Conversion of Host Cell Receptor into Virus Destructor by Immunodisc to Neutralize Diverse SARS-CoV-2 Variants.

The decreasing efficacy of antiviral drugs due to viral mutations highlights the challenge of developing a single agent targeting multiple strains. Using host cell viral receptors as competitive inhibitors is promising, but their low potency and membrane-bound nature have limited this strategy. In this study, the authors show that angiotensin-converting enzyme 2 (ACE2) in a planar membrane patch can effectively neutralize all tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that emerged during the COVID-19 pandemic. The ACE2-incorporated membrane patch implemented using nanodiscs replicated the spike-mediated membrane fusion process outside the host cell, resulting in virus lysis, extracellular RNA release, and potent antiviral activity. While neutralizing antibodies became ineffective as the SARS-CoV-2 evolved to better penetrate host cells the ACE2-incorporated nanodiscs became more potent, highlighting the advantages of using receptor-incorporated nanodiscs for antiviral purposes. ACE2-incorporated immunodisc, an Fc fusion nanodisc developed in this study, completely protected humanized mice infected with SARS-CoV-2 after prolonged retention in the airways. This study demonstrates that the incorporation of viral receptors into immunodisc transforms the entry gate into a potent virucide for all current and future variants, a concept that can be extended to different viruses.

In situ photo-crosslinkable hyaluronic acid-based hydrogel embedded with GHK peptide nanofibers for bioactive wound healing.

A versatile hydrogel was developed for enhancing bioactive wound healing by introducing the amphiphilic GHK peptide (GHK-C16) into a photo-crosslinkable tyramine-modified hyaluronic acid (HA-Ty). GHK-C16 self-assembled into GHK nanofibers (GHK NF) in HA-Ty solution, which underwent in situ gelation after the wound area was filled with precursor solution. Blue light irradiation (460-490 nm), with riboflavin phosphate as a photoinitiator, was used to trigger crosslinking, which enhanced the stability of the highly degradable hyaluronic acid and enabled sustained release of the nanostructured GHK derivatives. The hydrogels provided a microenvironment that promoted the proliferation of dermal fibroblasts and the activation of cytokines, leading to reduced inflammation and increased collagen expression during wound healing. The complexation of Cu2+ into GHK nanofibers resulted in superior wound healing capabilities compared with non-lipidated GHK peptide with a comparable level of growth factor (EGF). Additionally, nanostructured Cu-GHK improved angiogenesis through vascular endothelial growth factor (VEGF) activation, which exerted a synergistic therapeutic effect. Furthermore, in vivo wound healing experiments revealed that the Cu-GHK NF/HA-Ty hydrogel accelerated wound healing through densely packed remodeled collagen in the dermis and promoting the growth of denser fibroblasts. HA-Ty hydrogels incorporating GHK NF also possessed improved mechanical properties and a faster wound healing rate, making them suitable for advanced bioactive wound healing applications. STATEMENT OF SIGNIFICANCE: By combining photo-crosslinkable tyramine-modified hyaluronic acid with self-assembled Cu-GHK-C16 peptide nanofibers (Cu-GHK NF), the Cu-GHK NF/HA-Ty hydrogel offers remarkable advantages over conventional non-structured Cu-GHK for wound healing. It enhances cell proliferation, migration, and collagen remodeling-critical factors in tissue regeneration. The incorporation of GHK nanofibers complexed with copper ions imparts potent anti-inflammatory effects, promoting cytokine activation and angiogenesis during wound healing. The Cu-GHK NF/hydrogel's unique properties, including in situ photo-crosslinking, ensure high customization and potency in tissue regeneration, providing a cost-effective alternative to growth factors. In vivo experiments further validate its efficacy, demonstrating significant wound closure, collagen remodeling, and increased fibroblast density. Overall, the Cu-GHK NF/HA-Ty hydrogel represents an advanced therapeutic option for wound healing applications.

M13KO7 bacteriophage enables Potato Virus Y detection.

IMPORTANCE: In this study, we confirmed the binding of M13KO7 to Potato virus Y (PVY) using enzyme-linked immunosorbent assay. M13KO7 is a "bald" bacteriophage in which no recombinant antibody is displayed. M13KO7 is easy to propagate by using Escherichia coli, making this method more reasonable in economic perspective. Based on this study, we suggest that M13KO7 detection system has applicability as a novel biological tool for the detection of PVY.

Synthesis and physicochemical characterization of acyl myricetins as potential anti-neuroexocytotic agents.

Acyl myricetins (monopropionyl-, dipropionyl-, and monooctanoyl-myricetin, termed as MP1, MP2, and MO1, respectively) were synthesized through enzymatic or non-enzymatic esterification reaction of myricetin aglycone. Structure study indicated the hydroxyl group at C4' in B-ring was highly susceptible to acylation. Over its parental myricetin, acylated compounds showed enhanced lipophilicity (from 7.4- to 26.3-fold) and oxidative stability (from 1.9- to 3.1-fold) on the basis of logP and decay rate, respectively. MO1, presenting the physicochemical superiority compared to the others, provided lowest EC50 value of 2.51 µM on inhibition of neutrotransmitter release and CC50 value of 59.0 µM, leading to widest therapeutic window. All myricetin esters did not show any irritation toxicity when assessed with a chicken embryo assay. This study describes information on acylation of myricetin that has not yet been explored, and suggests that MO1 has membrane fusion-arresting and anti-neuroexocytotic potential for industrial application due to its enhanced biological properties.

Bifunctional hetero di-disc for broad-spectrum influenza neutralization.

Nanodiscs containing sialic acid, which binds the hemagglutinin of the influenza virus, rupture the viral envelope and entrap viral ribonucleoproteins in the endolysosome. While nanodiscs are potent antiviral platforms, ganglioside GD1a containing α2,3-sialic acid does not cover all virus strains. When two nanodiscs containing different receptors 6'-sialyllactose and GD1a were mixed, one nanodisc inhibited the function of the other. A nanodisc loaded with two different receptors exhibited a biased activity toward only one receptor precluding the generation of a multifunctional nanodisc. Here, we suggest hetero di-disc, in which two nanodiscs loaded with each receptor were conjugated through protein trans-splicing for a broad-spectrum antiviral. The hetero di-disc showed strong antiviral activity in vitro and in vivo. Our results suggested that hetero di-discs not only expanded the inhibitory spectrum of nanodiscs but also enabled nanodisc-based delivery of multiple ligands without interference.

Nanodisc-Mediated Conversion of Virustatic Antiviral Antibody to Disrupt Virus Envelope in Infected Cells.

Many antibody-based antivirals, including broadly neutralizing antibodies (bnAbs) against various influenza virus strains, suffer from limited potency. A booster of the antiviral activity of an antibody is expected to facilitate development of antiviral therapeutics. In this study, a nanodisc (ND), a discoidal lipid bilayer encircled by membrane scaffold proteins, is engineered to provide virucidal properties to antibodies, thereby augmenting their antiviral activity. NDs carrying the Fc-binding peptide sequence form an antibody-ND complex (ANC), which can co-endocytose into cells infected with influenza virus. ANC efficiently inhibits endosome escape of viral RNA by dual complimentary mode of action. While the antibody moiety in an ANC inhibits hemagglutinin-mediated membrane fusion, its ND moiety destroys the viral envelope using free hemagglutinins that are not captured by antibodies. Providing virus-infected host cells with the ability to self-eliminate by the synergistic effect of ANC components dramatically amplifies the antiviral efficacy of a bnAb against influenza virus. When the efficacy of ANC is assessed in mouse models, administration of ANCs dramatically reduces morbidity and mortality compared to bnAb alone. This study is the first to demonstrate the novel nanoparticle ANC and its role in combating viral infections, suggesting that ANC is a versatile platform applicable to various viruses.

Synthesis of Caffeoyl-Prolyl-Histidyl-Xaa Derivatives and Evaluation of Their Activities and Stability upon Long-Term Storage.

Antioxidants play a critical role in the treatment of degenerative diseases and delaying the aging of dermal tissue. Caffeic acid (CA) is a representative example of the antioxidants found in plants. However, CA is unsuitable for long-term storage because of its poor stability under ambient conditions. Caffeoyl-Pro-His-NH2 (CA-Pro-His-NH2, CA-PH) exhibits the highest antioxidant activity, free radical scavenging and lipid peroxidation inhibition activity among the histidine-containing CA-conjugated dipeptides reported to date. The addition of short peptides to CA, such as Pro-His, is assumed to synergistically enhance its antioxidative activity. In this study, several caffeoyl-prolyl-histidyl-Xaa-NH2 derivatives were synthesized and their antioxidative activities evaluated. CA-Pro-His-Asn-NH2 showed enhanced antioxidative activity and higher structural stability than CA-PH, even after long-term storage. CA-Pro-His-Asn-NH2 was stable for 3 months, its stability being evaluated by observing the changes in its NMR spectra. Moreover, the solid-phase synthetic strategy used to prepare these CA-Pro-His-Xaa-NH2 derivatives was optimized for large-scale production. We envision that CA-Pro-His-Xaa-NH2 derivatives can be used as potent dermal therapeutic agents and useful cosmetic ingredients.

Development of End-Spliced Dimeric Nanodiscs for the Improved Virucidal Activity of a Nanoperforator.

Lipid-bilayer nanodiscs (NDs) wrapped in membrane scaffold proteins (MSPs) have primarily been used to study membrane proteins of interest in a physiological environment. Recently, NDs have been employed in broader applications including drug delivery, cancer immunotherapy, bio-imaging, and therapeutic virucides. Here, we developed a method to synthesize a dimeric nanodisc, whose MSPs are circularly end-spliced, with long-term thermal stability and resistance to aggregation. The end-spliced nanodiscs (esNDs) were assembled using MSPs that were self-circularized inside the cytoplasm ofEscherichia colivia highly efficient protein trans-splicing. The esNDs demonstrated a consistent size and 4-5-fold higher stability against heat and aggregation than conventional NDs. Moreover, cysteine residues on trans-spliced circularized MSPs allowed us to modulate the formation of either monomeric nanodiscs (essNDs) or dimeric nanodiscs (esdNDs) by controlling the oxidation/reduction conditions and lipid-to-protein ratios. When the esdNDs were used to prepare an antiviral nanoperforator that induced the disruption of the viral membrane upon contact, antiviral activity was dramatically increased, suggesting that the dimerization of nanodiscs led to cooperativity between linked nanodiscs. We expect that controllable structures, long-term stability, and aggregation resistance of esNDs will aid the development of novel versatile membrane-mimetic nanomaterials with flexible designs and improved therapeutic efficacy.

Tannic acid-functionalized HEPA filter materials for influenza virus capture.

Influenza, one of the most contagious and infectious diseases, is predominantly transmitted through aerosols, leading to the development of filter-based protective equipment. Though the currently available filters are effective at removing submicron-sized particulates, filter materials with enhanced virus-capture efficiency are still in demand. Coating or chemically modifying filters with molecules capable of binding influenza viruses has received attention as a promising approach for the production of virus-capturing filters. For this purpose, tannic acid (TA), a plant-derived polyphenol, is a promising molecule for filter functionalization because of its antiviral activities and ability to serve as a cost-efficient adhesive for various materials. This study demonstrates the facile preparation of TA-functionalized high-efficiency particulate air (HEPA) filter materials and their efficiency in influenza virus capture. Polypropylene HEPA filter fabrics were coated with TA via a dipping/washing process. The TA-functionalized HEPA filter (TA-HF) exhibits a high in-solution virus capture efficiency of up to 2,723 pfu/mm2 within 10 min, which is almost two orders of magnitude higher than that of non-functionalized filters. This result suggests that the TA-HF is a potent anti-influenza filter that can be used in protective equipment to prevent the spread of pathogenic viruses.

Mechanisms of Resorcinol Antagonism of Benzo[a]pyrene-Induced Damage to Human Keratinocytes.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and ubiquitous environmental toxin with known harmful effects to human health. Abnormal phenotypes of keratinocytes are closely associated with their exposure to B[a]P. Resorcinol is a component of argan oil with reported anticancer activities, but its mechanism of action and potential effect on B[a]P damage to the skin is unknown. In this study, we investigated the effects of resorcinol on B[a]P-induced abnormal keratinocyte biology and its mechanisms of action in human epidermal keratinocyte cell line HaCaT. Resorcinol suppressed aryl hydrocarbon receptor (AhR) activity as evidenced by the inhibition of B[a]P-induced xenobiotic response element (XRE)-reporter activation and cytochrome P450 1A1 (CYP1A1) expression. In addition, resorcinol attenuated B[a]P-induced nuclear translocation of AhR, and production of ROS and pro-inflammatory cytokines. We also found that resorcinol increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activity. Antioxidant response element (ARE)-reporter activity and expression of ARE-dependent genes NAD(P)H dehydrogenase [quinone] 1 (NQO1), heme oxygenase-1 (HO-1) were increased by resorcinol. Consistently, resorcinol treatment induced nuclear localization of Nrf2 as seen by Western analysis. Knockdown of Nrf2 attenuated the resorcinol effects on ARE signaling, but knockdown of AhR did not affect resorcinol activation of Nrf2. This suggests that activation of antioxidant activity by resorcinol is not mediated by AhR. These results indicate that resorcinol is protective against effects of B[a]P exposure. The mechanism of action of resorcinol is inhibition of AhR and activation of Nrf2-mediated antioxidant signaling. Our findings suggest that resorcinol may have potential as a protective agent against B[a]P-containing pollutants.

Filamentous anti-influenza agents wrapping around viruses.

Owing to the emerging resistance to current anti-influenza therapies, strategies for blocking virus-cell interaction with agents that mimic interactions with host cell receptors are garnering interest. In this context, a multivalent presentation of sialyl groups on various types of scaffold materials such as dendrimers, liposomes, nanoparticles, and natural/synthetic polymers has been investigated for the inhibition of influenza A virus infection. However, the development of versatile antiviral agents based on monodisperse scaffolds capable of precise molecular design remains challenging. Whether an anisotropically extended filamentous nanostructure can serve as an effective scaffold for maximum inhibition of viral cell attachment has not been investigated. In this study, the preparation of a series of sialyllactose-conjugated filamentous bacteriophages (SLPhages), with controlled loading levels, ligand valencies, and two types of sialyllactose (α2,3' and α2,6'), is demonstrated. With optimal ligand loading and valency, SLPhages showed inhibitory activity (in vitro) against influenza A viruses at concentrations of tens of picomolar. This remarkable inhibition is due to the strong interaction between the SLPhage and the virus; this interaction is adequately potent to compensate for the cost of the bending and wrapping of the SLPhage around the influenza virus. Our study may open new avenues for the development of filamentous anti-viral agents, in which virus-wrapping or aggregation is the primary feature responsible for the blocking of cell entry.

Self-Assembled Multi-Epitope Peptide Amphiphiles Enhance the Immune Response against Enterovirus 71.

Subunit vaccines consist of non-genetic material, such as peptides or proteins. They are considered safe because they have fewer side effects; however, they have low immunogenicity when used alone. We aimed to enhance the immune response of peptide-based vaccines by using self-assembled multimeric peptide amphiphiles (PAs). We designed two epitope PAs by conjugating epitope peptides from Enterovirus 71 (EV71) virus particle (VP) 1 and VP3 capsid proteins with different fatty acid chain lengths (VP1PA and VP3PA). These PAs self-assembled into supramolecular structures at a physiological pH, and the resulting structures were characterized using atomic force microscopy. Multi-epitope PAs (m-PAs) consisted of a 1:1 mixture of VP1PA and VP3PA solutions. To evaluate immunogenicity, m-PA constructs were injected with adjuvant subcutaneously into female Balb/c mice. Levels of antigen-specific immunoglobulin G (IgG) and IgG1 in m-PA-injected mice serum samples were analyzed using ELISA and Western blotting. Additionally, cytokine production stimulated by each antigen was measured in splenocytes cultured from immunized mice groups. We found that m-PA showed improved humoral and cellular immune responses compared to the control and peptide groups. The sera from m-PA immunized mice group could neutralize EV71 infection and protect host cells. Thus, self-assembled m-PAs can promote a protective immune response and can be developed as a potential platform technology to produce peptide vaccines against infectious viral diseases.

Large-Scale Assembly of Peptide-Based Hierarchical Nanostructures and Their Antiferroelectric Properties.

An effective strategy is developed to create peptide-based hierarchical nanostructures through the meniscus-driven self-assembly in a large area and fabricate antiferroelectric devices based on these nanostructures for the first time. The diphenylalanine hierarchical nanostructures (FF-HNs) are self-assembled by vertically pulling a substrate from a diphenylalanine (FF) solution dissolved in a miscible solvent under precisely controlled conditions. Owing to the unique structural properties of FF nanostructures, including high crystallinity and α-helix structures, FF-HNs possess a net electrical dipole moment, which can be switched in an external electric field. The mass production of antiferroelectric devices based on FF-HNs can be successfully achieved by means of this biomimetic assembly technique. The devices show an evident antiferroelectric to ferroelectric transition under dark conditions, while the ferroelectricity is found to be tunable by light. Notably, it is discovered that the modulation of antiferroelectric behaviors of FF-HNs under glutaraldehyde exposure is due to the FF molecules that are transformed into cyclophenylalanine by glutaraldehyde. This work provides a stepping stone toward the mass production of self-assembled hierarchical nanostructures based on biomolecules as well as the mass fabrication of electronic devices based on biomolecular nanostructures for practical applications.

Robust Magnetized Graphene Oxide Platform for In Situ Peptide Synthesis and FRET-Based Protease Detection.

Graphene oxide (GO)/peptide complexes as a promising disease biomarker analysis platform have been used to detect proteolytic activity by observing the turn-on signal of the quenched fluorescence upon the release of peptide fragments. However, the purification steps are often cumbersome during surface modification of nano-/micro-sized GO. In addition, it is still challenging to incorporate the specific peptides into GO with proper orientation using conventional immobilization methods based on pre-synthesized peptides. Here, we demonstrate a robust magnetic GO (MGO) fluorescence resonance energy transfer (FRET) platform based on in situ sequence-specific peptide synthesis of MGO. The magnetization of GO was achieved by co-precipitation of an iron precursor solution. Magnetic purification/isolation enabled efficient incorporation of amino-polyethylene glycol spacers and subsequent solid-phase peptide synthesis of MGO to ensure the oriented immobilization of the peptide, which was evaluated by mass spectrometry after photocleavage. The FRET peptide MGO responded to proteases such as trypsin, thrombin, and ß-secretase in a concentration-dependent manner. Particularly, ß-secretase, as an important Alzheimer's disease marker, was assayed down to 0.125 ng/mL. Overall, the MGO platform is applicable to the detection of other proteases by using various peptide substrates, with a potential to be used in an automated synthesis system operating in a high throughput configuration.

Reduction of focal sweating by lipid nanoparticle-delivered myricetin.

Myricetin-a flavonoid capable of inhibiting the SNARE complex formation in neurons-reduces focal sweating after skin-application when delivers as encapsulated in lipid nanoparticles (M-LNPs). The stability of M-LNP enables efficient delivery of myricetin to sudomotor nerves located underneath sweat glands through transappendageal pathways while free myricetin just remained on the skin. Furthermore, release of myricetin from M-LNP is accelerated through lipase-/esterase-induced lipolysis in the skin-appendages, enabling uptake of myricetin by the surrounding cells. The amount of sweat is reduced by 55% after application of M-LNP (0.8 mg kg-1) on the mouse footpad. This is comparable to that of subcutaneously injected anticholinergic agents [0.25 mg kg-1 glycopyrrolate; 0.8 U kg-1 botulinum neurotoxin-A-type (BoNT/A)]. M-LNP neither shows a distal effect after skin-application nor induced cellular/ocular toxicity. In conclusion, M-LNP is an efficient skin-applicable antiperspirant. SNARE-inhibitory small molecules with suitable delivery systems have the potential to replace many BoNT/A interventions for which self-applications are preferred.

Envelope-deforming antiviral peptide derived from influenza virus M2 protein.

Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity () of M2 AH was not related to antiviral activity, modification of the hydrophobic moment (<µH>) of M2 AH dramatically altered the antiviral activity of this peptide. M2 MH, a derivative of M2 AH with a <µH> of 0.874, showed a half maximal inhibitory concentration (IC50) of 53.3 nM against the A/PR/8/34 strain (H1N1), which is 16-times lower than that of M2 AH. The selectivity index (IC50/CC50), where CC50 is the half maximal cytotoxic concentration, was 360 for M2 MH and 81 for M2 AH. Dynamic light scattering spectroscopy and electron microscopy revealed that M2 AH-derived peptides did not disrupt liposomes but altered the shape of viruses. This result suggests that the shape of virus envelope was closely related to its activity. Thus, we propose that deforming without rupturing the membranes may achieve a high selectivity index for peptide antivirals.

Virucidal nano-perforator of viral membrane trapping viral RNAs in the endosome.

Membrane-disrupting agents that selectively target virus versus host membranes could potentially inhibit a broad-spectrum of enveloped viruses, but currently such antivirals are lacking. Here, we develop a nanodisc incorporated with a decoy virus receptor that inhibits virus infection. Mechanistically, nanodiscs carrying the viral receptor sialic acid bind to influenza virions and are co-endocytosed into host cells. At low pH in the endosome, the nanodiscs rupture the viral envelope, trapping viral RNAs inside the endolysosome for enzymatic decomposition. In contrast, liposomes containing a decoy receptor show weak antiviral activity due to the lack of membrane disruption. The nanodiscs inhibit influenza virus infection and reduce morbidity and mortality in a mouse model. Our results suggest a new class of antivirals applicable to other enveloped viruses that cause irreversible physical damage specifically to virus envelope by viruses' own fusion machine. In conclusion, the lipid nanostructure provides another dimension for antiviral activity of decoy molecules.

Growth of Au and ZnS nanostructures via engineered peptide and M13 bacteriophage templates.

We demonstrate directed nucleation of Au and ZnS patterns on templates comprised of functional peptides and an M13 bacteriophage. We discuss the control over nucleation in terms of the interplay between enhanced ion binding and reduced interfacial energy resulting from the presence of the templates.

Cellulose Nanocrystal-Based Colored Thin Films for Colorimetric Detection of Aldehyde Gases.

We demonstrate a controllable and reliable process for manifesting color patterns on solid substrates using cellulose nanocrystals (CNCs) without the use of any other chemical pigments. The color can be controlled by adjusting the assembly conditions of the CNC solution during a dip-and-pull process while aiding the close packing of CNCs on a solid surface with the help of ionic-liquid (1-butyl-3-methylimidazolium) molecules that screen the repelling electrostatic charges between CNCs. By controlling the pulling speed from 3 to 9 µm/min during the dip-and-pull process, we were able to control the film thickness from 100 to 300 nm, resulting in films with different colors in the visible range. The optical properties were in good agreement with the finite-difference time-domain simulation results. By functionalizing these films with amine groups, we developed colorimetric sensors that can change in color when exposed to aldehyde gases such as formaldehyde or propanal. A principal component analysis showed that we can differentiate between different aldehyde gases and other interfering molecules. We expect that our approach will enable inexpensive and rapid volatile organic compound detection with on-site monitoring capabilities.

Engineered Phage Matrix Stiffness-Modulating Osteogenic Differentiation.

Herein, we demonstrate an engineered phage mediated matrix for osteogenic differentiation with controlled stiffness by cross-linking the engineered phage displaying Arg-Gly-Asp (RGD) and His-Pro-Gln (HPQ) with various concentrations of streptavidin or polymer, poly(diallyldimethylammonium)chloride (PDDA). Osteogenic gene expressions showed that they were specifically increased when MC3T3 cells were cultured on the stiffer phage matrix than the softer one. Our phage matrixes can be easily functionalized using chemical/genetic engineering and used as a stem cell tissue matrix stiffness platform for modulating differential cell expansion and differentiation.