Facemask acne attenuation through modulation of indirect microbiome interactions.

During the COVID-19 pandemic, facemasks played a pivotal role in preventing person-person droplet transmission of viral particles. However, prolonged facemask wearing causes skin irritations colloquially referred to as 'maskne' (mask + acne), which manifests as acne and contact dermatitis and is mostly caused by pathogenic skin microbes. Previous studies revealed that the putative causal microbes were anaerobic bacteria, but the pathogenesis of facemask-associated skin conditions remains poorly defined. We therefore characterized the role of the facemask-associated skin microbiota in the development of maskne using culture-dependent and -independent methodologies. Metagenomic analysis revealed that the majority of the facemask microbiota were anaerobic bacteria that originated from the skin rather than saliva. Previous work demonstrated direct interaction between pathogenic bacteria and antagonistic strains in the microbiome. We expanded this analysis to include indirect interaction between pathogenic bacteria and other indigenous bacteria classified as either 'pathogen helper (PH)' or 'pathogen inhibitor (PIn)' strains. In vitro screening of bacteria isolated from facemasks identified both strains that antagonized and promoted pathogen growth. These data were validated using a mouse skin infection model, where we observed attenuation of symptoms following pathogen infection. Moreover, the inhibitor of pathogen helper (IPH) strain, which did not directly attenuate pathogen growth in vitro and in vivo, functioned to suppress symptom development and pathogen growth indirectly through PH inhibitory antibacterial products such as phenyl lactic acid. Taken together, our study is the first to define a mechanism by which indirect microbiota interactions under facemasks can control symptoms of maskne by suppressing a skin pathogen.

Delivery of endolysin across outer membrane of Gram-negative bacteria using translocation domain of botulinum neurotoxin.

The emergence of multidrug-resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the botulinum neurotoxin translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machineries to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 min with only 0.5 µM against an Acinetobacter baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target-binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.

3-Fucosyllactose-mediated modulation of immune response against virus infection.

Viral pathogens, particularly influenza and SARS-CoV-2, pose a significant global health challenge. Given the immunomodulatory properties of human milk oligosaccharides, in particular 2'-fucosyllactose and 3-fucosyllactose (3-FL), we investigated their dietary supplementation effects on antiviral responses in mouse models. This study revealed distinct immune modulations induced by 3-FL. RNA-sequencing data showed that 3-FL increased the expression of interferon receptors, such as Interferon Alpha and Beta Receptor (IFNAR) and Interferon Gamma Receptor (IFNGR), while simultaneously downregulating interferons and interferon-stimulated genes, an effect not observed with 2'-fucosyllactose supplementation. Such modulation enhanced antiviral responses in both cell culture and animal models while attenuating pre-emptive inflammatory responses. Nitric oxide concentrations in 3-FL-supplemented A549 cells and mouse lung tissues were elevated exclusively upon infection, reaching 5.8- and 1.9-fold increases over control groups, respectively. In addition, 3-FL promoted leukocyte infiltration into the site of infection upon viral challenge. 3-FL supplementation provided protective efficacy against lethal influenza challenge in mice. The demonstrated antiviral efficacy spanned multiple influenza strains and extended to SARS-CoV-2. In conclusion, 3-FL is a unique immunomodulator that helps protect the host from viral infection while suppressing inflammation prior to infection.

Design of PD-L1-Targeted Lipid Nanoparticles to Turn on PTEN for Efficient Cancer Therapy.

Lipid nanoparticles (LNPs) exhibit remarkable mRNA delivery efficiency, yet their majority accumulate in the liver or spleen after injection. Tissue-specific mRNA delivery can be achieved through modulating LNP properties, such as tuning PEGylation or varying lipid components systematically. In this paper, a streamlined method is used for incorporating tumor-targeting peptides into the LNPs; the programmed death ligand 1 (PD-L1) binding peptides are conjugated to PEGylated lipids via a copper-free click reaction, and directly incorporated into the LNP composition (Pep LNPs). Notably, Pep LNPs display robust interaction with PD-L1 proteins, which leads to the uptake of LNPs into PD-L1 overexpressing cancer cells both in vitro and in vivo. To evaluate anticancer immunotherapy mediated by restoring tumor suppressor, mRNA encoding phosphatase and tensin homolog (PTEN) is delivered via Pep LNPs to PTEN-deficient triple-negative breast cancers (TNBCs). Pep LNPs loaded with PTEN mRNA specifically promotes autophagy-mediated immunogenic cell death in 4T1 tumors, resulting in effective anticancer immune responses. This study highlights the potential of tumor-targeted LNPs for mRNA-based cancer therapy.

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.

De novo biosynthesis of 2'-fucosyllactose by bioengineered Corynebacterium glutamicum.

2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

Affinity Peptide-Tethered Suspension Hydrogel Sensor for Selective and Sensitive Detection of Influenza Virus.

Influenza viruses are known to cause pandemic flu outbreaks through both inter-human and animal-to-human transmissions. Therefore, the rapid and accurate detection of such pathogenic viruses is crucial for effective pandemic control. Here, we introduce a novel sensor based on affinity peptide-immobilized hydrogel microspheres for the selective detection of influenza A virus (IAV) H3N2. To enhance the binding affinity performance, we identified novel affinity peptides using phage display and further optimized their design. The functional hydrogel microspheres were constructed using the drop microfluidic technique, employing a structure composed of natural (chitosan) and synthetic (poly(ethylene glycol) diacrylate and PEG 6 kDa) polymers with the activation of azadibenzocyclooctyne for the subsequent click chemistry reaction. The binding peptide-immobilized hydrogel microsphere (BP-Hyd) was characterized by field emission scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy and exhibited selective detection capability for the IAV H3N2. To capture the detected IAV H3N2, a Cy3-labeled IAV hemagglutinin antibody was utilized. By incorporating the affinity peptide with hydrogel microspheres, we achieved quantitative and selective detection of IAV H3N2 with a detection limit of 1.887 PFU mL-1. Furthermore, the developed suspension sensor exhibited excellent reproducibility and showed reusability potential. Our results revealed that the BP-Hyd-based fluorescence sensor platform could be feasibly employed to detect other pathogens because the virus-binding peptides can be easily replaced with other peptides through phage display, enabling selective and sensitive binding to different targets.

Korean Red Ginseng Relieves Inflammation and Modulates Immune Response Induced by Pseudo-Type SARS-CoV-2.

Few studies have reported the therapeutic effects of Korean red ginseng (KRG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the positive effects of KRG on other viruses have been reported and the effects of KRG on pulmonary inflammatory diseases have also been studied. Therefore, this study investigated the therapeutic effects of KRG-water extract (KRG-WE) in a pseudo-type SARS-CoV-2 (PSV)-induced lung injury model. Constructing the pseudovirus, human angiotensin-converting enzyme 2 (hACE2) transgenic mice were infected via intranasal injection that had been orally administered with KRG-WE for six weeks. After 7-days post infection (dpi), the antiviral effects of KRG-WE were confirmed, followed by real-time polymerase chain reaction (PCR), western blot analysis, flow cytometric analysis, and an enzyme-linked immunoassay (ELISA). KRG-WE significantly inhibited an increase in immunoglobulin caused by PSV. Furthermore, KRG-WE effectively suppressed alveolar macrophages (AMs) inside the lungs and helped normalize the population of other immune cells. In addition, virus-induced gene expression and inflammatory signals such as nuclear factor-kappa B and other upstream molecules were downregulated. Moreover, KRG-WE also normalized gene expression and protein activity in the spleen. In conclusion, KRG-WE reduced AMs, normalized the immune response, and decreased the expression of inflammatory genes and activation of signaling pathway phosphorylation, thereby exhibiting anti-inflammatory effects and attenuating lung damage.

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein.

The COVID-19 pandemic has significantly impacted human health for three years. To mitigate the spread of SARS-CoV-2, the development of neutralizing antibodies has been accelerated, including the exploration of alternative antibody formats such as single-domain antibodies. In this study, we identified variable new antigen receptors (VNARs) specific for the receptor binding domain (RBD) of SARS-CoV-2 by immunizing a banded houndshark (Triakis scyllium) with recombinant wild-type RBD. Notably, the CoV2NAR-1 clone showed high binding affinities in the nanomolar range to various RBDs and demonstrated neutralizing activity against SARS-CoV-2 pseudoviruses. These results highlight the potential of the banded houndshark as an animal model for the development of VNAR-based therapeutics or diagnostics against future pandemics.

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.

Development mechanism of resistant population post-exposure to tigecycline in tigecycline-heteroresistant Acinetobacter baumannii strain.

Tigecycline heteroresistance is highly prevalent in Acinetobacter baumannii clinical isolates, reducing the efficacy of tigecycline treatment. This study investigated the population dynamics of A. baumannii with tigecycline heteroresistance to determine the origin of resistance that occurs over time after antibiotic exposure. Tigecycline heteroresistance was imitated by mixing tigecycline-susceptible and -resistant A. baumannii isolates in a 1:10-6 ratio, and confirmed using population analysis profiling. Growth curves and an in-vitro competition assay found no difference in bacterial fitness between tigecycline-resistant and -susceptible populations. The green fluorescent protein (GFP) expression system and flow cytometry were used to monitor the population dynamics of the heteroresistant population, while differentiating the resistant population from the susceptible population. The mimicked tigecycline heteroresistance was confirmed to be reproducible and stable without tigecycline. The GFP-expressing population (i.e. the resistant population) nearly went undetected because it only represented approximately 10-6 of the entire population. However, when the mimicked tigecycline-heteroresistant strain was treated with tigecycline, most subpopulations expressing GFP were detected. The surviving A. baumannii population, upon exposure to tigecycline, exhibited a high minimum inhibitory concentration for tigecycline, equivalent to that of tigecycline-resistant isolates that were used to mimic heteroresistance. These results indicate that the development of resistance in tigecycline-heteroresistant A. baumannii strains, resulting in decreased antibiotic efficacy, may depend on the selection of a pre-existing resistant subpopulation.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose.

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (KanR) should be dependent on FucT2 solubility. KanR was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

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.

Fine-tuning of MXene-nickel oxide-reduced graphene oxide nanocomposite bioelectrode: Sensor for the detection of influenza virus and viral protein.

Influenza viruses can cause epidemics through inter-human transmission, and the social consequences of viral transmission are incalculable. Current diagnostics for virus detection commonly relies on antibodies or nucleic acid as recognition reagent. However, a more advanced and general method for the facile development of new biosensors is increasing in demand. In this study, we report the fabrication of an ultra-sensitive peptide-based nanobiosensor using a nickel oxide (NiO)-reduced graphene oxide (rGO)/MXene nanocomposite to detect active influenza viruses (H1N1 and H5N2) and viral proteins. The sensing mechanism is based on the signal inhibition, the specific interaction between H1N1 (QMGFMTSPKHSV) and H5N1 (GHPHYNNPSLQL) binding peptides anchored on the NiO-rGO/MXene/glassy carbon electrode (GCE) surface and the viral surface protein hemagglutinin (HA) is the critical factor for the decrease in the peak current of the sensor. In this strategy, the NiO-rGO/MXene nanocomposite results in synergistic signal effects, including electrical conductivity, porosity, electroactive surface area, and active site availability when viruses are deposited on the electrode. Based on these observations, the results showed that the developed nanobiosensor was capable of highly sensitive and specific detection of their corresponding influenza viruses and viral proteins with a very low detection limit (3.63 nM of H1N1 and 2.39 nM for H5N1, respectively) and good recovery. The findings demonstrate that the proposed NiO-rGO/MXene-based peptide biosensor can provide insights for developing a wide range of clinical screening tools for detecting affected patients.

Dual synergistic response for the electrochemical detection of H1N1 virus and viral proteins using high affinity peptide receptors.

Identifying alternatives to antibodies as bioreceptors to test samples feasibly is crucial for developing next-generation in vitro diagnostic methods. Here, we aimed to devise an analytical method for detecting H1N1 viral proteins (hemagglutinin [HA] and neuraminidase [NA]) as well as the complete H1N1 virus with high sensitivity and selectivity. By applying biopanning of M13 peptide libraries, high affinity peptides specific for HA or NA were successfully identified. After selection, three different synthetic peptides that incorporated gold-binding motifs were designed and chemically synthesized on the basis of the original sequence identified phage display technique with or without two repeat. Their binding interactions were characterized by enzyme-linked immunosorbent assay (ELISA), square wave voltammetry (SWV), Time of flight-secondary ion mass spectroscopy (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). The binding constants (Kd) of HA BP1, HA BP2 and NA BP1 peptides were found to be 169.72 nM, 70.02 nM and 224.49 nM for HA or NA proteins by electrochemical measurements (SWV). The single use of HA BP2 peptide enabled the detection of either H1N1 viral proteins or the actual H1N1 virus, while NA BP1 peptide exhibited lower binding for real H1N1 virus particles. Moreover, the use of both HA BP1 and BP2 as a divalent capturing reagent improved sensor performance as well as the strength of the electrochemical signal, thereby exhibiting a dual synergistic effect for the electrochemical detection of H1N1 antigens with satisfactory specificity and sensitivity (limit of detection of 1.52 PFU/mL).

Synaptotagmin 11 scaffolds MKK7-JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis.

BACKGROUND: Identifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC. METHODS: In vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient's survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis. RESULTS: SYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells. CONCLUSION: SYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

Neuron-recognizable characteristics of peptides recombined using a neuronal binding domain of botulinum neurotoxin.

Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2' and EGFP-RBD', exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBD' to SV2C-LD was determined to be 5.45 µM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2', respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBD' to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2', respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBD'. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.

Chlorogenic Acid Isomers Isolated from Artemisia lavandulaefolia Exhibit Anti-Rosacea Effects In Vitro.

Rosacea is a chronic inflammatory disease affecting facial skin. It is associated with immune and vascular dysfunction mediated via increased expression and activity of cathelicidin and kallikrein 5 (KLK5), a serine protease of stratum corneum. Therefore, KLK5 inhibitors are considered as therapeutic agents for improving the underlying pathophysiology and clinical manifestation of rosacea. Here, we isolated the active constituents of Artemisia lavandulaefolia (A. lavandulaefolia) and investigated their inhibitory effect on KLK5 protease activity. Using bioassay-guided isolation, two bioactive compounds including chlorogenic acid isomers, 3,5-dicaffeoylquinic acid (isochlorogenic acid A) (1), and 4,5-dicaffeoylquinic acid (isochlorogenic acid C) (2) were isolated from A. lavandulaefolia. In this study, we evaluated the effects of isochlorogenic acids A and C on dysregulation of vascular and immune responses to rosacea, and elucidated their molecular mechanisms of action. The two chlorogenic acid isomers inhibit KLK5 protease activity, leading to reduced conversion of inactive cathelicidin into active LL-37. This inhibition of LL-37 production by isochlorogenic acids A and C reveals the efficacy of suppressing the expression of inflammatory mediators induced by LL-37 in immune cells such as macrophages and mast cells. In addition, both isomers of chlorogenic acid directly inhibited the proliferation and migration of vascular endothelial cells induced by LL-37.

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.