Platycodi Radix Extract Prevents Hepatic Steatosis by Enhancing Bile Acid Synthesis in a High-Fat Diet-Induced Fatty Liver Mouse Model.

We aimed to identify the mechanism underlying the preventive effects of non-alcoholic fatty liver disease (NAFLD) through Platycodi Radix consumption using liver proteomic and bioinformatic analysis. C57BL/6J mice were categorized into three groups: those receiving a standard chow diet (NCD), those on a high-fat diet (HFD), and those on an HFD supplemented with 5% Platycodi Radix extract (PRE). After a 12-week period, PRE-fed mice exhibited a noteworthy prevention of hepatic steatosis. Protein identification and quantification in liver samples were conducted using LC-MS/MS. The identified proteins were analyzed through Ingenuity Pathway Analysis software, revealing a decrease in proteins associated with FXR/RXR activation and a concurrent increase in cholesterol biosynthesis proteins in the PRE-treated mouse liver. Subsequent network analysis predicted enhanced bile acid synthesis from these proteins. Indeed, the quantity of bile acids, which was reduced in HFD conditions, increased in the PRE group, accompanied by an elevation in the expression of synthesis-related proteins. Our findings suggest that the beneficial effects of PRE in preventing hepatic steatosis may be mediated, at least in part, through the modulation of FXR/RXR activation, cholesterol biosynthesis, and bile acid synthesis pathways.

Ultrasensitive biosensing platform for Mycobacterium tuberculosis detection based on functionalized graphene devices.

Tuberculosis (TB) has high morbidity as a chronic infectious disease transmitted mainly through the respiratory tract. However, the conventional diagnosis methods for TB are time-consuming and require specialists, making the diagnosis of TB with point-of-care (POC) detection difficult. Here, we developed a graphene-based field-effect transistor (GFET) biosensor for detecting the MPT64 protein of Mycobacterium tuberculosis with high sensitivity as a POC detection platform for TB. For effective conjugation of antibodies, the graphene channels of the GFET were functionalized by immobilizing 1,5-diaminonaphthalene (1,5-DAN) and glutaraldehyde linker molecules onto the graphene surface. The successful immobilization of linker molecules with spatial uniformity on the graphene surface and subsequent antibody conjugation were confirmed by Raman spectroscopy and X-ray photoelectron spectroscopy. The GFET functionalized with MPT64 antibodies showed MPT64 detection with a detection limit of 1 fg/mL in real-time, indicating that the GFET biosensor is highly sensitive. Compared to rapid detection tests (RDT) and enzyme-linked immunosorbent assays, the GFET biosensor platform developed in this study showed much higher sensitivity but much smaller dynamic range. Due to its high sensitivity, the GFET biosensor platform can bridge the gap between time-consuming molecular diagnostics and low-sensitivity RDT, potentially aiding in early detection or management of relapses in infectious diseases.

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Serum proteomics of severe fever with thrombocytopenia syndrome patients.

BACKGROUND: Dabie bandavirus, also termed as severe fever with thrombocytopenia syndrome virus (SFTSV), was first isolated in China in 2010. At this time, the virus was found to have spread to South Korea, Japan, and other countries. A high case fatality rate is reported for SFTS, ranging from 12-50% within various sources. Several omics for clinical studies among SFTS patients as well as studies of cultured SFTSV have attempted to characterize the relevant molecular biology and epidemiology of the disease. However, a global serum proteomics analysis among SFTS patients has not yet been reported to date. METHODS: In the current study, we evaluated comparative serum proteomics among SFTS patients (eight recovered patients and three deceased patients) with the goal of identifying the protein expression patterns associated with the clinical manifestations of SFTS. RESULTS: The proteomic results in the current study showed that the coagulation factor proteins, protein S and protein C, were statistically significantly downregulated among the deceased patients. Downregulation of the complement system as well as prolonged neutrophil activation were also observed. Additionally, the downstream proteins of tumour necrosis factor alpha, neutrophil-activating cytokine, and interleukin-1ß, an inflammatory cytokine, were overexpressed. CONCLUSIONS: Thrombocytopenia and multiple organ failure are the major immediate causes of death among SFTS patients. In this study, serum proteomic changes related to thrombocytopenia, abnormal immune response, and inflammatory activation were documented in SFTS patients. These findings provide useful information for understanding the clinical manifestations of SFTS.

Proteomics-based diagnostic peptide discovery for severe fever with thrombocytopenia syndrome virus in patients.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging infectious virus which causes severe hemorrhage, thrombocytopenia, and leukopenia, with a high fatality rate. Since there is no approved therapeutics or vaccines for SFTS, early diagnosis is essential to manage this infectious disease. METHODS: Here, we tried to detect SFTS virus in serum samples from SFTS patients by proteomic analysis. Firstly, in order to obtain the reference MS/MS spectral data of SFTS virus, medium from infected Vero cell culture was used for shotgun proteomic analysis. Then, tryptic peptides in sera from SFTS patients were confirmed by comparative analysis with the reference MS/MS spectral data of SFTS virus. RESULTS: Proteomic analysis of culture medium successfully discovered tryptic peptides from all the five antigen proteins of SFTS virus. The comparative spectral analysis of sera of SFTS patients revealed that the N-terminal tryptic peptide of the nucleocapsid (N) protein is the major epitope of SFTS virus detected in the patient samples. The prevalence of the peptides was strongly correlated with the viral load in the clinical samples. CONCLUSIONS: Proteomic analysis of SFTS patient samples revealed that nucleocapsid (N) protein is the major antigen proteins in sera of SFTS patients and N-terminal tryptic peptide of the N protein might be a useful proteomic target for direct detection of SFTS virus. These findings suggest that proteomic analysis could be an alternative tool for detection of pathogens in clinical samples and diagnosis of infectious diseases.

Computational Method-Based Optimization of Carbon Nanotube Thin-Film Immunosensor for Rapid Detection of SARS-CoV-2 Virus.

The recent global spread of COVID-19 stresses the importance of developing diagnostic testing that is rapid and does not require specialized laboratories. In this regard, nanomaterial thin-film-based immunosensors fabricated via solution processing are promising, potentially due to their mass manufacturability, on-site detection, and high sensitivity that enable direct detection of virus without the need for molecular amplification. However, thus far, thin-film-based biosensors have been fabricated without properly analyzing how the thin-film properties are correlated with the biosensor performance, limiting the understanding of property-performance relationships and the optimization process. Herein, the correlations between various thin-film properties and the sensitivity of carbon nanotube thin-film-based immunosensors are systematically analyzed, through which optimal sensitivity is attained. Sensitivities toward SARS-CoV-2 nucleocapsid protein in buffer solution and in the lysed virus are 0.024 [fg/mL]-1 and 0.048 [copies/mL]-1, respectively, which are sufficient for diagnosing patients in the early stages of COVID-19. The technique, therefore, can potentially elucidate complex relationships between properties and performance of biosensors, thereby enabling systematic optimization to further advance the applicability of biosensors for accurate and rapid point-of-care (POC) diagnosis.

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.

Identification of a Novel Antigen for Serological Diagnosis of Scrub Typhus.

Scrub typhus is an acute infectious disease caused by the bacterium Orientia tsutsugamushi, which is widely distributed in northern, southern, and eastern Asia. Early diagnosis is essential because the average case fatality rate is usually >10% but can be as high as 45% if antimicrobial treatment is delayed. Although an O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used for serological diagnosis of scrub typhus, the 56-kDa TSA shows variations among O. tsutsugamushi strains, which may lead to poor diagnostic results. Therefore, the discovery of new antigenic proteins may improve diagnostic accuracy. In this study, we identified an O. tsutsugamushi 27 kDa antigen through an immunoinformatic approach and verified its diagnostic potential using patient samples. Compared with the O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity with similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa antigen shows potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.

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.

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Application of multi-omics technology for the elucidation of anti-pneumococcal activity of 3-acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivative against Streptococcus pneumoniae.

Streptococcus pneumoniae is one of Gram-positive pathogen that causes invasive pneumococcal disease. Nowadays, many S. pneumoniae strains are resistant to commonly used antibiotics such as ß-lactams and macrolides. 3-Acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivatives are known as novel chemicals having anti-pneumococcal activity against S. pneumoniae. The underlying mechanism of the anti-pneumococcal activity of this inhibitor remains unknown. Therefore, we tried to find the anti-pneumococcal mechanism of APDQ230122, one of the APDQ derivatives active against S. pneumoniae. We performed transcriptomic analysis (RNA-Seq) and proteomic analysis (LC-MS/MS analysis) to get differentially expressed genes (DEG) and differentially expressed proteins (DEP) of S. pneumoniae 521 treated with sub-inhibitory concentrations of APDQ230122 and elucidated the comprehensive expression changes of genes and proteins using multi-omics analysis. As a result, genes or proteins of peptidoglycan biosynthesis and DNA replication were significantly down-regulated. Electron microscopy analysis revealed that the structure of peptidoglycan was damaged by APDQ230122 in a chemical concentration-dependent manner. Therefore, we suggest peptidoglycan biosynthesis is a major target of APDQ230122. Multi-omics analysis can provide us useful information to elucidate anti-pneumococcal activity of APDQ230122.

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.

Comparative Analysis of Primer-Probe Sets for RT-qPCR of COVID-19 Causative Virus (SARS-CoV-2).

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within 8 months of the outbreak, more than 10,000,000 cases of COVID-19 have been confirmed worldwide. Since human-to-human transmission occurs easily and the rate of human infection is rapidly increasing, sensitive and early diagnosis is essential to prevent a global outbreak. Recently, the World Health Organization (WHO) announced various primer-probe sets for SARS-CoV-2 developed at different institutions: China Center for Disease Control and Prevention (China CDC, China), Charité (Germany), The University of Hong Kong (HKU, Hong Kong), National Institute of Infectious Diseases in Japan (Japan NIID, Japan), National Institute of Health in Thailand (Thailand NIH, Thailand), and US CDC (USA). In this study, we compared the ability to detect SARS-CoV-2 RNA among seven primer-probe sets for the N gene and three primer-probe sets for the Orf1 gene. The results revealed that "NIID_2019-nCOV_N" from the Japan NIID and "ORF1ab" from China CDC represent a recommendable performance of RT-qPCR analysis for SARS-CoV-2 molecular diagnostics without nonspecific amplification and cross-reactivity for hCoV-229E, hCoV-OC43, and MERS-CoV RNA. Therefore, the appropriate combination of NIID_2019-nCOV_N (Japan NIID) and ORF1ab (China CDC) sets should be selected for sensitive and reliable SARS-CoV-2 molecular diagnostics.

Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor.

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for containing the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clinical samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was determined using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concentrations of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clinical transport medium. In addition, the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 × 101 pfu/mL) and clinical samples (LOD: 2.42 × 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunological diagnostic method for COVID-19 that requires no sample pretreatment or labeling.

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.

Biocompatible liquid-type carbon nanodots (C-paints) as light delivery materials for cell growth and astaxanthin induction of Haematococcus pluvialis.

In this study, we aimed to demonstrate the feasibility of the application of biocompatible liquid type fluorescent carbon nanodots (C-paints) to microalgae by improving microalgae productivity. C-paints were prepared by a simple process of ultrasound irradiation using polyethylene glycol (PEG) as a passivation agent. The resulting C-paints exhibited a carbonyl-rich surface with good uniformity of particle size, excellent water solubility, photo-stability, fluorescence efficiency, and good biocompatibility (<10.0 mg mL-1 of C-paints concentration). In the practical application of C-paints to microalgae culture, the most effective and optimized condition leading to growth promoting effect was observed at a C-paints concentration of 1.0 mg mL-1 (>20% higher than the control cell content). A C-paints concentration of 1-10.0 mg mL-1 induced an approximately >1.8 times higher astaxanthin content than the control cells. The high light delivery effect of non-cytotoxic C-paints was applied as a stress condition for H. pluvialis growth and was found to play a major role in enhancing productivity. Notably, the results from this study are an essential approach to improve astaxanthin production, which can be used in various applications because of its therapeutic effects such as cancer prevention, anti-inflammation, immune stimulation, and treatment of muscle-soreness.

Ultrastructural visualization of Orientia tsutsugamushi in biopsied eschars and monocytes from scrub typhus patients in South Korea.

Scrub typhus, which is caused by Orientia tsutsugamushi, is a public health problem in the Asian-Pacific region and is the third most frequently reported infectious disease in South Korea. While ultrastructural studies have been performed on O. tsutsugamushi in murine fibroblasts, its variable locations in patients have hampered similar studies in humans. Two patients with scrub typhus agreed to provide an eschar biopsy and peripheral blood, respectively. Transmission electron microscopy was performed separately on the necrotic crust and perifocal skin of the eschar, the peripheral blood, and the infected murine L cells. O. tsutsugamushi was located within or adjacent to the outermost layer of the perifocal inflamed skin of the eschar but not in the necrotic centre. O. tsutsugamushi in peripheral blood monocytes exhibited the characteristic features of O. tsutsugamushi in L cells, namely, nearly round shaped bacteria with a size of 1-2 µm and a double membrane bearing a clear halo-like outer layer. The findings confirmed that the bacterium was predominantly located in the inflamed skin around the eschar and that the bacterium had the same ultrastructural features in human monocytes as in L cells. These findings suggest that the perifocal area, not the necrotic centre, should be sampled for diagnosis.

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.

Studies on mass production and highly solar light photocatalytic properties of gray hydrogenated-TiO2 sphere photocatalysts.

In this paper, it is first reported that gray hydrogenated TiO2 sphere photocatalysts (H-TiO2) with high reactivity to solar light are mass produced within a few minutes using an underwater discharge plasma modified sol-gel method at room temperature and atmospheric pressure. This plasma modified system is an easy one-step in-situ synthetic process and the crystallinity, hydrogenation, and spherical structure of H-TiO2 are achieved by the synergy effect between the continuous reaction of highly energetic atomic and molecular species generated from the underwater plasma and surface tension of water. The resultant H-TiO2 demonstrated high anatase/rutile bicrystallinity and extended optical absorption spectrum from the ultraviolet (UV) to visible range. Furthermore, various defects including oxygen vacancies and hydroxyl species on the TiO2 surface permitted the enhancement of the photocatalytic performance. It was demonstrated that H-TiO2 photocatalysts showed significant degradation efficiencies for reactive black 5 (RB 5), rhodamine B (Rho B), and phenol (Ph) under solar light irradiation, up to approximately 5 times higher than that of commercial anatase TiO2 (C-TiO2), which resulted in good water purification. Notably, it was also possible to cultivate HepG2 cells using such well-purified water (to degrees up to 76%), with minimal cytotoxicity. Considering all these results, we believe that this novel plasma technology is promising for important environmental applications.