A chimeric MERS-CoV virus-like particle vaccine protects mice against MERS-CoV challenge.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. The lack of approved vaccines or antivirals currently constitutes a barrier in controlling disease outbreaks and spread. METHODS: In this study, using a mammalian expression system, which is advantageous for maintaining correct protein glycosylation patterns, we constructed chimeric MERS-CoV virus-like particles (VLPs) and determined their immunogenicity and protective efficacy in mice. RESULTS: Western blot and cryo-electron microscopy analyses demonstrated that MERS-CoV VLPs were efficiently produced in cells co-transfected with MERS-CoV spike (S), envelope, membrane and murine hepatitis virus nucleocapsid genes. We examined their ability as a vaccine in a human dipeptidyl peptidase 4 knock-in C57BL/6 congenic mouse model. Mice immunized with MERS VLPs produced S-specific antibodies with virus neutralization activity. Furthermore, MERS-CoV VLP immunization provided complete protection against a lethal challenge with mouse-adapted MERS-CoV and improved virus clearance in the lung. CONCLUSIONS: Overall, these data demonstrate that MERS-CoV VLPs have excellent immunogenicity and represent a promising vaccine candidate.

Heat shock protein 70 could enhance porcine epidemic diarrhoea virus replication by interacting with membrane proteins.

In this study, we investigated the role of heat shock protein 70 (HSP70) in porcine epidemic diarrhoea virus (PEDV) replication. We found that PEDV infection induced strong HSP70 overexpression in the very early stage of infection. We also confirmed that HSP70 overexpression increased the speed of PEDV replication, resulting in the generation of more virions. In contrast, knockout of HSP70 in cells significantly downregulated PEDV protein expression, resulting in a significant reduction in PEDV replication. Most importantly, we confirmed that among the structural proteins of PEDV, membrane (M) proteins have this important role. We found that membrane proteins control cellular HSP70 expression in PEDV-infected cells. We confirmed HSP70/M complex formation by both immunoprecipitation and immunofluorescence assays. Additionally, PEDV M overexpression induced strong HSP70 expression. All our results clearly confirmed that in PEDV-infected cells, the M protein plays a very important role in PEDV replication in collaboration with HSP70.

Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella.

Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs' accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs' production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.

Determination of the Absolute Configuration of Polyhydroxy Compound Ostreol B Isolated from the Dinoflagellate Ostreopsis cf. ovata.

Following isolation of the polyhydroxy compound, ostreol B, from cultivated cells of the toxic dinoflagellate Ostreopsis cf. ovata collected in South Korea, 1D and 2D NMR spectroscopy were employed to determine the planar chemical structure of this compound, which contained a tetrahydropyran ring, two terminal double bonds, and 21 hydroxyl groups. The absolute configurations of all stereogenic carbon centers in ostreol B were then determined through a combination of the J-based configuration analysis, rotating frame Overhauser effect correlations, and the modified Mosher method following cleavage of the 1,2-diol bonds. Ostreol B was also found to exhibit moderate cytotoxicity in HepG2, Neuro-2a and HCT-116 cells.

Characterization of a New Trioxilin and a Sulfoquinovosyl Diacylglycerol with Anti-Inflammatory Properties from the Dinoflagellate Oxyrrhis marina.

Two new compounds-a trioxilin and a sulfoquinovosyl diacylglycerol (SQDG)-were isolated from the methanolic extract of the heterotrophic dinoflagellate Oxyrrhis marina cultivated by feeding on dried yeasts. The trioxilin was identified as (4Z,8E,13Z,16Z,19Z) -7(S),10(S),11(S)-trihydroxydocosapentaenoic acid (1), and the SQDG was identified as (2S)-1-O-hexadecanosy-2-O-docosahexaenoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol (2) by a combination of nuclear magnetic resonance (NMR) spectra, mass analyses, and chemical reactions. The two compounds were associated with docosahexaenoic acid, which is a major component of O. marina. The two isolated compounds showed significant nitric oxide inhibitory activity on lipopolysaccharide-induced RAW264.7 cells. Compound 2 showed no cytotoxicity against hepatocarcinoma (HepG2), neuroblastoma (Neuro-2a), and colon cancer (HCT-116) cells, while weak cytotoxicity was observed for compound 1 against Neuro-2a cells.

Insights into the Pathogenesis and Development of Recombinant Japanese Encephalitis Virus Genotype 3 as a Vaccine.

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development.

Development of saxitoxin biosynthesis gene sxtB-targeted qPCR assay for the quantification of toxic dinoflagellates Alexandrium catenella (group I) and A. pacificum (group IV) occurring in the Korean coast.

Toxic dinoflagellate Alexandrium can produce saxitoxins (STXs) and cause paralytic shellfish poisoning (PSP), and thus they are monitored for environmental safety management. Microscopic discrimination of dinoflagellates is difficult to distinguish between toxic and non-toxic species due to their similar morphology. Meanwhile, an alternative quantitative PCR (qPCR) assay is sensitive, rapid, and cost-effective for harmful species monitoring. Herein, we developed a novel qPCR assay to detect the STXs biosynthesis gene sxtB of Alexandrium catenella and A. pacificum, the leading cause of PSP outbreaks in Asian coasts and worldwide. The newly designed sxtB TaqMan probes target the species without any positive signal in other relative dinoflagellates. Deming regression analysis revealed that the sxtB copy number of A. catenella and A. pacificum was 3.6 and 4.1 copies per cell, respectively. During the blooming periods (April 13th-14th, 2020), only A. catenella cells were detected through the qPCR assay, ranging from 5.0 × 10 to 2.5 × 104 eq cells L-1. In addition, sxtB qPCR quantified more accurately compared to large subunit (LSU) rRNA targeting qPCR assay that overestimate cell density. Besides, the sensitivity of sxtB was higher compared to the microscope when the species were rarely present (5.0 × 102 cells L-1). These suggest that the sxtB qPCR assay can be applied to toxic Alexandrium monitoring in the Korean coast, even in the early stage of bloomings.

Quantification of Alexandrium catenella (Group I) using sxtA4-based digital PCR for screening of paralytic shellfish toxins in Jinhae-Masan Bay, Korea.

We employed a detection method to quantify Alexandrium catenella (Group I), one of the causative species for paralytic shellfish poisoning (PSP) in Jinhae-Masan Bay, Korea, targets sxtA4, via chip-based digital PCR. Additionally, we explored the dynamics of Alexandrium during the spring of 2022 using an rDNA-based quantitative PCR (qPCR) assay to enhance the performance of the dPCR assay. In matching dPCR results with PSP monitoring reports, we optimized a cell regulatory threshold of 102 cells L-1, the maximum cell density when shellfish harvesting was permitted, for the dPCR assay. This threshold functioned similar to the PST threshold used in mouse bioassays (MBAs). Furthermore, we validated a total concordance rate of 83.8 % between the two assays for 2020-2022, reaching a maximum of 96.2 % in 2020. Thus, the result of dPCR could complement MBAs, facilitating the early detection of PSP outbreaks.

A one-step immunoassay based on switching peptides for diagnosis of porcine epidemic diarrhea virus (PEDV) using screened Fv-antibodies.

In this study, a one-step immunoassay for porcine epidemic diarrhea virus (PEDV) based on Fv-antibodies and switching peptides was developed, and the assay results of PEDV were obtained by just mixing samples without any further reaction or washing steps. The Fv-antibodies with binding affinity to the spike protein of PEDV were screened from the Fv-antibody library using the receptor-binding domain (RBD) of the spike protein as a screening probe. Screened Fv-antibodies with binding affinities to the RBD antigen were expressed, and the binding constants (KD) were calculated to be 83-142 nM. The one-step immunoassay for the detection of PEDV was configured as a displacement immunoassay using a fluorescence-labeled switching peptide. The one-step immunoassay based on switching peptides was performed using PEDV, and the limit of detection (LOD) values for PEDV detection were estimated to be Ct = 39.7-36.4. Compared with the LOD value for a conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay showed a remarkably improved LOD for the detection of PEDV. Finally, the interaction between the screened Fv-antibodies and the PEDV RBD was investigated using docking simulations and compared with the amino acid sequences of the receptors on host cells, such as aminopeptidase N (APN) and angiotensin-converting enzyme-2 (ACE-2).

Distribution, compositional characteristics, and historical pollution records of microplastics in tidal flats of South Korea.

Studies on distribution of microplastics (MPs) in sediments of tidal flats are relatively scarce compared to other coastal areas. In this study, spatial and vertical distributions and compositions of MPs in tidal flat sediments along the west coast of Korea were investigated. The abundance of MPs in surface and core sediments ranged from 20 to 325 and 14 to 483 particles per 50 g dry weight, respectively. Polypropylene (51%) and polyethylene (36%) were the most dominant MPs; the size was <0.3 mm, and the shape was mostly fragments followed by fibers. The abundance of MPs in sediments has increased rapidly since the 1970s, and recently showed a slight decrease. Surface morphology of MPs analyzed using a scanning electron microscope revealed that the MPs in tidal flats were highly weathered mechanically and/or oxidatively. The results of this study provide valid baseline data on distributions of MPs in tidal flats.

Phenolic compounds in the freshwater environment in South Korea: Occurrence and tissue-specific distribution.

In this study, we investigated the occurrence and distribution of phenolic compounds, including phenol, cresols, chlorophenols, nitrophenol, and bromophenols, in freshwater environments. We also focused on phenolic compounds in crucian carp (Carassius auratus) tissues, specifically the muscle, gills, brain, blood, liver, and gonads, to assess their potential bioaccumulation in fish and human health risks. Phenolic compounds were found to be widespread in various freshwater environments throughout South Korea. Phenol was predominant in all matrices, with median concentrations of 57.0 ng/L in freshwater, 54.3 ng/g dry weight (dw) in sediment, and ranging from 71 ng/g wet weight (ww) to 621 ng/g ww in crucian carp tissues. Cresols were the second most dominant compound, with m-cresol exhibiting the highest prevalence. Most of the compounds detected in crucian carp samples were also detected in freshwater and sediment, whereas pentachlorophenol and 2,4,6-tribromophenol were exclusively detected in crucian carp tissues. A high bioaccumulation potential in the liver was observed for most phenolic compounds [median log bioconcentration factor (BCF): 3.2-3.7]. Interestingly, only m-cresol showed high bioaccumulation potential in the gills (median log BCF: 3.1). The estimated daily intake of phenolic compounds suggested that it does not pose an immediate concern for human exposure owing to crucian carp consumption. These findings enhance our understanding of the exposure status, distribution, and bioaccumulation potency of phenolic compounds in aquatic ecosystems and emphasize the importance of ongoing monitoring and risk assessment efforts.

Self-Attention LSTM-FCN model for arrhythmia classification and uncertainty assessment.

This paper presents ArrhyMon, a self-attention-based LSTM-FCN model for arrhythmia classification from ECG signal inputs. ArrhyMon targets to detect and classify six different types of arrhythmia apart from normal ECG patterns. To the best of our knowledge, ArrhyMon is the first end-to-end classification model that successfully targets the classification of six detailed arrhythmia types and compared to previous work does not require additional preprocessing and/or feature extraction operations separate from the classification model. ArrhyMon's deep learning model is designed to capture and exploit both global and local features embedded in ECG sequences by integrating fully convolutional network (FCN) layers and a self-attention-based long and short-term memory (LSTM) architecture. Moreover, to enhance its practicality, ArrhyMon incorporates a deep ensemble-based uncertainty model that generates a confidence-level measure for each classification result. We evaluate ArrhyMon's effectiveness using three publicly available arrhythmia datasets (i.e., MIT-BIH, Physionet Cardiology Challenge 2017 and 2020/2021) to show that ArrhyMon achieves state-of-the-art classification performance (average accuracy 99.63%), and that confidence measures show close correlation with subjective diagnosis made from practitioners.

The future of butyric acid in industry.

In this paper, the different applications of butyric acid and its current and future production status are highlighted, with a particular emphasis on the biofuels industry. As such, this paper discusses different issues regarding butyric acid fermentations and provides suggestions for future improvements and their approaches.

Porcine Epidemic Diarrhea Virus Infection Induces Autophagosome Formation but Inhibits Autolysosome Formation during Replication.

In this study, we investigated the correlation between the mechanism involved in porcine epidemic diarrhea virus (PEDV) replication and autophagic flux. In this study, we found that as PEDV replicated, production of LC3-II was significantly induced up to 24 h post-infection (hpi). Interestingly, although there was significant production of LC3-II, greater p62 accumulation was simultaneously found. Pretreatment with rapamycin significantly induced PEDV replication, but autolysosome formation was reduced. These results were confirmed by the evaluation of ATG5/ATG12 and LAMP1/LAMP2. Taken together, we conclude that PEDV infection induces autophagosome formation but inhibits autolysosome formation during replication.

Metagenomic Analysis of the Species Composition and Seasonal Distribution of Marine Dinoflagellate Communities in Four Korean Coastal Regions.

Biomonitoring of dinoflagellate communities in marine ecosystems is essential for efficient water quality management and limiting ecosystem disturbances. Current identification and monitoring of toxic dinoflagellates, which cause harmful algal blooms, primarily involves light or scanning electron microscopy; however, these techniques are limited in their ability to monitor dinoflagellates and plankton, leaving an incomplete analysis. In this study, we analyzed the species composition and seasonal distribution of the dinoflagellate communities in four Korean coastal regions using 18S rRNA amplicon sequencing. The results showed significantly high diversity in the dinoflagellate communities in all regions and seasons. Furthermore, we found seasonally dominant species and causative species of harmful algal blooms (Cochlodinium sp., Alexandrium sp., Dinophysis sp., and Gymnodinium sp.). Moreover, dominant species were classified by region and season according to the difference in geographical and environmental parameters. The molecular analysis of the dinoflagellate community based on metagenomics revealed more diverse species compositions that could not be identified by microscopy and revealed potentially harmful or recently introduced dinoflagellate species. In conclusion, metagenomic analysis of dinoflagellate communities was more precise and obtained results faster than microscopic analysis, and could improve the existing monitoring techniques for community analysis.

Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

Alexandrium pacificum, which produces the paralytic shellfish toxin (PST) saxitoxin (STX), is one of the causative species of paralytic shellfish poisoning outbreaks in coastal areas of Korea. In this study, we developed a chip-based digital PCR (dPCR) method for A. pacificum detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an A. pacificum strain isolated in 2017, species-specific primers targeting sxtA4 (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of A. pacificum. Cell abundance in field samples, estimated by a chip-based dPCR, was compared with the PST content, and measured using a mouse bioassay. A comparison with shellfish PST concentrations indicated that cell concentrations above 500 cells L-1, as measured using the dPCR assay, may cause shellfish PST concentrations to exceed the allowed limits for PSTs. Concordance rates between dPCR and PST results were 62.5% overall in 2018-2021, reaching a maximum of 91.7% in 2018-2019. The sensitivity of the dPCR assay was higher than that of microscopy and sxtA4-based qPCRs. Absolute quantification by chip-based dPCRs targeting sxtA4 in A. pacificum exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.

Evaluation of Radiation Resistance of Polystyrene Using Molecular Dynamics Simulation.

The scission rates of polystyrene and fluorinated polystyrene irradiated in an irradiation facility with Co-60 γ-rays were determined using molecular dynamics simulation and gel permeation chromatography (GPC) molecular weight distributions. The prediction was based on the assumption that γ-ray energy is transferred to the initial velocity of the primary knock-on atom. We employed a molecular dynamics simulation procedure to compute the changes in bond length between the connections for selected values of the absorbed dose and compared the calculated values with measurements made on the irradiated samples. The samples were exposed to four different absorbed doses of 25, 50, 75, and 100 kGy. The scission process and scission ratio were simulated with LAMMPS with ReaxFF potential for each bond, and we compared the simulation results with the experimental data especially measuring average molecular weight to evaluate the effect of fluorination on radiation enhancement.

Disentangling the clinical data chaos: User-centered interface system design for trauma centers.

This paper presents a year-long study of our project, aiming at (1) understanding the work practices of clinical staff in trauma intensive care units (TICUs) at a trauma center, with respect to their usage of clinical data interface systems, and (2) developing and evaluating an intuitive and user-centered clinical data interface system for their TICU environments. Based on a long-term field study in an urban trauma center that involved observation-, interview-, and survey-based studies to understand our target users and their working environment, we designed and implemented MediSenseView as a working prototype. MediSenseView is a clinical-data interface system, which was developed through the identification of three core challenges of existing interface system use in a trauma care unit-device separation, usage inefficiency, and system immobility-from the perspectives of three staff groups in our target environment (i.e., doctors, clinical nurses and research nurses), and through an iterative design study. The results from our pilot deployment of MediSenseView and a user study performed with 28 trauma center staff members highlight their work efficiency and satisfaction with MediSenseView compared to existing clinical data interface systems in the hospital.

Differential Diagnosis for Highly Pathogenic Avian Influenza Virus Using Nanoparticles Expressing Chemiluminescence.

Highly pathogenic avian influenza (HPAI) virus is a causative agent of systemic disease in poultry, characterized by high mortality. Rapid diagnosis is crucial for the control of HPAI. In this study, we aimed to develop a differential diagnostic method that can distinguish HPAI from low pathogenic avian influenza (LPAI) viruses using dual split proteins (DSPs). DSPs are chimeras of an enzymatic split, Renilla luciferase (RL), and a non-enzymatic split green fluorescent protein (GFP). Nanoparticles expressing DSPs, sialic acid, and/or transmembrane serine protease 2 (TMPRSS2) were generated, and RL activity was determined in the presence of HPAI or LPAI pseudotyped viruses. The RL activity of nanoparticles containing both DSPs was approximately 2 × 106 RLU, indicating that DSPs can be successfully incorporated into nanoparticles. The RL activity of nanoparticles containing half of the DSPs was around 5 × 101 RLU. When nanoparticles containing half of the DSPs were incubated with HPAI pseudotyped viruses at low pH, RL activity was increased up to 1 × 103 RLU. However, LPAI pseudotyped viruses produced RL activity only in the presence of proteases (trypsin or TMPRSS2), and the average RL activity was around 7 × 102 RLU. We confirmed that nanoparticle fusion assay also diagnoses authentic viruses with specificity of 100% and sensitivity of 91.67%. The data indicated that the developed method distinguished HPAI and LPAI, and suggested that the diagnosis using DSPs could be used for the development of differential diagnostic kits for HPAI after further optimization.

Predicting speech discrimination scores from pure-tone thresholds-A machine learning-based approach using data from 12,697 subjects.

Diagnostic tests for hearing impairment not only determines the presence (or absence) of hearing loss, but also evaluates its degree and type, and provides physicians with essential data for future treatment and rehabilitation. Therefore, accurately measuring hearing loss conditions is very important for proper patient understanding and treatment. In current-day practice, to quantify the level of hearing loss, physicians exploit specialized test scores such as the pure-tone audiometry (PTA) thresholds and speech discrimination scores (SDS) as quantitative metrics in examining a patient's auditory function. However, given that these metrics can be easily affected by various human factors, which includes intentional (or accidental) patient intervention, there are needs to cross validate the accuracy of each metric. By understanding a "normal" relationship between the SDS and PTA, physicians can reveal the need for re-testing, additional testing in different dimensions, and also potential malingering cases. For this purpose, in this work, we propose a prediction model for estimating the SDS of a patient by using PTA thresholds via a Random Forest-based machine learning approach to overcome the limitations of the conventional statistical (or even manual) methods. For designing and evaluating the Random Forest-based prediction model, we collected a large-scale dataset from 12,697 subjects, and report a SDS level prediction accuracy of 95.05% and 96.64% for the left and right ears, respectively. We also present comparisons with other widely-used machine learning algorithms (e.g., Support Vector Machine, Multi-layer Perceptron) to show the effectiveness of our proposed Random Forest-based approach. Results obtained from this study provides implications and potential feasibility in providing a practically-applicable screening tool for identifying patient-intended malingering in hearing loss-related tests.