca Rezende E Mello, Bryan D Fleming, Alex Renn, Catherine Z Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T Eastman, Zina Itkin, Thomas B Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D Hall, Mario J Borgnia, and Anton Simeonov
- Subjects
Medicine ,Science - Abstract
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.
- Published
- 2022
- Full Text
- View/download PDF
4. Liquid Crystal Nanoparticle Conjugates for Scavenging Reactive Oxygen Species in Live Cells
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Okhil K. Nag, Jawad Naciri, Kwahun Lee, Eunkeu Oh, Bethany Almeida, and James B. Delehanty
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liquid crystal nanoparticles ,ROS scavenger ,oxidative stress ,TEMPO ,lipid peroxidation ,reactive oxygen species ,Medicine ,Pharmacy and materia medica ,RS1-441 - Abstract
The elevated intracellular production of or extracellular exposure to reactive oxygen species (ROS) causes oxidative stress to cells, resulting in deleterious irreversible biomolecular reactions (e.g., lipid peroxidation) and disease progression. The use of low-molecular weight antioxidants, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), as ROS scavengers fails to achieve the desired efficacy because of their poor or uncontrolled cellular uptake and off-target effects, such as dysfunction of essential redox homeostasis. In this study, we fabricated a liquid crystal nanoparticle (LCNP) conjugate system with the fluorescent dye perylene (PY) loaded in the interior and poly (ethylene glycol) (PEG) decorated on the surface along with multiple molecules of TEMPO (PY-LCNP-PEG/TEMPO). PY-LCNP-PEG/TEMPO exhibit enhanced cellular uptake, and efficient ROS-scavenging activity in live cells. On average, the 120 nm diameter PY-LCNPs were conjugated with >1800 molecules of TEMPO moieties on their surface. PY-LCNP-PEG/TEMPO showed significantly greater reduction in ROS activity and lipid peroxidation compared to free TEMPO when the cells were challenged with ROS generating agents, such as hydrogen peroxide (H2O2). We suggest that this is due to the increased local concentration of TEMPO molecules on the surface of the PY-LCNP-PEG/TEMPO NPs, which efficiently bind to the plasma membrane and enter cells. Overall, these results demonstrate the enhanced capability of TEMPO-conjugated LCNPs to protect live cells from oxidative stress by effectively scavenging ROS and reducing lipid peroxidation.
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- 2022
- Full Text
- View/download PDF
5. Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers
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Katherine E. Rogers, Okhil K. Nag, Kimihiro Susumu, Eunkeu Oh, and James B. Delehanty
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Pharmacology ,Organic Chemistry ,Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,Biotechnology - Published
- 2023
6. Seedless Synthesis of Disulfide-Grafted Gold Nanoflowers with Size and Shape Control and Their Photothermally Mediated Cell Perforation
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Ajmeeta Sangtani, Kwahun Lee, Okhil K. Nag, Kimihiro Susumu, R. Joseph Weiblen, Mijin Kim, Igor Vurgaftman, Sz-Chian Liou, James B. Delehanty, and Eunkeu Oh
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General Chemical Engineering ,Materials Chemistry ,General Chemistry - Published
- 2022
7. Benzaldehyde Lyase Kinetic Improvements, Potential Channeling to Alcohol Dehydrogenase, and Substrate Scope when Immobilized on Semiconductor Quantum Dots
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Shelby Hooe, Joyce Breger, Scott Dean, Kimihiro Susumu, Eunkeu Oh, Scott Walper, Gregory A. Ellis, and Igor L. Medintz
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General Materials Science - Published
- 2022
8. Mechanistic Understanding of DNA Denaturation in Nanoscale Thermal Gradients Created by Femtosecond Excitation of Gold Nanoparticles
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David A. Hastman, Parth Chaturvedi, Eunkeu Oh, Joseph S. Melinger, Igor L. Medintz, Lela Vuković, and Sebastián A. Díaz
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Time Factors ,Materials Testing ,Temperature ,Metal Nanoparticles ,Biocompatible Materials ,General Materials Science ,DNA ,Gold ,Molecular Dynamics Simulation ,Nucleic Acid Denaturation - Abstract
There is significant interest in developing photothermal systems that can precisely control the structure and function of biomolecules through local temperature modulation. One specific application is the denaturation of double-stranded (ds) DNA through femtosecond (fs) laser pulse optical heating of gold nanoparticles (AuNPs); however, the mechanism of DNA melting in these systems is not fully understood. Here, we utilize 55 nm AuNPs with surface-tethered dsDNA, which are locally heated using fs laser pulses to induce DNA melting. By varying the dsDNA distance from the AuNP surface and the laser pulse energy fluence, this system is used to study how the nanosecond duration temperature increase and the steep temperature gradient around the AuNP affect dsDNA dehybridization. Through modifying the distance between the dsDNA and AuNP surface by 3.8 nm in total and the pulse energy fluence from 7.1 to 14.1 J/m
- Published
- 2022
9. Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease
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María Elisa Vázquez, Andrea Cecilia Mesías, Leonardo Acuña, Joseph Spangler, Brenda Zabala, Cecilia Parodi, Meghna Thakur, Eunkeu Oh, Scott Allan Walper, and Cecilia Pérez Brandán
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Microbiology (medical) ,Trypanosoma cruzi ,vaccine ,outer membrane vesicle - Abstract
BACKGROUND Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.
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- 2023
10. Enzyme Assembly on Nanoparticle Scaffolds Enhances Cofactor Recycling and Improves Coupled Reaction Kinetics
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Joyce C. Breger, Ellen R Goldman, Kimihiro Susumu, Eunkeu Oh, Christopher M Green, Shelby Hooe, Meghna Thakur, Igor Medintz, and Gregory A. Ellis
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General Materials Science - Abstract
Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme...
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- 2023
11. Fluorescent quantum dots enable SARS-CoV-2 antiviral drug discovery and development
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Kirill Gorshkov, Mason A. Wolak, Eunkeu Oh, and Kimihiro Susumu
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SARS-CoV-2 ,medicine.drug_class ,viruses ,Computational biology ,Biology ,medicine.disease_cause ,Antiviral Agents ,Small molecule ,Virus ,COVID-19 Drug Treatment ,Drug repositioning ,Immune system ,Quantum dot ,Biosafety level ,Quantum Dots ,Drug Discovery ,medicine ,Humans ,Antiviral drug ,Coronavirus - Abstract
Introduction SARS-CoV-2 is a highly infectious and deadly coronavirus whose study requires the use of a biosafety level 3 (BSL-3) containment facility to investigate viral biology and pathogenesis, which limits the study of live virus and slows progress towards finding suitable treatments for infection. While vaccines from several companies have proven very effective in combating the virus, few treatments exist for those who do succumb to the viral-induced systemic disease called COVID-19. Areas covered This short review focuses on fluorescent quantum dot-based modeling of SARS-CoV-2. New BSL-2 viral models are essential to find small molecules and biologics that may be effective in stopping viral infection as well as treating already infected individuals. Nanoparticles are invaluable tools for biological research as they can be used to both modeling pathogens and serve as a platform for developing vaccines. Expert opinion Visualizing viral activity with fluorescent quantum dots enables both biochemical and cell-based assays to detect virus-host receptor interactions, cellular activity after binding to cell plasma membrane, screening for interventions using small molecule drug repurposing, and testing of novel biologics. Quantum dots can also be used for diagnostic assays, vaccine development, and importantly, pan-antiviral drugs to address variants that may escape the immune response.
- Published
- 2021
12. Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors
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Divita Mathur, Meghna Thakur, Sebastián A. Díaz, Kimihiro Susumu, Michael H. Stewart, Eunkeu Oh, Scott A. Walper, and Igor L. Medintz
- Subjects
Biomedical Engineering ,General Medicine ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Article - Abstract
Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme’s cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.
- Published
- 2022
13. Microbial survival and growth on <scp>non‐corrodible</scp> conductive materials
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Leonard M. Tender, Sarah M. Glaven, Eunkeu Oh, Brian J. Eddie, Daniel A. Phillips, and Lina J. Bird
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Microbial fuel cell ,Bioelectric Energy Sources ,Open-circuit voltage ,Carbon fixation ,Electric Conductivity ,Biofilm ,Biology ,Chromatiaceae ,Microbiology ,Redox ,Corrosion ,Chemical engineering ,Biofilms ,Electrode ,Graphite ,Electrodes ,Oxidation-Reduction ,Ecology, Evolution, Behavior and Systematics - Abstract
Biofilms growing aerobically on conductive substrates are often correlated with a positive, sustained shift in their redox potential. This phenomenon has a beneficial impact on microbial fuel cells by increasing their overall power output but can be detrimental when occurring on stainless steel by enhancing corrosion. The biological mechanism behind this potential shift is unresolved and a metabolic benefit to cells has not been demonstrated. Here, biofilms containing the electroautotroph 'Candidatus Tenderia electrophaga' catalysed a shift in the open circuit potential of graphite and indium tin oxide electrodes by >100 mV. Biofilms on open circuit electrodes had increased biomass and a significantly higher proportion of 'Ca. Tenderia electrophaga' compared to those on plain glass. Addition of metabolic inhibitors showed that living cells were required to maintain the more positive potential. We propose a model to describe these observations, in which 'Ca. Tenderia electrophaga' drives the shift in open circuit potential through electron uptake for oxygen reduction and CO2 fixation. We further propose that the electrode is continuously recharged by oxidation of trace redox-active molecules in the medium at the more positive potential. A similar phenomenon is possible on natural conductive substrates in the environment.
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- 2021
14. Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures
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Christopher M, Green, Divita, Mathur, Kimihiro, Susumu, Eunkeu, Oh, Igor L, Medintz, and Sebastián A, Díaz
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Quantum Dots ,Histidine ,DNA ,Coloring Agents ,Luciferases ,Nanostructures - Abstract
DNA nanostructures self-assemble into almost any arbitrary architecture, and when combined with their capability to precisely position and orient dyes, nanoparticles, and biological moieties, the technology reaches its potential. We present a simple yet multifaceted conjugation strategy based on metal coordination by a multi-histidine peptide tag (Histag). The versatility of the Histag as a means to conjugate to DNA nanostructures is shown by using Histags to capture semiconductor quantum dots (QDs) with numerical and positional precision onto a DNA origami breadboard. Additionally, Histag-expressing enzymes, such as the bioluminescent luciferase, can also be captured to the DNA origami breadboard with similar precision. DNA nanostructure conjugation of the QDs or luciferase is confirmed through imaging and/or energy transfer to organic dyes integrated into the DNA nanostructure.
- Published
- 2022
15. High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells
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Kirill Gorshkov, Mason Wolak, Kimihiro Susumu, Eunkeu Oh, and Bruce Nguyen Tran
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HEK293 Cells ,General Immunology and Microbiology ,SARS-CoV-2 ,General Chemical Engineering ,General Neuroscience ,Quantum Dots ,Spike Glycoprotein, Coronavirus ,Humans ,Endocytosis ,General Biochemistry, Genetics and Molecular Biology - Abstract
The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.
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- 2022
16. Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures
- Author
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Christopher M. Green, Divita Mathur, Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz, and Sebastián A. Díaz
- Published
- 2022
17. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike
- Author
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Ying Fu, Juliana da Fonseca Rezende e Mello, Bryan D. Fleming, Alex Renn, Catherine Z. Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M. Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T. Eastman, Zina Itkin, Thomas B. Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D. Hall, Mario J. Borgnia, and Anton Simeonov
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Mutation ,Multidisciplinary ,biology ,SARS-CoV-2 ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,COVID-19 ,Spike Protein ,Human airway ,Single-Domain Antibodies ,Antibodies, Viral ,medicine.disease_cause ,Antibodies, Neutralizing ,Virology ,Article ,Neutralization ,Spike Glycoprotein, Coronavirus ,medicine ,biology.protein ,Humans ,Bacteriophages ,Spike (software development) ,Antibody ,Ex vivo ,Protein Binding - Abstract
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: A cost-effective, high-throughput, adaptable pipeline capable of identifying effective humanized nanobodies against SARS-CoV-2.
- Published
- 2021
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