SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 'highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19.

An Atelocollagen Injection Enhances the Healing of Nonoperatively Treated Achilles Tendon Tears: An Experimental Study in Rats.

Background: There is growing interest in nonoperative treatment for the management of Achilles tendon ruptures (ATRs). However, nonoperative treatment is limited by the risk of tendon reruptures and low satisfaction rates. Recently, atelocollagen injections have been reported to have beneficial effects on tendon healing. Purpose: To evaluate the beneficial effects of injected atelocollagen on Achilles tendon healing and investigate the mechanism of atelocollagen on tendon healing. Study Design: Controlled laboratory study. Methods: Percutaneous tenotomy of the right Achilles tendon in 66 rats was performed. The animals were equally divided into the noninjection group (NG) and the collagen injection group (CG). At 1, 3, and 6 weeks, the Achilles functional index, cross-sectional area, load to failure, stiffness, stress, and the modified Bonar score were assessed. Transmission electron microscopy, western blotting, and immunohistochemistry were also performed. Results: The Achilles functional index (-6.8 vs -43.0, respectively; P = .040), load to failure (42.1 vs 27.0 N, respectively; P = .049), and stiffness (18.8 vs 10.3 N/mm, respectively; P = .049) were higher in the CG than those in the NG at 3 weeks. There were no significant differences in histological scores between the 2 groups. Transmission electron microscopy analysis showed that the mean diameter of collagen fibrils in the CG was greater than that in the NG at 3 weeks (117.2 vs 72.6 nm, respectively; P < .001) and 6 weeks (202.1 vs 144.0 nm, respectively; P < .001). Western blot analysis showed that the expression of collagen type I in the CG was higher than that in the NG at 1 week (P = .005) and 6 weeks (P = .001). Conclusion: An atelocollagen injection had beneficial effects on the healing of nonoperatively treated Achilles tendon injuries. The Achilles tendon of CG rats exhibited better functional, biomechanical, and morphological outcomes compared with NG rats. The molecular data indicated that the mechanism of atelocollagen injections may be associated with an increased amount of collagen type I. Clinical Relevance: An atelocollagen injection might be a good adjuvant option for the nonoperative treatment of ATRs.

An amphiphilic material arginine-arginine-bile acid promotes α-synuclein amyloid formation.

Amyloid generation plays essential roles in various human diseases, biological functions, and nanotechnology. However, developing efficient chemical and biological candidates for regulating amyloid fibrillation remains difficult because information on the molecular actions of modulators is insufficient. Thus, studies are needed to understand how the intermolecular physicochemical properties of the synthesised molecules and amyloid precursors influence amyloidogenesis. In this study, we synthesised a novel amphiphilic sub-nanosized material, arginine-arginine (RR)-bile acid (BA), by conjugating positively charged RR to hydrophobic BA. The effects of RR-BA on amyloid formation were investigated on α-synuclein (αSN) in Parkinson's disease and on K18 and amyloid-ß (1-42) (Aß42) in Alzheimer's disease. RR-BA showed no appreciable effect on the kinetics of K18 and Aß42 amyloid fibrillation because of their weak and non-specific interactions. However, RR-BA specifically bound to αSN with moderate binding affinity through electrostatic interactions between the positively charged RR and the negatively charged cluster in the C-terminus of αSN. In addition, hydrophobic BA in the αSN-RR-BA complex transiently condensed αSN for primary nucleation, thereby accelerating αSN amyloid fibrillation. We propose an electrostatic binding and hydrophobic condensation model of RR-BA-driven amyloid formation of αSN, which will contribute to the rational design and development of molecules for controlling amyloid aggregation in diverse fields.

Bean Extract-Based Gargle for Efficient Diagnosis of Active COVID-19 Infection Using Rapid Antigen Tests.

The antigen-based rapid diagnostic test (Ag-RDT) using saliva specimens is fast, noninvasive, and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. We evaluated a novel Beanguard gargle (BG)-based virus collection method that can be applied to Ag-RDT as an alternative to the current RT-PCR with an NPS for early diagnosis of COVID-19. This clinical trial comprised 102 COVID-19-positive patients hospitalized after a governmental screening process and 100 healthy individuals. Paired NPS and BG-based saliva specimens from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs, whose diagnostic performance for detecting SARS-CoV-2 was compared. BG-Ag-RDTs showed high sensitivity (97.8%) and specificity (100%) in 45 patients within 6 days of illness and detected all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic active COVID-19 cases, both BG-Ag-RDTs and BG-RT-PCR showed sensitivities and specificities of 100%. Sensitivities of BG-Ag-RDT and BG-RT-PCR toward salivary viral detection were highly concordant, with no discrimination between symptomatic (97.0%), asymptomatic (100%), or SARS-CoV-2 variant (100%) cases. The intermolecular interactions between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from the bean extract (BE), were observed in terms of physicochemical properties. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Using BG-based saliva specimens remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables noninvasive, rapid, and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management. IMPORTANCE An Ag-RDT is less likely to be accepted as an initial test method for early diagnosis owing to its low sensitivity. However, our self-collection method, Ag-RDT using BG-based saliva specimens, showed significantly enhanced detection sensitivity and specificity toward SARS-CoV-2 including the Alpha and Delta variants in all patients tested within 6 days of illness. The method represents an attractive alternative to nasopharyngeal swabs for the early diagnosis of symptomatic and asymptomatic COVID-19 cases. The evidence suggests that the method could have a potential for mass screening and monitoring of COVID-19 cases.

Tauroursodeoxycholic Acid Decreases Keloid Formation by Reducing Endoplasmic Reticulum Stress as Implicated in the Pathogenesis of Keloid.

Keloids are a common form of pathologic wound healing and are characterized by an excessive production of extracellular matrix. This study examined the major contributing mechanism of human keloid pathogenesis using transcriptomic analysis. We identified the upregulation of mitochondrial oxidative stress response, protein processing in the endoplasmic reticulum, and TGF-ß signaling in human keloid tissue samples compared to controls, based on ingenuity pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Electron microscopic examinations revealed an increased number of dysmorphic mitochondria and expanded endoplasmic reticulum (ER) in human keloid tissue samples than that in controls. Western blot analysis performed using human tissues suggested noticeably higher ER stress signaling in keloids than in normal tissues. Treatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, significantly decreased scar formation in rabbit models, compared to normal saline and steroid injections. In summary, our findings demonstrate the contributions of mitochondrial dysfunction and dysregulated ER stress signaling in human keloid formation and the potential of TUDCA in the treatment of keloids.

Dataset of single nucleotide polymorphisms and comprehensive proteomic analysis of Streptococcus equi subsp. equi ATCC 39506.

Streptococcus equi subspecies equi (S. equi) is an opportunistic pathogen and a major causative agent of equine strangles, a contagious respiratory infection in horses and other equines. In this study, we provide the dataset associated with our research publication "Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infections" [1]. We describe the genomic differences between S. equi 4047 and S. equi ATCC 39506 and outline the comprehensive proteome information of various fractions, including the whole cell lysate, membrane proteome, secretory proteome, and extracellular vesicle proteome. In addition, we included a dataset of highly immunoreactive proteins identified through immunoprecipitation. The specifications table provides a detailed summary of the gene annotation and quantitative information obtained for each proteome. The proteomics data were analyzed using shotgun proteomics with LTQ Velos and Q Exactive mass spectrometry in the data-dependent acquisition mode. We have deposited the acquired data, including the mass spectrometry raw files and exported MASCOT search results, in the PRIDE public repository under the accession numbers PXD025152 and PXD025527.

Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infection.

Streptococcus equi subspecies equi is a pathogenic bacterium that causes strangles, a highly contagious respiratory infection in horses and other equines. The limitations of current vaccines against S. equi infection warrants the development of an affordable, safe, and effective vaccine. Because gram-positive extracellular vesicles (EVs) transport various immunogenic antigens, they are attractive vaccine candidates. Here, we purified the EVs of S. equi ATCC 39506 and evaluated them as a vaccine candidate against S. equi infection in mice. As an initial step, comparative proteomic analysis was performed to characterize the functional features of the EVs. Reverse vaccinology and knowledge-based annotations were then used to screen potential vaccine candidates (PVCs) for S. equi ATCC 39506. Finally, 32 PVCs were found to be enriched in the EV fraction, suggesting the usefulness of this fraction as a vaccine. Importantly, a significantly higher survival rate after S. equi infection was detected in mice immunized with S. equi-derived EVs via the intraperitoneal route than in mice immunized with heat-killed bacteria. Of note, immunoprecipitation-mass spectrometry results validated various immunogenic antigens within the EV proteome. In conclusion, our results suggest that S. equi-derived EVs can serve as a vaccine candidate against S. equi infection.

Sulfamoylbenzamide-based Capsid Assembly Modulators for Selective Inhibition of Hepatitis B Viral Replication.

As the spread of infections caused by hepatitis B virus (HBV) threatens public health worldwide, investigations from multiple perspectives and of various mechanisms of action are urgently required to increase the HBV cure rate. Targeting the encapsidation of the nuclear capsid protein (core protein, HBc) has emerged as an attractive strategy for inhibiting the viral assembly process; however, a drug targeting this mechanism has not yet been approved. We synthesized novel sulfamoylbenzamides (SBAs) as capsid assembly modulators of HBV and found that the effects and safety profiles of compounds 3 and 8 have potential therapeutic applicability against HBV. The formation of tubular particles was time-dependent in the presence of 3, indicating a new mode of protein assembly by SBA compounds. Our findings provide a new entity for developing safe and efficient treatments for HBV infection.

Three-Dimensional TEM Study of Dendrimer-Encapsulated Pt Nanoparticles for Visualizing Structural Characteristics of the Whole Organic-Inorganic Hybrid Nanostructure.

Here, we report three-dimensional (3-D) visualization of dendrimer-encapsulated Pt nanoparticles (Pt DENs) by using 3-D electron tomography to reveal intricate structural characteristics of their whole organic-inorganic hybrid nanostructure. We reconstructed the 3-D spatial volume of Pt DENs by back-projecting a tilt series of two-dimensional (2-D) projections of Pt nanoparticles encapsulated inside dendrimers negatively stained with uranyl acetate. The direct 3-D visualization of Pt DENs elucidated their encapsulation characteristics with the spatial imaging of Pt nanoparticles embraced inside dendrimers in three dimensions. The encapsulation characteristics of Pt DENs were further verified with selective electrochemical poisoning experiments. In addition, quantitative 3-D structural characterization of Pt DENs provided more accurate and precise size distributions of nanoparticles than those obtained from conventional 2-D transmission electron microscopy analysis relying only on a 3-D structure projected on a 2-D plane.

Transmission Electron Microscopy Confirmation of Orientia tsutsugamushi in Human Bile.

Scrub typhus, the third most frequently reported infectious disease in South Korea, causes serious public health problems. In 2019, we collected a bile specimen from a patient with scrub typhus through percutaneous transhepatic gallbladder drainage and performed transmission electron microscopy to confirm the ultrastructure of Orientia tsutsugamushi.

Analysis of the Extracellular Proteome of Colistin-Resistant Korean Acinetobacter baumannii Strains.

We analyzed the extracellular proteome of colistin-resistant Korean Acinetobacter baumannii (KAB) strains to identify proteome profiles that can be used to characterize extensively drug-resistant KAB strains. Four colistin-resistant KAB strains with colistin resistance associated with point mutations in pmrB and pmrC genes were analyzed. Analysis of the extracellular proteome of these strains revealed the presence of 506 induced common proteins, which were hence considered as the core extracellular proteome. Class C ADC-30 and class D OXA-23 ß-lactamases were abundantly induced in these strains. Porins (CarO and CarO-like porin), outer membrane proteins (OmpH and BamABDE), transport protein (AdeK), receptor (TonB), and several proteins of unknown function were among the specifically induced proteins. Based on the sequence homology analysis of proteins from the core proteome and those of other A. baumannii strains and pathogenic bacterial species as well as further in silico screening, we propose that CarO-like porin is an A. baumannii-specific protein and that two tryptic peptides that originate from CarO-like porin detected by tandem mass spectrometry are peptide makers of this protein.

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, Acinetobacter baumannii DU202.

BACKGROUND: Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis. METHODS: Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed. RESULTS: OMV secretion was increased > twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which ß-lactamase OXA-23, various proteases, outer membrane proteins, ß-barrel assembly machine proteins, peptidyl-prolyl cis-trans isomerases and inherent prophage head subunit proteins were significantly upregulated. CONCLUSION: In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.

Genomic characterization of extensively drug-resistant Acinetobacter baumannii strain, KAB03 belonging to ST451 from Korea.

Extensively drug-resistant (XDR) Acinetobacter baumannii strains have emerged rapidly worldwide. The antibiotic resistance characteristics of XDR A. baumannii strains show regional differences; therefore, it is necessary to analyze both genomic and proteomic characteristics of emerging XDR A. baumannii clinical strains isolated in Korea to elucidate their multidrug resistance. Here, we isolated new sequence type of XDR A. baumannii clinical strain (KAB03) from Korean hospitals and performed comprehensive genome analyses. The strain belongs to new sequence type, ST451. Single nucleotide polymorphism (SNP) analysis with other types of A. baumannii strains revealed that KAB03 has unique SNP pattern in the regions of gyrB and gpi of MLST profiles. A. baumannii KAB03 harbours three antibiotic resistance islands (AbGRI1, 2, and 3). AbGRI1 harbours two copies of Tn2006 containing blaOXA-23, which play an important role in antibiotic resistance. AbGRI2 possesses aminoglycoside resistant gene aph(3')-Ic and class A ß-lactamase blaTEM. AbGIR3 has macrolide resistant genes and aminoglycoside resistant gene armA. A. baumannii KAB03 harbours mutations in pmrB and pmrC, which are believed to confer colistin resistance. In addition, proteomic and transcriptional analysis of KAB03 confirmed that ß-lactamases (ADC-73 and OXA-23), Ade efflux pumps (AdeIJK), outer membrane proteins (OmpA and OmpW), and colistin resistance genes (PmrCAB) were major proteins responsible for antibiotic resistance. Our proteogenomic results provide valuable information for multi-drug resistance in emerging XDR A. baumannii strains belonging to ST451.

Correction: Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-degrading Bacterium Burkholderia sp. K24.

[This corrects the article DOI: 10.1371/journal.pone.0154233.].

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24.

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the ß-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.