A sequential liquid dispensing method in a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of foodborne pathogens.

This study proposes a sequential liquid dispensing method using a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of nucleic acid targets across multiple samples in a single operation. A pair of passive valves integrated into each microchamber enabled the liquid to fill towards the center of rotation against the centrifugal force, facilitating the complete removal of air inside the microchamber. Liquid manipulation can be achievable without any surface coating of the device by exploiting the inherent hydrophobicity of the polymer. Furthermore, design guidelines for the optimization of microfluidic devices are clarified. Consequently, our proposed method allows direct liquid dispensing into the reaction chambers without cross-contamination while simultaneously metering the sample/reagent volume for the colorimetric loop-mediated isothermal amplification (LAMP) reaction. In addition, we demonstrated the simultaneous detection of four foodborne pathogens (Salmonella spp., Vibrio parahaemolyticus, Campylobacter spp., and norovirus genogroup II (GII)) across four samples in a centrifugal microfluidic device within 60 min. Furthermore, the device exhibited high quantitation (R 2 > 0.98) of the DNA concentration in the sample. Our proposed method enables a more compact design by eliminating the need for metering chambers and offers a point-of-care testing platform with high simplicity as it operates at a constant rotational speed.

Ciclesonide Inhibits SARS-CoV-2 Papain-Like Protease in Vitro.

The emergence of coronavirus disease 2019 (COVID-19), a novel identified pneumonia resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has significantly impacted and posed significant challenges to human society. The papain-like protease (PLpro) found in the nonstructural protein 3 of SARS-CoV-2 plays a vital role in viral replication. Moreover, PLpro disrupts the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 from host proteins. Consequently, PLpro has emerged as a promising drug target against SARS-CoV-2 infection. Computational studies have reported that ciclesonide can bind to SARS-CoV-2 PLpro. However, the inhibitory effects of ciclenoside on the PLpro have not been experimentally evaluated. Here, we evaluated the inhibitory effects of synthetic glucocorticoids (sGCs), including ciclesonide, on SARS-CoV-2 PLpro in vitro assay. Ciclesonide significantly inhibited the enzymatic activity of PLpro, compared with other sGCs and its IC50 was 18.4 ± 1.89 µM. These findings provide insights into the development of PLpro inhibitors.

Inhibitory effects of senkyuchachosan on SARS-CoV-2 papain-like protease activity in vitro.

Papain-like protease (PLpro) enzyme plays a vital role in viral replication as it breaks down polyproteins and disrupts the host's immune response. There are few reports on Kampo formulas that focus on PLpro activity. In this study, we evaluated the inhibitory effects of senkyuchachosan, a traditional Japanese medicine, on PLpro of SARS-CoV-2, the virus responsible for causing COVID-19. We purified the PLpro enzyme and conducted in vitro enzymatic assays using specific substrates. Among the nine crude drugs present in senkyuchachosan, four (Cyperi Rhizoma, Schizonepetae Spica, Menthae Herba, and Camelliae sinensis Folium [CsF]) strongly inhibited PLpro activity. CsF, derived from Camellia sinensis (green tea), contains polyphenols, including catechins and tannins. To confirm that the PLpro inhibitory effects of senkyuchachosan predominantly stem from tannins, the tannins were removed from the decoction using polyvinylpolypyrrolidone (PVPP). The inhibitory effect of senkyuchachosan on PLpro activity was reduced by the removal of PVPP. In addition, the tannin fraction obtained from the CsF extracts showed significant PLpro inhibitory effects. These findings lay the groundwork for the potential development of therapeutic agents that target SARS-CoV-2 infection by intervening in proteolytic cleavage of the virus.

A Tricyclic Aromatic Polyketide Isolated from the Marine-Derived Fungus Curvularia aeria.

A novel tricyclic polyketide, curvulanone (1), was isolated from the marine-derived fungus Curvularia aeria. The structure of 1 was determined by NMR and single-crystal X-ray crystallography. 1 had a cyclopentabenzopyranone with 3-acetic acid structure that is rarely found in natural compounds. Monoamine oxidase and sirtuin 1 inhibitory test was exhibited and 1 showed their inhibitory activity.

Inhibition of furin-like enzymatic activities and SARS-CoV-2 infection by osthole and phenolic compounds with aryl side chains.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread as a pandemic and caused damage to people's lives and countries' economies. The spike (S) protein of SARS-CoV-2 contains a cleavage motif, Arg-X-X-Arg, for furin and furin-like enzymes at the boundary of the S1/S2 subunits. Given that cleavage plays a crucial role in S protein activation and viral entry, the cleavage motif was selected as the target. Our previous fluorogenic substrate study showed that osthole, a coumarin compound, inhibits furin-like enzyme activity. In this study, we examined the potential activities of 15 compounds with a structure-activity relationship with osthole, and evaluated their protective ability against SARS-CoV-2 infection. Of the 15 compounds tested, compounds C1 and C2 exhibited the inhibitory effects of osthole against furin-like enzymatic activity; however, little or no inhibitory effects against furin activity were observed. We further examined the inhibition of SARS-CoV-2 activity by compounds C1 and C2 using a Vero E6 cell line that expresses the transmembrane protease serine 2 (TMPRSS2). Compounds C1, C2, and osthole effectively inhibited SARS-CoV-2 infection. Therefore, osthole and its derivatives can potentially be used as therapeutic agents against SARS-CoV-2.

Development of Novel Monoclonal Antibodies Against Nattokinase.

Nattokinase is a protease produced by Bacillus subtilis var. natto that exhibits various beneficial biological effects. Thus, a reliable assay to determine nattokinase levels is needed. In this study, we developed novel mouse monoclonal antibodies (mAbs) that recognize nattokinase, and created a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure nattokinase levels. The ELISA was developed using a combination of new mouse antinattokinase mAbs used as capture antibodies coated onto 96-well plates, with a peroxidase-conjugated antibody used for detection. This ELISA enabled detection of nattokinase at 1 ng/mL. We believe that the novel mAbs developed in this study will be useful in future for elucidating nattokinase function.

Inhibitory effect of Ephedra herba on human norovirus infection in human intestinal organoids.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases worldwide with public health concern, yet no antiviral therapies have been developed. In this study, we aimed to screen crude drugs, which are components of Japanese traditional medicine, ''Kampo'' to see their effects on HuNoV infection using a reproducible HuNoV cultivation system, stem-cell derived human intestinal organoids/enteroids (HIOs). Among the 22 crude drugs tested, Ephedra herba significantly inhibited HuNoV infection in HIOs. A time-of-drug addition experiment suggested that this crude drug more preferentially targets post-entry step than entry step for the inhibition. To our knowledge, this is the first anti-HuNoV inhibitor screen targeting crude drugs, and Ephedra herba was identified as a novel inhibitor candidate that merits further study.

Anti-inflammatory Effect of a Combination of Cannabidiol and Morinda citrifolia Extract on Lipopolysaccharide-stimulated RAW264 Macrophages.

BACKGROUND/AIM: The inflammatory response plays an important role in the activation and progression of many inflammation-related diseases. Cannabis sativa and Morinda citrifolia have long been used in folk medicine to treat inflammation. Cannabidiol is the most abundant non-psychoactive phytocannabinoid in C. sativa and exhibits anti-inflammatory activity. The objective of this study was to examine the anti-inflammatory effect of cannabidiol in combination with M. citrifolia and compare its effects with those of cannabidiol alone. MATERIALS AND METHODS: RAW264 cells stimulated with lipopolysaccharide (200 ng/ml) were treated with cannabidiol (0-10 µM), M. citrifolia seed extract (0-100 µg/ml), or a combination of both for 8 or 24 h. Following the treatments, nitric oxide production in the activated RAW264 cells and the expression of inducible nitric oxide synthase were assessed. RESULTS: Our results showed that combination of cannabidiol (2.5 µM) and M. citrifolia seed extract (100 µg/ml) exhibited more efficient inhibition of nitric oxide production than cannabidiol treatment alone in lipopolysaccharide-stimulated RAW264 cells. The combination treatment also reduced the expression of inducible nitric oxide synthase. CONCLUSION: These results suggest that the anti-inflammatory effect of combined treatment with cannabidiol and M. citrifolia seed extract causes a reduction in the expression of inflammatory mediators.

Gene knockdown in HaCaT cells by small interfering RNAs entrapped in grapefruit-derived extracellular vesicles using a microfluidic device.

Small interfering RNAs (siRNAs) knockdown the expression of target genes by causing mRNA degradation and are a promising therapeutic modality. In clinical practice, lipid nanoparticles (LNPs) are used to deliver RNAs, such as siRNA and mRNA, into cells. However, these artificial nanoparticles are toxic and immunogenic. Thus, we focused on extracellular vesicles (EVs), natural drug delivery systems, for the delivery of nucleic acids. EVs deliver RNAs and proteins to specific tissues to regulate various physiological phenomena in vivo. Here, we propose a novel method for the preparation siRNAs encapsulated in EVs using a microfluidic device (MD). MDs can be used to generate nanoparticles, such as LNPs, by controlling flow rate to the device, but the loading of siRNAs into EVs using MDs has not been reported previously. In this study, we demonstrated a method for loading siRNAs into grapefruit-derived EVs (GEVs), which have gained attention in recent years for being plant-derived EVs developed using an MD. GEVs were collected from grapefruit juice using the one-step sucrose cushion method, and then GEVs-siRNA-GEVs were prepared using an MD device. The morphology of GEVs and siRNA-GEVs was observed using a cryogenic transmission electron microscope. Cellular uptake and intracellular trafficking of GEVs or siRNA-GEVs to human keratinocytes were evaluated by microscopy using HaCaT cells. The prepared siRNA-GEVs encapsulated 11% of siRNAs. Moreover, intracellular delivery of siRNA and gene suppression effects in HaCaT cells were achieved using these siRNA-GEVs. Our findings suggested that MDs can be used to prepare siRNA-EV formulations.

Effect of Maillard reaction on the quality of clarified butter, ghee.

In Ayurveda, a traditional Indian medicine system, clarified butter is called ghee and is used for food and medicinal purposes. Since butter is subjected to heat to prepare ghee, the heating process affects the ghee quality, such as oxidation, flavor, nutritional value, and biological activity. Therefore, this study focused on the Maillard reaction progress and free-radical scavenging activity with temperature and time during ghee preparation. First, ghee was prepared at low to high temperatures, and its quality (milk fat content, retinol, α-tocopherol, peroxide value, Maillard reaction progress, and free radical scavenging activity) was evaluated. Maillard reaction progress was enhanced at medium and high temperatures (120-160 â„ƒ), and the free radical-scavenging activity of ghee corresponded to the Maillard reaction progress. Since ghee is often reheated during use, we further evaluated the effect of the reheating process. The reheating process did not alter the Maillard reaction progress or the free radical scavenging activity. Our findings serve as good quality control measures for ghee preparation.

1H-NMR-Based Metabolomics for the Classification of the Roots of Paeonia lactiflora, a Constituent of Kampo Medicines.

The root of Paeonia lactiflora (PAEONIAE RADIX) is a constituent of the traditional Japanese medicines (Kampo) and is known to have various effects. Peony roots cultivated in Japan and China are available in the Japanese market for medicinal use. In this study, the chemical diversity of ten available peony roots in the market that differed in their cultivation area was investigated using 1H-NMR metabolomics techniques. Principal component analysis and hierarchical cluster analysis of the 1H-NMR spectra of the peony roots methanolic extracts revealed a clear difference between the metabolic profiles of Japanese and Chinese peony roots. By preparative procedures using chromatography based on 1H-NMR spectra measurements, oxypaeoniflorin and (+)-catechin were found to be specific compounds for Japanese peony root. All peony roots used in this study were listed in the Japanese Pharmacopoeia. Therefore, the differences in the constituents of these peony roots might be attributed to growing conditions than differences in species. Cultivation conditions also influence the quality of natural medicines.

Degradative Effect of Nattokinase on Spike Protein of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic and has inflicted enormous damage on the lives of the people and economy of many countries worldwide. However, therapeutic agents against SARS-CoV-2 remain unclear. SARS-CoV-2 has a spike protein (S protein), and cleavage of the S protein is essential for viral entry. Nattokinase is produced by Bacillus subtilis var. natto and is beneficial to human health. In this study, we examined the effect of nattokinase on the S protein of SARS-CoV-2. When cell lysates transfected with S protein were incubated with nattokinase, the S protein was degraded in a dose- and time-dependent manner. Immunofluorescence analysis showed that S protein on the cell surface was degraded when nattokinase was added to the culture medium. Thus, our findings suggest that nattokinase exhibits potential for the inhibition of SARS-CoV-2 infection via S protein degradation.

A microfluidic diagnostic device with air plug-in valves for the simultaneous genetic detection of various food allergens.

The identification of accidental allergen contamination in processed foods is crucial for risk management strategies in the food processing industry to effectively prevent food allergy incidents. Here, we propose a newly designed passive stop valve with high pressure resistance performance termed an "air plug-in valve" to further improve microfluidic devices for the detection of target nucleic acids. By implementing the air plug-in valve as a permanent stop valve, a maximal allowable flow rate of 70 µL/min could be achieved for sequential liquid dispensing into an array of 10 microchambers, which is 14 times higher than that achieved with the previous valve arrangement using single-faced stop valves. Additionally, we demonstrate the simultaneous detection of multiple food allergens (wheat, buckwheat, and peanut) based on the colorimetric loop-mediated isothermal amplification assay using our diagnostic device with 10 microchambers compactly arranged in a 20-mm-diameter circle. After running the assays at 60 °C for 60 min, any combination of the three types of food allergens and tea plant, which were used as positive and negative control samples, respectively, yielded correct test results, without any cross-contamination among the microchambers. Thus, our diagnostic device will provide a rapid and easy sample-to-answer platform for ensuring food safety and security.

Antifungal activity of dehydrocurvularin for Candida spp. through the inhibition of adhesion to human adenocarcinoma cells.

Cell adhesion plays a crucial role in candidiasis through invasion of the human body and obtaining resistance to drugs by forming biofilms. Cell adhesion thus is a critical target for combating candidiasis by preventing the entry of fungal hyphae into the epithelium. We report here that dehydrocurvularin (1), isolated from the marine-derived fungus Curvularia aeria, exhibited anti-fungal activities for Candida albicans and Candida auris. This compound also prevented the adherence of C. albicans to human adenocarcinoma cells. Real-time RT-PCR analysis showed that exposure to 1 results in decreased expression of HWP1, EFG1, and ECE1, genes involved in Candida adhesion to epithelial cells and hyphal morphogenesis.

Fruit body formation and intra-species DNA polymorphism in Japanese Wolfiporia cocos strains.

Poria, the dried sclerotium of Wolfiporia cocos, is a medicinal mushroom that is widely used in traditional Japanese medicine. The fruit body of W. cocos is rarely found in the natural environment in Japan, therefore an optimized technique for fruit body formation is essential for producing new strains through crossbreeding and for biological research. Here, we developed a cultivation technique for fruit body formation of W. cocos using three strains collected from different areas of Japan. When mycelia were cultured on sawdust-based medium after liquid medium culture, all strains successfully formed fruit bodies as a brown honeycomb-like structure. Furthermore, we analyzed single nucleotide polymorphisms of the three strains using the STE3-like pheromone receptor protein gene, STE3.2, and found a genetic marker for discriminating one strain from the others. The results are expected to promote extensive studies on crossbreeding and domestic production of W. cocos.

Curcumae Longae Rhizoma and Saussureae Radix Inhibit Nitric Oxide Production and Cannabinoid Receptor 2 Down-regulation.

BACKGROUND/AIM: The cannabinoid 2 (CB2) receptor is an important regulator of immunoinflammatory responses. Crude drugs commonly used in Japanese traditional Kampo medicine have displayed anti-inflammatory effects; however, few studies have reported that these effects are mediated via CB2 receptor signaling. Therefore, this study aimed to elucidate CB2 receptor-related anti-inflammatory regulation in crude drugs. MATERIALS AND METHODS: The ethanol extracts of 34 crude drugs listed in the Japanese Pharmacopeia were tested, and the inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production were evaluated in murine macrophage RAW 264 cells. RESULTS: The extracts of Curcumae Longae Rhizoma (dried rhizome of Curcuma longa) and Saussureae Radix (dried root of Saussurea lappa) significantly inhibited NO production and attenuated the LPS-induced decrease in CB2 receptor mRNA expression. CONCLUSION: Curcumae Longae Rhizoma and Saussureae Radix can modulate the CB2-receptor-related anti-inflammatory regulation in macrophages.

Comparison of various commercially available cinnamon barks using NMR metabolomics and the quantification of coumarin by quantitative NMR methods.

Cinnamon bark is an important spice worldwide. In this study, the chemical diversity of various commercially available cinnamon barks that differed in their production areas and utility applications (culinary spice or medicines) were investigated by the use of 1H NMR metabolomics. Our results indicated that principle component analysis (PCA) and hierarchical cluster analysis (HCA) of the 1H NMR spectra of the cinnamon bark methanolic extracts including the deduction of their species by nucleotide sequence analysis enabled differentiation of the cinnamon barks according to their species, production areas and utility applications. The constituents of Vietnam cinnamon were found to differ significantly from the other samples investigated based on PCA score plots and HCA constellation dendrograms. Coumarin was found to be a key compound for the discrimination of Vietnamese cinnamon by multivariate analysis of the 1H NMR spectral data and direct comparison of the 1H NMR spectra. In addition, coumarin was quantified using quantitative NMR methods. As a result, coumarin was contained in Vietnamese cinnamon at a higher level compared to other cinnamons. This study indicated that 1H NMR metabolomics could deduce spices, utility, and producing area of commercially available cinnamon barks. Furthermore, combining quantitative 1H NMR methods with 1H NMR metabolomics enable quantification of coumarin in cinnamon bark on a single measurement.

Inhibitory effect of honokiol on furin-like activity and SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a pandemic and has caused damage to the lives of the people and economy of countries. However, the therapeutic reagents against SARS-CoV-2 remain unclear. The spike (S) protein of SARS-CoV-2 contains a cleavage motif at the S1/S2 boundary, known to be cleaved by furin. As cleavage is essential for S protein activation and viral entry, furin was selected as the target compound. In this study, we examined the inhibitory effects of two lignans (honokiol and magnolol) on furin-like enzymatic activity using a fluorogenic substrate with whole-cell lysates. Of two compounds tested, honokiol partially inhibited furin-like enzymatic activity. We further examined the anti-SARS-CoV-2 activity of honokiol using VeroE6 cell line, which is stably expressing a transmembrane protease serine 2 (TMPRSS2). It was shown that honokiol exhibited remarkable inhibition of SARS-CoV-2 infection. Therefore, honokiol and crude drugs which contain honokiol such as Magnolia species have a potential therapeutic reagents for SARS-CoV-2.

Anti-Inflammatory Effects of Morinda citrifolia Extract against Lipopolysaccharide-Induced Inflammation in RAW264 Cells.

Leaves of Morinda citrifolia (noni) have been used in Polynesian folk medicine for the treatment of pain and inflammation, and their juice is very popular worldwide as a functional food supplement. This study aimed to demonstrate that M. citrifolia seed extract exerts anti-inflammatory effects on RAW264 cells stimulated by lipopolysaccharide. To confirm the inhibitory effect of M. citrifolia seed extract, we assessed the production of nitric oxide (NO) and inflammatory cytokines. The M. citrifolia seed extract showed a significant inhibition of NO production, with no effect on cell viability, and was more active than M. citrifolia seed oil, leaf extract, and fruit extract. The M. citrifolia seed extract was found to reduce the expression of inducible NO synthase and tumor necrosis factor-alpha of pro-inflammatory cytokines. These results suggest that the anti-inflammatory effect of M. citrifolia seed extract is related to a reduction in the expression of inflammatory mediators and support its potential therapeutic use.

Screening for inhibitory effects of crude drugs on furin-like enzymatic activities.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a cleavage motif R-X-X-R for furin-like enzymes at the boundary of the S1/S2 subunits. The cleavage of the site by cellular proteases is essential for S protein activation and virus entry. We screened the inhibitory effects of crude drugs on in vitro furin-like enzymatic activities using a fluorogenic substrate with whole-cell lysates. Of the 124 crude drugs listed in the Japanese Pharmacopeia, aqueous ethanolic extract of Cnidii Monnieris Fructus, which is the dried fruit of Cnidium monnieri Cussion, significantly inhibited the furin-like enzymatic activities. We further fractionated the plant extract and isolated the two active compounds with the inhibitory activity, namely, imperatorin and osthole, whose IC50 values were 1.45 mM and 9.45 µM, respectively. Our results indicated that Cnidii Monnieris Fructus might exert inhibitory effects on furin-like enzymatic activities, and that imperatorin and osthole of the crude drug could be potential inhibitors of the motif cleavage.