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This study investigated the effects of salmon nasal cartilage proteoglycan (PG), which shows anti‐inflammatory properties, on obesity induced by high‐fat diet (HFD) in a mouse model. Mice were fed either a HFD or normal diet (ND), with or without PG, for 8–12 weeks. After 12 weeks, the body weight of mice fed with PG‐free HFD was 54.08 ± 4.67 g, whereas that of mice fed with HFD containing PG was 41.83 ± 4.97 g. The results suggest that the increase in body weight was attenuated in mice fed with HFD containing PG. This effect was not observed in mice fed with ND. The PG administration suppressed the elevation of serum lipids (the level of serum lipids ranged between 54% and 69% compared to 100% in mice fed with PG‐free HFD) and the upregulated mRNA expression of sterol regulatory element‐binding protein‐1c (SREBP‐1c), which is a transcription factor that acts as a master regulator of lipogenic gene expression in the liver (the expression level was 77.5% compared to 100% in mice fed with PG‐free HFD). High leptin levels in mice fed with PG‐free HFD were observed during fasting (average at 14,376 ng/ml), and they did not increase after refeeding (average of 14,263 ng/ml), whereas serum leptin levels in mice fed with HFD containing PG were low during fasting (average of 6481 ng/ml) and increased after refeeding (average 13,382 ng/ml). These results suggest that PG feeding has an anti‐obesity effect and that the regulation of SREBP‐1c and leptin secretion play a role in this effect.

The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 10

Aerosols or saliva containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can contaminate living environments, and viruses can be indirectly transmitted. To understand the survival potential of the virus, the viral titers of bovine coronavirus (BCoV), as a model virus, and SARS-CoV-2 were measured on porous and non-porous surfaces. The amount of infectious BCoV recovered remained relatively high on non-porous substrates. However, it quickly decreased on several non-porous surfaces such as nitrile rubber. The time taken to reach the limit of detection on non-woven masks, as a porous substrate, was longer than that of non-porous substrates. On porous substrates other than non-woven masks, the amount of virus recovered quickly decreased, and then remained at a low level. Representative substrates were tested with SARS-CoV-2. The decrease in the amount of infectious virus recovered was similar to that of BCoV, although that of SARS-CoV-2 was more rapid. RNA derived from SARS-CoV-2 was also detected using real-time PCR, and it remained on surfaces much longer than infectious virus, on all substrates. Therefore, it is important to measure the viral titer to avoid the overestimation of infectious virus contamination in the environments. Our results suggest that the surface structure was not directly related to viral survivability.

Escherichia albertii is an emerging foodborne pathogen. Owing to its distribution in river water, it is important to determine the presence of E. albertii in aquaculture-related foods. In this study, we investigated the distribution of E. albertii in retail oyster samples. A total of 427 raw oyster samples (385 Pacific oysters, and 42 Japanese rock oysters) were enriched in modified Escherichia coli broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42 °C. The cultures were used for E. albertii -specific nested PCR assay, as well as for E. albertii isolation using deoxycholate hydrogen sulfide lactose agar (DHL), DHL supplemented with rhamnose and xylose (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). The population of E. albertii in nested PCR-positive samples was determined using the most probable number (MPN) method. E. albertii isolates were subjected to biochemical and genetic characterization. E. albertii was detected in 5 of 315 (1.6%) Pacific oyster samples (one piece each), 2 of 70 (2.9 %) Pacific oyster samples (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples procured from four geographically distant regions. A total of 64 E. albertii strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and the MPN value was under the detection limit (< 3 MPN/10 g). A specific season or month for detecting E. albertii was not observed in this study, suggesting that the pathogen is present in seawater. All the E. albertii isolates, except one, were positive for the virulence factor eae, indicating that these isolates have the potential to infect humans.

Extracellular vesicles (EVs) released from bacteria are enclosed particles carrying biological active molecules. They have been shown to play a role in bacterial communications and delivery of virulence factors to the host cells. Staphylococcus aureus is an opportunistic pathogen causing a variety of infections ranging from impetigo to septicaemia. The EVs released from S. aureus have a high potential to be used for vaccine development against S. aureus infections. However, it is important to clearly understand the impact of SaEVs on the host’s immune response. Our study demonstrated that purified EVs from a clinical isolated methicillin-resistant S. aureus (SaEVs) significantly stimulated proinflammatory cytokine production in mouse immune cells and induced host cell death. An impairment of cytokine production in the Toll-like receptor (TLR)-silenced macrophages suggested that SaEVs stimulate proinflammatory response via TLRs 2, 4 and 9. In mouse infection model, the results demonstrated that SaEV immunization did not provide protective effect. In contrast, all SaEV-immunized mice died within Day 1 after methicillin-resistant S. aureus (MRSA) infection. After MRSA infection for 3 h, the production of IL-6, TNF-α and IL-17 in the spleen of SaEV-immunized mice was significantly higher than that of control mice. On Day 5 after the second immunization, total IgE in the serum was significantly enhanced, and a high titre of Th2-related cytokines was remarkably induced after ex vivo stimulation of the spleen cells with SaEVs. These results suggested that MRSA-derived EVs act as an immunostimulant that induces inflammatory response and IgE-mediated hypersensitivity after MRSA infection.

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced by Staphylococcus aureus is a causative agent of toxic shock syndrome (TSS) that is frequently associated with tampon use. It has long been suggested that TSS is induced when TSST-1 circulates through the body. However, the systemic distribution of TSST-1 from vagina or uterus has never been demonstrated. In this study, a mouse cervicovaginal infection model was established. Transcervical inoculation with a virulence strain of S. aureus and its derivative TSST-1-deficient mutant demonstrated that TSST-1 distributed to the bloodstream and spleen, and promoted systemic inflammation without bacteremia. Transcervical administration with the wild-type toxin and a superantigen-deficient mutant of TSST-1 (mTSST-1) demonstrated that the superantigenic activity of TSST-1 was essential to stimulate the systemic inflammation. Furthermore, this activity was not promoted by co-transcervical inoculation with lipopolysaccharides. The circulating TSST-1 and systemic inflammation rapidly reduced at 48 h after administration, suggesting that persistence of S. aureus in the uterus may be involved in long-term complications of TSS. Transcervical inoculation with mTSST-1-producing S. aureus showed that this toxin promoted bacterial number, uterine tissue damage, and localization of bacterial cells around uterine cavity. The results suggest that TSST-1 enhances S. aureus burden in uterine cavity, the secreted TSST-1 distributes into circulation system, and then systemic inflammation is induced.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable causative agents of emetic food poisoning in humans. New types of SEs and SE-like (SEl) toxins have been reported. Several epidemiological investigations have shown that the SEs and SEl genes, particularly, SEK, SEL, SEM, SEN and SEO genes, are frequently detected in strains isolated from patients with food poisoning. The purpose of the present study was to evaluate the emetic activity of recently identified SEs using a small emetic animal model, the house musk shrew. The emetic activity of these SEs in house musk shrews was evaluated by intraperitoneal administration and emetic responses, including the number of shrews that vomited, emetic frequency and latency of vomiting were documented. It was found that SEs induce emetic responses in these animals. This is the first time to demonstrate that SEK, SEL, SEM, SEN and SEO possess emetic activity in the house musk shrew.

Staphylococcal enterotoxins (SEs) are extracellular proteins, produced mainly by Staphylococcus aureus, which cause staphylococcal food poisoning (SFP) when ingested. Here, a novel SE was identified from two strains, which were identified as the causative microbes of the SFP outbreak that occurred in Tokyo in 2004. Both strains harbored the SEA gene, but its production was lower than that of other SEA-producing SFP isolates. Whole-genome sequencing analysis demonstrated that both strains harbored a SE-like gene besides sea. Phylogenetic analysis revealed that the amino acid sequence deduced from the SE-like gene belonged to the SEB group. Therefore, this gene was presumed to be a novel SE gene and termed “SE02.” The stability of SE02 against heating and proteolytic digestions was a little different from that of SEA. SE02 has both superantigenic and emetic bioactivities. Namely, SE02 activated mouse splenocytes and exhibited emetic activity in the common marmoset. SE02 mRNA was highly expressed in both isolates during the exponential phase of cultivation. In addition, SE02 protein was produced at 20 °C and 25 °C, which reflects the actual situation of SFP. SE02 appears to be a novel emetic toxin that was likely the causative toxin in combination with SEA in the SFP outbreak.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are known as causative agents of emetic food poisoning. We previously demonstrated that SEA binds with submucosal mast cells and evokes mast cell degranulation in a small emetic house musk shrew model. Notably, primates have been recognized as the standard model for emetic assays and analysis of SE emetic activity. However, the mechanism involved in SEA-induced vomiting in primates has not yet been elucidated. In the present study, we established common marmosets as an emetic animal model. Common marmosets were administered classical SEs, including SEA, SEB and SEC, and exhibited multiple vomiting responses. However, a non-emetic staphylococcal superantigen, toxic shock syndrome toxin-1, did not induce emesis in these monkeys. These results indicated that the common marmoset is a useful animal model for assessing the emesis-inducing activity of SEs. Furthermore, histological analysis uncovered that SEA bound with submucosal mast cells and induced mast cell degranulation. Additionally, ex vivo and in vivo pharmacological results showed that SEA-induced histamine release plays a critical role in the vomiting response in common marmosets. The present results suggested that 5-hydroxytryptamine also plays an important role in the transmission of emetic stimulation on the afferent vagus nerve or central nervous system. We conclude that SEA induces histamine release from submucosal mast cells in the gastrointestinal tract and that histamine contributes to the SEA-induced vomiting reflex via the serotonergic nerve and/or other vagus nerve., Author summary Staphylococcal enterotoxin A (SEA) is a bacterial toxin that has been recognized as a leading causative agent of staphylococcal food poisoning since 1930. The primary symptoms of staphylococcal food poisoning are nausea and emesis, which develop up to 1–6 h after ingestion of the causative foods contaminated by the bacteria. In the present study, we established the common marmoset as an emetic animal model and investigated the mechanisms of SEA-induced emesis in the primate model. Common marmosets that received SEA showed multiple emetic responses. We observed that SEA bound with submucosal mast cells in the intestinal tract and induced mast cell degranulation. Furthermore, SEA promoted histamine release from mast cells. We also demonstrated that histamine plays an important role in the SEA-induced emetic response in common marmosets. We conclude that SEA induces histamine release from submucosal mast cells in the intestinal tract and that the stimulation is transmitted from intestine to the brain via nerves, causing emesis. Our study provides a novel insight into functions of submucosal mast cells in the digestive tract.

Aims To elucidate an entry site of staphylococcal enterotoxin A (SEA), which is a major toxin for staphylococcal foodborne poisoning, into gastrointestinal tissue using a house musk shrew model. Methods and Results House musk shrews were per orally administered with recombinant SEA and localization of SEA in gastrointestinal tissues was investigated by immunohistochemistry and immunoelectron microscopy 30 min after administration. SEA was detected in a subset of intestinal epithelial cells and lamina propria in the villi of jejunum and ileum. This observation was also found in gastrointestinal loops. Morphological characteristics of the SEA-immunopositive cells indicated that goblet cells are an entry site of SEA.SEA entered mucus-expelling goblet cells and the induction of mucus secretion by alyll isothiocyanate resulted in an intensive SEA signal. These results suggest that mucus secretion by goblet cells is important for the translocation of SEA. Conclusions SEA can translocate across intestinal epithelia via mucus-expelling goblet cells. Significance and Impacts of the Study An entry site of SEA during translocation across the gastrointestinal mucosal barrier was investigated. This study was the first to demonstrate the significance of goblet cells as an entry site of this bacterial toxin.

Proteoglycan (PG) is a complex glycohydrate, which is widely distributed in the extracellular matrix. It has been reported that daily oral administration of PG (extracted from salmon nasal cartilage) modulates the severity of proinflammatory cytokine responses in mouse experimental colitis, autoimmune encephalomyelitis, collagen‑induced arthritis and obesity‑induced inflammation. The present study investigated the effect of salmon nasal cartilage PG on allergic responses using a mouse model of papain‑induced respiratory inflammation. Low titers of immunoglobulin E were identified in the sera of the PG‑administered mice. Oral administration of PG attenuated eosinophil infiltration in the lung. In the acute model of papain‑induced allergic inflammation, PG‑administered mice exhibited low titers of epithelium‑derived and T helper 2‑associated cytokines. The results of the present study demonstrated that salmon cartilage PG has an immunomodulatory effect on intranasally delivered papain. These results suggest a potential role for PG as a prophylactic agent which may attenuate allergic respiratory inflammation.

Obesity is associated with chronic inflammation of adipose tissue and causes development of type 2 diabetes. M1 macrophage population was increased in adipose tissue of obese mouse. M1 macrophages induce insulin resistance through the secretion of proinflammatory cytokines. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in various mouse inflammatory diseases. In this study, we examined the effect of PG on type 2 diabetes using high-fat-diet (HFD) induced obese mouse model. Oral PG administration enhanced the population of small adipocytes (area less than 1000 μm2) without body and tissue weight gain. In addition, PG administration suppressed mRNA expression of TNF-α, IL-6 and CXCL2 in adipose tissue. The proportion of M1 macrophages was decreased by PG administration. In addition, PG administration suppressed hyperglycemia after intraperitoneal glucose injection. Fasted serum insulin level was decreased in PG-administered mice. Moreover, insulin-stimulated phosphorylation of Akt was enhanced in the liver and gastrocnemius skeletal muscle of PG-administered mice. These data suggested that PG administration improves hyperglycemia and insulin sensitivity in obese mice by modulation of M1 macrophages which secrete proinflammatory cytokines in adipose tissue and activation of Akt in liver and skeletal muscle.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable causative agents of emetic food poisoning in humans. New types of SEs and SE-like (SEl) toxins have been reported. Several epidemiological investigations have shown that the SEs and SEl genes, particularly, SEK, SEL, SEM, SEN and SEO genes, are frequently detected in strains isolated from patients with food poisoning. The purpose of the present study was to evaluate the emetic activity of recently identified SEs using a small emetic animal model, the house musk shrew. The emetic activity of these SEs in house musk shrews was evaluated by intraperitoneal administration and emetic responses, including the number of shrews that vomited, emetic frequency and latency of vomiting were documented. It was found that SEs induce emetic responses in these animals. This is the first time to demonstrate that SEK, SEL, SEM, SEN and SEO possess emetic activity in the house musk shrew.

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.