COVID-19 Therapeutics: An Update on Effective Treatments Against Infection With SARS-CoV-2 Variants.

The coronavirus disease 2019 (COVID-19) pandemic is one of the most consequential global health crises in over a century. Since its discovery in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to mutate into different variants and sublineages, rendering previously potent treatments and vaccines ineffective. With significant strides in clinical and pharmaceutical research, different therapeutic strategies continue to be developed. The currently available treatments can be broadly classified based on their potential targets and molecular mechanisms. Antiviral agents function by disrupting different stages of SARS-CoV-2 infection, while immune-based treatments mainly act on the human inflammatory response responsible for disease severity. In this review, we discuss some of the current treatments for COVID-19, their mode of actions, and their efficacy against variants of concern. This review highlights the need to constantly evaluate COVID-19 treatment strategies to protect high risk populations and fill in the gaps left by vaccination.

Efficient Anti-Tumor Immunotherapy Using Tumor Epitope-Coated Biodegradable Nanoparticles Combined With Polyinosinic-Polycytidylic Acid and an Anti-PD1 Monoclonal Antibody.

Vaccination with tumor peptide epitopes associated with MHC class I molecules is an attractive approach directed at inducing tumor-specific CTLs. However, challenges remain in improving the therapeutic efficacy of peptide epitope vaccines, including the low immunogenicity of peptide epitopes and insufficient stimulation of innate immune components in vivo. To overcome this, we aimed to develop and test an innovative strategy that elicits potent CTL responses against tumor epitopes. The essential feature of this strategy is vaccination using tumor epitope-loaded nanoparticles (NPs) in combination with polyinosinic-polycytidylic acid (poly-IC) and anti-PD1 mAb. Carboxylated NPs were prepared using poly(lactic-co-glycolic acid) and poly(ethylene/maleic anhydride), covalently conjugated with anti-H-2Kb mAbs, and then attached to H-2Kb molecules isolated from the tumor mass (H-2b). Native peptides associated with the H-2Kb molecules of H-2Kb-attached NPs were exchanged with tumor peptide epitopes. Tumor peptide epitope-loaded NPs efficiently induced tumor-specific CTLs when used to immunize tumor-bearing mice as well as normal mice. This activity of the NPs significantly was increased when co-administered with poly-IC. Accordingly, the NPs exerted significant anti-tumor effects in mice implanted with EG7-OVA thymoma or B16-F10 melanoma, and the anti-tumor activity of the NPs was significantly increased when applied in combination with poly-IC. The most potent anti-tumor activity was observed when the NPs were co-administered with both poly-IC and anti-PD1 mAb. Immunization with tumor epitope-loaded NPs in combination with poly-IC and anti-PD1 mAb in tumor-bearing mice can be a powerful means to induce tumor-specific CTLs with therapeutic anti-tumor activity.

A Novel Bacterium, Butyricimonas virosa, Preventing HFD-Induced Diabetes and Metabolic Disorders in Mice via GLP-1 Receptor.

Knowledge of the impact of the gut microbiota on human health has increased, and modulation of the bacterial community is now considered a therapeutic target for various diseases. Certain novel bacterial species have probiotic properties associated with improvement in obesity and related metabolic disorders. The relative abundance of Butyricimonas spp. is correlated with metabolic parameters; however, the physiological role of Butyricimonas in metabolic improvement is unclear. In this study, live and heat-killed Butyricimonas virosa were administered to mice with high-fat diet (HFD)-induced obesity. Both live and heat-killed B. virosa ameliorated HFD-impaired body weight, serum glucose level, insulin resistance, and liver steatosis. Moreover, activation of the glucagon-like peptide-1 receptor (GLP-1R) and peroxisome proliferator-activated receptor α (PPARα) was observed in the liver, and the expression levels of insulin receptor substrate (IRS)-1, IRS-2, Toll-like receptor 5 (TLR5), and zonula occludens-1 (ZO-1) were upregulated in the ileum. Finally, we demonstrated that the effect of B. virosa treatment on glucose regulation may be linked to the upregulation of GLP-1R in the liver and is not a result of colonization of the gut by B. virosa or B. virosa-produced butyrate. Our results provide a rationale for the development of Butyricimonas spp.-based therapeutics and prophylactics for hyperglycemia.

MUL1-RING recruits the substrate, p53-TAD as a complex with UBE2D2-UB conjugate.

The RING domain of MUL1 (RINGMUL1 ) alone mediates ubiquitylation of the p53-transactivation domain (TADp53 ). To elucidate the mechanism underlying the simultaneous recruitment of UBE2D2 and the substrate TADp53 by RINGMUL1 , we determined the complex structure of RINGMUL1 :UBE2D2 and studied the interaction between RINGMUL1 and TADp53 in the presence of UBE2D2-UB thioester (UBE2D2~UB) mimetics. The RINGMUL1 -binding induced the closed conformation of UBE2D2S22R/C85S -UBK48R oxyester (UBE2D2RS -UBR OE ), and strongly accelerated its hydrolysis, which was suppressed by the additional N77A-mutation of UBE2D2. Interestingly, UBE2D2S22R/N77A/C85S -UBK48R oxyester (UBE2D2RAS -UBR OE ) already formed a closed conformation in the absence of RINGMUL1 . Although TADp53 exhibited weak binding for RINGMUL1 or UBE2D2 alone, its binding affinity was enhanced and even further for RINGMUL1 :UBE2D2 and RINGMUL1 :UBE2D2RAS -UBR OE , respectively. The recognition of TADp53 by RINGMUL1 as a complex with UBE2D2~UB is related to the multivalency of the binding events and underlies the ability of RINGMUL1 to ubiquitylate the intrinsically disordered protein, TADp53 .

Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

Tertiary RNA Folding-Targeted Drug Screening Strategy Using a Protein Nanopore.

Bacterial riboswitch RNAs are attractive targets for novel antibiotics against antibiotic-resistant superbacteria. Their binding to cognate metabolites is essential for the regulation of bacterial gene expression. Despite the importance of RNAs as therapeutic targets, the development of RNA-targeted, small-molecule drugs is limited by current biophysical methods. Here, we monitored the specific interaction between the adenine-sensing riboswitch aptamer domain (ARS) and adenine at the single-molecule level using α-hemolysin (αHL) nanopores. During adenine-induced tertiary folding, adenine-bound ARS intermediates exhibited characteristic nanopore events, including a two-level ionic current blockade and a ∼ 5.6-fold longer dwell time than that of free RNA. In a proof-of-concept experiment, tertiary RNA folding-targeted drug screening was performed using a protein nanopore, which resulted in the discovery of three new ARS-targeting hit compounds from a natural compound library. Taken together, these results reveal that αHL nanopores are a valuable platform for ultrasensitive, label-free, and single-molecule-based drug screening against therapeutic RNA targets.

Induction of Peptide-specific CTL Activity and Inhibition of Tumor Growth Following Immunization with Nanoparticles Coated with Tumor Peptide-MHC-I Complexes.

Tumor peptides associated with MHC class I molecules or their synthetic variants have attracted great attention for their potential use as vaccines to induce tumor-specific CTLs. However, the outcome of clinical trials of peptide-based tumor vaccines has been disappointing. There are various reasons for this lack of success, such as difficulties in delivering the peptides specifically to professional Ag-presenting cells, short peptide half-life in vivo, and limited peptide immunogenicity. We report here a novel peptide vaccination strategy that efficiently induces peptide-specific CTLs. Nanoparticles (NPs) were fabricated from a biodegradable polymer, poly(D,L-lactic-co-glycolic acid), attached to H-2Kb molecules, and then the natural peptide epitopes associated with the H-2Kb molecules were exchanged with a model tumor peptide, SIINFEKL (OVA257-268). These NPs were efficiently phagocytosed by immature dendritic cells (DCs), inducing DC maturation and activation. In addition, the DCs that phagocytosed SIINFEKL-pulsed NPs potently activated SIINFEKL-H-2Kb complex-specific CD8+ T cells via cross-presentation of SIINFEKL. In vivo studies showed that intravenous administration of SIINFEKL-pulsed NPs effectively generated SIINFEKL-specific CD8+ T cells in both normal and tumor-bearing mice. Furthermore, intravenous administration of SIINFEKL-pulsed NPs into EG7.OVA tumor-bearing mice almost completely inhibited the tumor growth. These results demonstrate that vaccination with polymeric NPs coated with tumor peptide-MHC-I complexes is a novel strategy for efficient induction of tumor-specific CTLs.

Efficacy of Local Minocycline Agents in Treating Peri-Implantitis: An Experimental In Vivo Study in Beagle Dogs.

BACKGROUND: Local delivery agents (LDA) have the advantage of delivering the antibiotics at high concentrations to the targeted sites. However, the constant flow of gingival crevicular fluids and saliva may restrict their efficacy. Therefore, the drug sustainability and pharmacodynamic properties of any proposed LDA should be evaluated. METHODS: Four dental implants were placed unilaterally in the edentulous mandible of six beagle dogs. Peri-implantitis were experimentally induced using silk-ligatures. Each implant was randomly allocated to receive one of the following four treatments: (i) MC (Chitosan-alginate (CA) minocycline), (ii) MP (CA-without minocycline), (iii) PG (Polyacrylate-glycerin minocycline), and (iv) Control (mechanical debridement only). Mechanical therapies and LDAs were administered into the gingival sulcus two times at a 4-week interval. Drug sustainability as well as clinical, radiographical, and immunohistochemical (IHC) analyses were conducted to evaluate the efficacies of treatments. RESULTS: Reduced mean probing depth was observed in all of the test groups after the second delivery. A minimal marginal bone level change was observed during the treatment period (MP (-0.06 ± 0.53 mm) to PG (-0.25 ± 0.42 mm)). The distribution of IHC cell marker analysis of all targeted antibodies ranged from 6.34% to 11.33%. All treatment outcomes between the test groups were comparable. A prolonged retention of LDA was observed from CA microspheres (MC and MP) at both administrations (p < 0.017) and prolonged sustainability of bacteriostatic effect was observed from MC compared to PG after the second administration (p < 0.05). CONCLUSIONS: Prolonged retention of CA microspheres was observed and the longer bacteriostatic effect was observed from the MC group. Mechanical debridement with adjunct LDA therapy may impede peri-implantitis progression, however, prolonged drug action did not lead to improved treatment outcome.

Adjuvanticity of Processed Aloe vera gel for Influenza Vaccination in Mice.

The effectiveness of current influenza vaccines is considered suboptimal, and 1 way to improve the vaccines is using adjuvants. However, the current pool of adjuvants used in influenza vaccination is limited due to safety concerns. Aloe vera, or aloe, has been shown to have immunomodulatory functions and to be safe for oral intake. In this study, we explored the potential of orally administered processed Aloe vera gel (PAG) as an adjuvant for influenza vaccines in C57BL/6 mice. We first evaluated its adjuvanticity with a split-type pandemic H1N1 (pH1N1) Ag by subjecting the mice to lethal homologous influenza challenge. Oral PAG administration with the pH1N1 Ag increased survival rates in mice to levels similar to those of alum and MF59, which are currently used as adjuvants in influenza vaccine formulations. Similarly, oral PAG administration improved the survival of mice immunized with a commercial trivalent influenza vaccine against lethal homologous and heterologous virus challenge. PAG also increased hemagglutination inhibition and virus neutralization Ab titers against homologous and heterologous influenza strains following immunization with the split-type pH1N1 Ag or the commercial trivalent vaccine. Therefore, this study demonstrates that PAG may potentially be used as an adjuvant for influenza vaccines.

Locally Applied Slow-Release of Minocycline Microspheres in the Treatment of Peri-Implant Mucositis: An Experimental In Vivo Study.

BACKGROUND: The objective of this is preclinical investigation was to evaluate the differential drug sustainability and pharmacodynamic properties of two local minocycline microsphere carriers: chitosan-coated alginate (CA) and poly(meth)acrylate-glycerin (PG). METHODS: Four dental implants were placed unilaterally in the edentulous mandible of six beagle dogs. Each implant was randomly assigned to receive one of the following four treatments: (i) CA (CA-based minocycline), (ii) placebo (CA substrate without minocycline), (iii) PG (PG-based minocycline) and (iv) control (mechanical debridement only). After inducing peri-implant mucositis, the randomly assigned treatments were administered into the gingival sulcus twice at a 4-week interval using a plastic-tipped syringe. Drug sustainability and pharmacodynamic (clinical, radiographical and cell marker intensity) evaluations were performed after each administration. RESULTS: The CA microspheres remained longer around the healing abutment compared to the PG microspheres at both administrations and a longer bacteriostatic effect was observed from CA (7.0 ± 5.7 days) compared to PG (1.2 ± 2.6 days). The efficacy of the applied therapies based on clinical, radiographical and histological analyses were comparable across all treatment groups. CONCLUSIONS: CA microspheres showed longer carrier and bacteriostatic effect sustainability when compared to PG microspheres, however, longer drug sustainability did not lead to improved treatment outcomes.

The RING domain of mitochondrial E3 ubiquitin ligase 1 and its complex with Ube2D2: crystallization and X-ray diffraction.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order to understand this novel ubiquitylation mechanism, it is necessary to determine the three-dimensional structures of MUL1-RING and of its complex with the cognate E2 enzyme. Here, Ube2D2 was validated as a functional E2 enzyme for the ubiquitylation of the p53 transactivation domain (p53-TAD) by MUL1-RING, and purification and crystallization processes for MUL1-RING and the MUL1-RING-Ube2D2 complex are reported.

Immunotherapy of Autoimmune Diseases with Nonantibiotic Properties of Tetracyclines.

Tetracyclines, which have long been used as broad-spectrum antibiotics, also exhibit a variety of nonantibiotic activities including anti-inflammatory and immunomodulatory properties. Tetracyclines bind to the 30S ribosome of the bacteria and inhibit protein synthesis. Unlike antimicrobial activity, the primary molecular target for the nonantibiotic activity of tetracycline remains to be clarified. Nonetheless, the therapeutic efficacies of tetracyclines, particularly minocycline and doxycycline, have been demonstrated in various animal models of autoimmune disorders, such as multiple sclerosis, rheumatoid arthritis, and asthma. In this study, we summarized the anti-inflammatory and immunomodulatory activities of tetracyclines, focusing on the mechanisms underlying these activities. In addition, we highlighted the on-going or completed clinical trials with reported outcomes.

Ethanol extract from Gynostemma pentaphyllum ameliorates dopaminergic neuronal cell death in transgenic mice expressing mutant A53T human alpha-synuclein.

Gynostemma (G.) pentaphyllum (Cucurbitaceae) contains various bioactive gypenosides. Ethanol extract from G. pentaphyllum (GP-EX) has been shown to have ameliorative effects on the death of dopaminergic neurons in animal models of Parkinson's disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- and 6-hydroxydopamine. PD patients exhibit multiple symptoms, so PD-related research should combine neurotoxin models with genetic models. In the present study, we investigated the ameliorative effects of GP-EX, including gypenosides, on the cell death of dopaminergic neurons in the midbrain of A53T α-synuclein transgenic mouse models of PD (A53T). Both GP-EX and gypenosides at 50 mg/kg per day were orally administered to the A53T mice for 20 weeks. α-Synuclein-immunopositive cells and α-synuclein phosphorylation were increased in the midbrain of A53T mice, which was reduced following treatment with GP-EX. Treatment with GP-EX modulated the reduced phosphorylation of tyrosine hydroxylase, extracellular signal-regulated kinase (ERK1/2), Bcl-2-associated death promoter (Bad) at Ser112, and c-Jun N-terminal kinase (JNK1/2) due to α-synuclein overexpression. In the A53T group, GP-EX treatment prolonged the latency of the step-through passive avoidance test and shortened the transfer latency of the elevated plus maze test. Gypenosides treatment exhibited the effects and efficacy similar to those of GP-EX. Taken together, GP-EX, including gypenosides, has ameliorative effects on dopaminergic neuronal cell death due to the overexpression of α-synuclein by modulating ERK1/2, Bad at Ser112, and JNK1/2 signaling in the midbrain of A53T mouse model of PD. Further studies are needed to investigate GP-EX as a treatment for neurodegenerative synucleinopathies, including PD. This study was approved by the Animal Ethics Committee of Chungbuk National University (approval No. CBNUA-956-16-01) on September 21, 2016.

Oral treatment with Aloe polysaccharide ameliorates ovalbumin-induced atopic dermatitis by restoring tight junctions in skin.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. A hallmark of AD is dry itchy skin that results from defects in the epidermal barrier function. Aloe vera is used widely to promote general health and is administered topically to treat skin conditions such as eczema, burns and wounds. However, effects of A vera on AD were not fully elucidated. In this study, we investigated the oral administration of processed A vera gel (PAG) containing low molecular weight Aloe polysaccharides to treat ovalbumin (OVA)-induced AD in mice. Oral administration of PAG suppressed total and OVA-specific IgE production in sera and decreased the epidermal thickness of skin. Numbers of Ki-67-positive cells were reduced by PAG treatment. Expression levels of tight junction genes, including those that encode ZO-1, Claudin-1 and Claudin-8, were decreased in AD skin lesions, whereas oral administration of PAG partially restored the expression levels of tight junction genes. In addition, IL-4 and IL-17A mRNA transcript levels were reduced in skin lesions after PAG treatment. Taken together, our findings suggest that oral administration of PAG ameliorated AD, normalized tight junction gene expression and suppressed inflammatory cytokines in AD skin.

Alterations in Gut Microbiota by Statin Therapy and Possible Intermediate Effects on Hyperglycemia and Hyperlipidemia.

Dysbiosis of the gut microbiota is a contributing factor for obesity-related metabolic diseases such as hyperglycemia and hyperlipidemia. Pharmacotherapy for metabolic diseases involves the modulation of gut microbiota, which is suggested to be a potential therapeutic target. In this study, the modulation of gut microbiota by statins (cholesterol-lowering drugs: atorvastatin and rosuvastatin) was investigated in an aged mouse model of high-fat diet-induced obesity, and the association between gut microbiota and immune responses was described. Atorvastatin and rosuvastatin significantly increased the abundance of the genera Bacteroides, Butyricimonas, and Mucispirillum. Moreover, the abundance of these genera was correlated with the inflammatory response, including levels of IL-1ß and TGFß1 in the ileum. In addition, oral fecal microbiota transplantation with fecal material collected from rosuvastatin-treated mouse groups improved hyperglycemia. From these results, the effect of statins on metabolic improvements could be explained by altered gut microbiota. Our findings suggest that the modulation of gut microbiota by statins has an important role in the therapeutic actions of these drugs.

Processed Aloe vera gel attenuates non-steroidal anti-inflammatory drug (NSAID)-induced small intestinal injury by enhancing mucin expression.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known for exerting numerous adverse effects on the gastrointestinal tract such as bleeding, ulceration, and perforation, thereby limiting their use. Most previous studies have focused on NSAID-induced gastropathy. However, improved diagnostic techniques have recently highlighted NSAID-induced small intestinal ulcers, which have so far been underestimated. While proton pump inhibitors are prescribed to control NSAID-induced gastropathy, few preventive strategies are existent for NSAID-induced small intestinal injury, thus requiring new methods to treat these enteropathies. Numerous studies have reported the beneficial biological effects of Aloe vera, such as wound healing, anti-cancer, immune modulation, anti-oxidant, anti-microbial, and gastroprotective effects. A previous report on the effect of Aloe vera against NSAID-induced ulcers studied only gastric ulcers and elucidated the results as an anti-inflammatory effect of Aloe vera. However, ulcer prevention cannot be justified entirely to be due to the anti-inflammatory effects of Aloe vera, since NSAIDs themselves also exert an anti-inflammatory reaction. We therefore investigated the anti-ulcer effects of Aloe vera on the small intestine, especially focusing on mucin expression. Our results indicate that processed Aloe vera gel (PAG) treatment attenuates not only the severity of intestinal ulcers but also bacterial translocation, by enhancing the mucus layer in the indomethacin-induced small intestinal damage mouse model. We further confirmed that PAG positively regulates the mucin expression in the LS174T human cell line, mainly via the ERK-dependent pathway. We propose that PAG application is a potential strategy for the alleviation of NSAID-induced small intestinal ulcers.

Downregulation of IL-18 Expression in the Gut by Metformin-induced Gut Microbiota Modulation.

IL-18 is a crucial pro-inflammatory cytokine that mediates chronic intestinal inflammation. Metformin, an anti-diabetic drug, was reported to have ameliorative effects on inflammatory bowel disease. Recently, the mechanism of action of metformin was explained as a modulation of gut microbiota. In this study, fecal microbiota transplantation (FMT) using fecal material from metformin-treated mice was found to upregulate the expression of GLP-1 and pattern-recognition receptors TLR1 and TLR4 for the improvement in hyperglycemia caused by a high-fat diet. Further, FMT downregulated the expression of the inflammatory cytokine IL-18. Within the genera Akkermansia, Bacteroides, and Butyricimonas, which were promoted by metformin therapy, Butyricimonas was found to be consistently abundant following FMT. Our findings suggest that modulation of gut microbiota is a key factor for the anti-inflammatory effects of metformin which is used for the treatment of hyperglycemia.

Lipophilic statins inhibit Zika virus production in Vero cells.

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family. ZIKV infection has been associated with neurological complications such as microcephaly in newborns and Guillain-Barré syndrome in adults; thus, therapeutic agents are urgently needed. Statins are clinically approved for lowering cholesterol levels to prevent cardiovascular disease but have shown potential as antiviral drugs. In this study, we explored the possibility of utilizing statins as anti-ZIKV drugs. We found that, generally, lipophilic statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, and simvastatin) could reduce ZIKV production in vitro and result in smaller foci of infection. Time-of-drug-addition assay revealed that early treatment with statins is more beneficial than late treatment; however, statins could not completely inhibit the entry stage of ZIKV infection. Furthermore, individual lipophilic statins differed in anti-ZIKV capacity, with fluvastatin being the most efficient at low concentrations. Taken together, this study shows that statins or their derivatives have the potential to be used as anti-ZIKV therapeutic agents.