Development of Three-Stage Bioaerosol Sampler for Size-Selective Sampling.

In this study, a three-stage bio-aerosol sampler with a sampling flow rate of 170 L/min was designed and fabricated for sampling the bio-aerosols released during human breathing and coughing, and its performance was evaluated. The sampler was constructed using a cyclone separator with a cutoff size of 2.5 µm as a preseparator, a multinozzle virtual impactor with a cutoff size of 0.34 µm as an aerosol concentrator, and a Bio-Sampler, which is a commercial product, for collecting bio-aerosols in a collection fluid. The collection efficiency of the sampler was evaluated through simulations and experiments. Only particles with sizes of 0.1-4 µm were selectively collected in the collection fluid. Bacteriophage bio-aerosols were sampled using the developed sampler and ACD-200 Bobcat sampler, which is a commercial product. The amounts of collected bacteriophages were compared using the polymerase chain reaction (PCR) technique. The sampling performance of the developed sampler was similar to that of the ACD-200 Bobcat sampler. Moreover, the developed sampler showed its ability to sample bio-aerosols of a specific size range and collect them directly in a collection fluid for the PCR analysis. Therefore, the developed sampler is expected to be useful for indoor environmental monitoring by effectively sampling the bio-aerosols released indoors during human breathing and coughing.

High-volume sampler for size-selective sampling of bioaerosols including viruses.

Owing to the recent global spread of the new coronavirus SARS-CoV-2, the development of technology to effectively detect viruses in crowded public places is urgently needed. In this study, a three-stage high-volume bioaerosol sampler was developed for the size-selective sampling of bioaerosols through the suction of air at a high flow rate of 1000 L/min. In stage 1, an omnidirectional inlet cyclone separator that can draw air from all directions was applied to collect bioaerosols larger than 10 µm in the collection fluid. In stage 2, an axial flow cyclone separator was used to collect bioaerosols sized between 2.5 and 10 µm in the collection fluid. In stage 3, bioaerosols smaller than 2.5 µm were collected on a filter and extracted in a solution through an elution process using a sodium phosphate buffer. To simulate the suspension of bioparticles including viruses that are attached to other particles in the atmosphere, the aerosol samples were prepared by coagulating aerosolized bacteriophages with Arizona test dust. Then, the coagulated particles were collected for 30 min using the developed bioaerosol sampler, and the samples collected in each stage were analyzed via polymerase chain reaction (PCR) method. The PCR analysis results confirmed that the high-volume bioaerosol sampler enables size-selective bioaerosol sampling even at a high airflow rate of 1000 L/min. The developed high-volume bioaerosol sampler will be useful in detecting viruses through PCR analysis because it can collect bioaerosols within a specific size range.

Enzymatic hydrolysis of anchovy fine powder at high and ambient pressure, and characterization of the hydrolyzates.

BACKGROUND: At specific conditions of high pressure, the stability and activity of some enzymes are reportedly known to increase. The aim of this study was to apply pressure-tolerant proteases to hydrolyzing anchovy fine powder (AFP) and to determine product characteristics of the resultant hydrolyzates. RESULTS: Anchovy fine powder enzyme hydrolyzates (AFPEHs) were produced at 300 MPa and ambient pressure using combinations of Flavourzyme 500MG, Alcalase 2.4L, Marugoto E and Protamex. When the same protease combination was used for hydrolysis, the contents of total soluble solids, total water-soluble nitrogen and trichloroacetic acid-soluble nitrogen in the AFPEHs produced at 300 MPa were conspicuously higher than those in the AFPEHs produced at ambient pressure. This result and electrophoretic characteristics indicated that the high-pressure process of this study accelerates protein hydrolysis compared with the ambient-pressure counterpart. Most peptides in the hydrolyzates obtained at 300 MPa had molecular masses less than 5 kDa. Functionality, sensory characteristics and the content of total free amino acids of selected hydrolyzates were also determined. CONCLUSION: The high-pressure hydrolytic process utilizing pressure-tolerant proteases was found to be an efficient method for producing protein hydrolyzates with good product characteristics.

Role of Junctin protein interactions in cellular dynamics of calsequestrin polymer upon calcium perturbation.

Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca(2+)-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca(2+) that were decondensed upon Ca(2+) depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca(2+) depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca(2+) concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca(2+) and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca(2+) homeostasis.

Extraction of ginsenosides from fresh ginseng roots (Panax ginseng C.A. Meyer) using commercial enzymes and high hydrostatic pressure.

A combination of high hydrostatic pressure (HHP) and enzymatic hydrolysis (HHP-EH) was applied for the extraction of ginsenosides from fresh ginseng roots (Panax ginseng C.A. Myer). The highest yield of ginsenosides was obtained by using a mixture of three enzymes (Celluclast + Termamyl + Viscozyme) along with HHP (100 MPa, at 50 °C for 12 h) in comparison to control samples (no enzymes, atmosphere pressure, P < 0.05). Total ginsenosides increased by 184% while Rg1 + Rb1 increased by 273%. Application of these conditions significantly increased total ginsenosides by 49% and Rg1 + Rb1 by 103% compared to HHP treatment alone (P < 0.05). The effect of HHP on increased yield of ginsenosides is likely due in part, to acceleration of enzyme activity. Thus HHP-EH significantly improves the extraction of ginsenosides from fresh ginseng roots.

Draft genome sequencing of Bacillus sp. strain M2-6, isolated from the roots of Korean ginseng, Panax ginseng C. A. Meyer, after high-hydrostatic-pressure processing.

A bacterium, designated M2-6, was isolated from Korean ginseng, Panax ginseng C. A. Meyer, roots after high-hydrostatic-pressure processing. On the basis of 16 rRNA gene phylogeny, the isolate was presumptively identified as a Bacillus sp. Here we report the draft genome sequence of Bacillus sp. strain M2-6 (= KACC 16563).

Draft genome sequence of Staphylococcus saprophyticus subsp. saprophyticus M1-1, isolated from the gills of a Korean rockfish, Sebastes schlegeli Hilgendorf, after high hydrostatic pressure processing.

A bacterium designated M1-1 was isolated from the gills of a Korean rockfish, Sebastes schlegeli Hilgendorf, after high hydrostatic pressure processing. Studies of 16S rRNA phylogeny and comparative genomics demonstrated that the isolate belongs to Staphylococcus saprophyticus subsp. saprophyticus. Here, we report the draft genome sequence of S. saprophyticus subsp. saprophyticus M1-1 (KACC 16562).

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP).

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

In vitro selection of Escherichia coli O157:H7-specific RNA aptamer.

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes life-threatening symptoms in humans worldwide. To rapidly and properly identify the pathogen and avoid its toxic effects, ligands which can directly and specifically bind to the virulent E. coli O157:H7 serotype should be identified. In this study, a RNA aptamer-based ligand which can specifically distinguish the pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX method. To this end, an RNA library was first incubated with the E. coli K12 strain, and the RNAs binding to the strain were discarded. The precluded RNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. This was evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer that distinguishes between the virulent serotype and the nonpathogenic strain specifically bound to an O157:H7-specific lipopolysaccharide which includes the O antigen. This novel O157:H7-specific aptamer could be of potential application as a diagnostic ligand against the pathogen-related food borne illness.

Cytosolic malate dehydrogenase regulates senescence in human fibroblasts.

Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated ß-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.

Rapid detection of food pathogens using RNA aptamers-immobilized slide.

The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.

A novel isothermal method for amplification of long specific amplicon from linear template.

Isothermal nucleic acid amplification methods have been successfully developed and applied for diagnostic purpose, especially for detection of pathogens. However, amplicon size of such methods is relatively short (< 500 bp) to limit their application for long amplicon production that can be used for various downstream applications including genomic surveillance of pathogens. To fill the gap, we developed a method for specific amplification of kilobases-long target sequence from RNA templates. This method, named CREA, utilizes sequence specific recombination of Cre recombinase to generate circular intermediate template for subsequent RCA reaction. CREA with SARS-CoV-2 spike template could amplify ~ 2.9 kb target and up to ~ 1.9 kb amplicon was able to produce in sufficient amount for general cloning. Each step of CREA procedure was thoroughly analyzed to provide directions for further optimizations. Furthermore, we evaluated a variation of CREA which utilized DNA ligase.

Noninvasive in vivo imaging of CD4 cells in simian-human immunodeficiency virus (SHIV)-infected nonhuman primates.

Since the earliest days of the HIV epidemic, the number of CD4(+) T cells per unit volume of blood has been recognized as a major prognostic factor for the development of AIDS in persons with HIV infection. It has also been generally accepted that approximately 2% of total body lymphocytes circulate in the blood. In the present study, we have used a nondepleting humanized anti-CD4 monoclonal antibody labeled with the gamma emitter indium-111 to visualize the CD4(+) T-cell pool in vivo in nonhuman primates with simian HIV infection. A strong correlation was noted between radiotracer uptake in spleen, tonsil, axillary lymph nodes, and peripheral blood CD4 T-cell counts (rho = 0.75, 0.93, and 0.85, respectively, P < .005). The relationship between radiotracer retention in lymphoid tissues and CD4(+) T-cell counts in the circulation was governed by an exponential law. These data provide an estimate for the total number of lymphocytes in the body as being between 1.9 and 2.9 x 10(12) and suggest that the partition between peripheral blood and lymphoid tissue is between 0.3% and 0.5%.

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons.

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H(2)O(2)) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca(2+)-free medium or treated with BAPTA showed reduced glutamate-induced H(2)O(2) generation, indicating that H(2)O(2) generation is Ca(2+)-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H(2)O(2) generation and that activation of NMDA and AMPA receptors is involved in this H(2)O(2) generation. The Nox4 siRNA reduced NMDA-induced H(2)O(2) production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H(2)O(2) production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.

Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic materials. Here, we provide a protocol to perform reverse transcription LAMP targeting SARS-CoV-2. We adopted both real-time fluorescence detection and end-point colorimetric detection approaches. Our protocol would be useful for screening diagnosis of COVID-19 and be a baseline for development of improved POCT NAAT.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.

A zebrafish model of nondystrophic myotonia with sodium channelopathy.

Nondystrophic myotonias are disorders of Na+ (Nav1.4 or SCN4A) and Cl- (CLCN1) channels in skeletal muscles, and frequently show phenotype heterogeneity. The molecular mechanism underlying their pathophysiology and phenotype heterogeneity remains unclear. As zebrafish models have been recently exploited for studies of the pathophysiology and phenotype heterogeneity of various human genetic diseases, a zebrafish model may be useful for delineating nondystrophic myotonias. Here, we generated transgenic zebrafish expressing a human mutant allele of SCN4A, referred to as Tg(mylpfa:N440K), and needle electromyography revealed increased number of myotonic discharges and positive sharp waves in the muscles of Tg(mylpfa:N440K) than in controls. In addition, forced exercise test at a water temperature of 24 °C showed a decrease in the distance moved, time spent in and number of visits to the zone with stronger swimming resistance. Finally, a forced exercise test at a water temperature of 18 °C exhibited a higher number of dive-bombing periods and drifting-down behavior than in controls. These findings indicate that Tg(mylpfa:N440K) is a good vertebrate model of exercise- and cold-induced human nondystrophic myotonias. This zebrafish model may contribute to provide insight into the pathophysiology of myotonia in sodium channelopathy and could be used to explore a new therapeutic avenue.

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.

Wnt-PLC-IP3-Connexin-Ca2+ axis maintains ependymal motile cilia in zebrafish spinal cord.

Ependymal cells (ECs) are multiciliated neuroepithelial cells that line the ventricles of the brain and the central canal of the spinal cord (SC). How ependymal motile cilia are maintained remains largely unexplored. Here we show that zebrafish embryos deficient in Wnt signaling have defective motile cilia, yet harbor intact basal bodies. With respect to maintenance of ependymal motile cilia, plcδ3a is a target gene of Wnt signaling. Lack of Connexin43 (Cx43), especially its channel function, decreases motile cilia and intercellular Ca2+ wave (ICW) propagation. Genetic ablation of cx43 in zebrafish and mice diminished motile cilia. Finally, Cx43 is also expressed in ECs of the human SC. Taken together, our findings indicate that gap junction mediated ICWs play an important role in the maintenance of ependymal motile cilia, and suggest that the enhancement of functional gap junctions by pharmacological or genetic manipulations may be adopted to ameliorate motile ciliopathy.

Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.

NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.