Systemic immune responses do not affect significant immune responses in the skin.

Fish skin plays an important role in defending against pathogens in water, primarily through the secretion of skin mucus containing various immune-related factors. Local immune responses in the skin activate systemic immune responses by inflammatory cytokines. However, it remains unclear whether immune responses in the skin occur after systemic immune responses caused by pathogen invasion into the fish body. This study aimed to clarify the relationship between systemic immune responses and skin responses after intraperitoneal injection of formalin-killed cells (FKC) of Vibrio anguillarum. Although systemic inflammatory responses were observed in the spleen after injection, expression changes in the skin did not show significant differences. In contrast, expression of hemoglobin subunit genes significantly increased in the skin after FKC injection, suggesting that erythrocytes infiltrate extravascularly.

Euphausia pacifica emulsified oil powder improves sleep quality in partially sleep-restricted healthy volunteers.

Although it is known that adequate sleep is crucial for maintaining a healthy lifestyle, approximately 30% of the general population has experienced insomnia. Thus, a better understanding of the relationship between food components and sleep quality is needed. North Pacific krill, Euphausia pacifica, is rich in marine n-3 polyunsaturated fatty acids in phospholipid form as well as 8R-hydroxy-eicosapentanoic acid. Here, emulsified oil powder derived from this krill was used in a trial involving 64 participants to assess its potential to enhance sleep quality. Consumption of the powdered emulsified oil was found to reduce drowsiness upon waking and enhance fatigue recovery, and for participants aged 40 and above, an improvement in sleep cycle was observed. In conclusion, consumption of krill emulsified oil powder was effective in enhancing sleep quality for individuals with partial sleep restrictions.

Impact of antimicrobial stewardship implementation on the antibiotic use and susceptibility in a Japanese long-term care hospital.

BACKGROUND: Antimicrobial use (AMU) is closely related to the emergence of antimicrobial-resistant (AMR) bacteria. Meanwhile, long-term care hospitals (LTCHs) have been pointed out to be important reservoirs for AMR. However, evidence illustrating the association between AMU and AMR in LTCHs is lacking compared to that of acute care hospitals. METHODS: We evaluated the impact of an antimicrobial stewardship (AS) program implementation, in a LTCH on AMU and antibiotic susceptibility between three periods: the pre-AS-period (pre-AS); the first period after AS implementation (post-AS 1), in which initiated recommendation the blood culture collection and definitive therapy by AS team; and the second period (post-AS 2), implementation of a balanced use of antibiotics was added. RESULTS: After the AS implementation, a significant increase in the number of blood cultures collected was observed. Conversely, the AMU of piperacillin-tazobactam (PIPC/TAZ), which has activity against Pseudomonas aeruginosa, was increased and occupied 43.0% of all injectable AMU in post-AS 1 compared with that in pre-AS (35.5%). In the post-AS 2 period, we analyzed the %AUD and recommended hospital-wide PIPC/TAZ sparing; this resulted in the significant reduction in %AUD of PIPC/TAZ, which was associated with improved susceptibility of P. aeruginosa to PIPC/TAZ. CONCLUSIONS: These results suggest that AS programs aimed at implementing antibiotic sparing may lead to improve AMR, highlighting the necessity of correcting overuse of a single class of antibiotics and usefulness of AMU monitoring in the LTCH setting.

Type I interferon induced by polyinosinic-polycytidylic acid does not contribute to the efficacy of a formalin-killed cell vaccine against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus).

Polyinosinic-polycytidylic acid (poly I:C) is a type of pathogen-associated molecular pattern that can strongly induce the expression of type I interferon (I-IFN). Our previous study has demonstrated that the combination of poly I:C with a recombinant protein antigen not only stimulated the expression of I-IFN but also conferred protection against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus). In this study, our aim was to develop a better immunogenic and protective fish vaccine, for which we intraperitoneally coinjected P. olivaceus with poly I:C and formalin-killed cells (FKCs) of E. piscicida and compared the efficiency of protection against E. piscicida infection with that of FKC vaccine alone. Results showed that the expression levels of I-IFN, IFN-γ, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and the interferon-stimulated genes (ISGs) ISG15 and Mx were significantly increased in the spleen of fish inoculated with poly I:C + FKC. The results of ELISA showed that the levels of specific serum antibodies in the FKC and FKC + poly I:C groups were gradually increased until 28 days postvaccination and were significantly higher than those in the PBS and poly I:C groups. At 3 weeks after vaccination in the challenge test, the respective cumulative mortality rates of fish in the PBS, FKC, poly I:C, and poly I:C + FKC groups were 46.7%, 20.0%, 33.3%, and 13.3% under low-concentration challenge and 93.3%, 46.7%, 78.6%, and 53.3% under high-concentration challenge. This study showed that poly I:C may not provide an effective adjuvant effect with FKC vaccine for intracellular bacterial infections.

8(R)-Hydroxyeicosapentaenoic acid (8R-HEPE) induces transcription of cholesterol efflux receptors via activation of liver X receptor in macrophages.

Dyslipidemia is a risk factor for the development of atherosclerotic cardiovascular disease. 8-Hydroxyeicosapentaenoic acid (8-HEPE) from North Pacific krill (Euphausia pacifica) is known to reduce plasma low-density lipoprotein (LDL) cholesterol levels and increase plasma high-density lipoprotein cholesterol levels in LDL receptor knock-out mice fed a western diet. Moreover, 8-HEPE also reduces the area of aortic atherosclerosis in apoE knock-out mice fed the same diet. In this study, we examined the stereochemical-specific activity of 8-HEPE for inducing expression of cholesterol efflux receptors (Abca1 and Abcg1) in J774.1 cells. Our findings show 8R-HEPE induces the expression of Abca1 and Abcg1 via activation of liver X receptor, whereas 8S-HEPE elicits no such activity. These results suggest that 8R-HEPE derived from North Pacific krill may have beneficial effects against dyslipidemia.

Whole-genome analysis of red sea bream iridovirus spread in 2021 in Japan provided epidemiological and viral traits insight.

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

Model Synthetic Study of Tutin, a Picrotoxane-Type Sesquiterpene: Stereoselective Construction of a cis-Fused 5,6-Ring Skeleton.

Picrotoxinin, coriamyrtin, and tutin are representative natural products classified as picrotoxane-type sesquiterpenes and they function as strong neurotoxins. Because they possess a cis-fused 5,6-ring skeleton with a highly congested functionalization, organic chemistry researchers have pursued the development of a stereoselective synthesis method for such skeleton. This study aims to stereoselectively synthesize the cis-fused 5,6-ring skeleton with two tetrasubstituted carbons at both angular positions using a model compound. The results revealed that the desymmetrization of the 2-methyl-1,3-cyclopentanedione moiety via the DL-proline-mediated intramolecular aldol reaction of a pentanal derivative bearing an isopropenyl group and the five-membered ring at the 3- and 5-position, respectively, provided the desired cis-fused skeleton. This reaction can construct four contiguous stereogenic centers of the bicyclic skeleton with the two angular positions in good yield with high stereoselectivity. Further, this reaction was applied to the kinetic resolution of the racemate using L-proline, providing the enantiomeric pure aldol product with the desired skeleton. This method can be utilized for total synthesis of picrotoxane-type sesquiterpenes.

Total Synthesis of Macaranin B.

This study describes the total synthesis of macaranin B, a naturally occurring ellagitannin containing 1-O-galloyl and 3,6-O-macaranoyl groups in an axial-rich d-glucose. The key steps of the synthesis include an oxidative coupling reaction of galloyl groups with 1,2,4-orthoacetylglucose moiety and oxa-Michael addition/elimination using an orthoquinone monoketal. This facilitates the construction of the macaranoyl group and the first total synthesis of macaranin B.

A yeast-based screening system identified bakkenolide B contained in Petasites japonicus as an inhibitor of interleukin-2 production in a human T cell line.

Ca2+ signaling is related to various diseases such as allergies, diabetes, and cancer. We explored Ca2+ signaling inhibitors in natural resources using a yeast-based screening method and found bakkenolide B from the flower buds of edible wild plant, Petasites japonicus, using the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ). Bakkenolide B exhibited growth-restoring activity against the YNS17 strain and induced Li+ sensitivity of wild-type yeast cells, suggesting that it inhibits the calcineurin pathway. Additionally, bakkenolide B inhibited interleukin-2 production at gene and protein levels in Jurkat cells, a human T cell line, but not the in vitro phosphatase activity of human recombinant calcineurin, an upstream regulator of interleukin-2 production. Furthermore, bakkenolide A showed weak activity in YNS17 and Jurkat cells compared with bakkenolide B. These findings revealed new biological effects and the structure-activity relationships of bakkenolides contained in P. japonicus as inhibitors of interleukin-2 production in human T cells.

ß-Selective Glycosylation Using Axial-Rich and 2-O-Rhamnosylated Glucosyl Donors Controlled by the Protecting Pattern of the Second Sugar.

Herein, we describe two counterexamples of the previously reported ß/α-selectivity of 96/4 for glycosylation using ethyl 2-O-[2,3,4-tris-O-tert-butyldimethylsilyl (TBS)-α-L-rhamnopyranosyl]-3,4,6-tris-O-TBS-thio-ß-D-glucopyranoside as the glycosyl donor. Furthermore, we investigated the effects of protecting group on the rhamnose moieties in the glycosylation with cholestanol and revealed that ß-selectivity originated from the two TBS groups at the 3-O and 4-O positions of rhamnose. In contrast, the TBS group at the 2-O position of rhamnose hampered the ß-selectivity. Finally, the ß/α-selectivity during the glycosylation was enhanced to ≥99/1. The results obtained herein suggest that the protecting groups on the sugar connected to the 2-O of a glycosyl donor with axial-rich conformation can control the stereoselectivity of glycosylation.

Total Synthesis of Mallotusinin.

The total synthesis of mallotusinin, which bears a tetrahydroxydibenzofuranoyl (THDBF) bridge between the 2-oxygen and 4-oxygen of glucose on corilagin with a 3,6-O-(R)-hexahydroxydiphenoyl (HHDP) bridge, is described. The key features of the total synthesis are: 1) improvements of our previously reported method to synthesize corilagin; 2) establishment of the THDBF skeleton via an unusual intramolecular SN Ar reaction of an HHDP analogue, and 3) the application of a two-step bislactonization strategy for a HHDP bridge construction into the 2,4-O-THDBF bridge. Oxidative phenol coupling of 1,2,4-orthoacetyl-3,6-di-(4-O-benzylgalloyl)-α-d-glucopyranose and the orthoester cleavage of the coupling product without the pyranose-furanose ring transformation are key reactions for the improved synthesis of corilagin, which enabled the adequate supply of a corilagin precursor that was required to develop the mallotusinin synthesis. These established methods are expected to help develop the synthesis of other ellagitannins with a bridge between the two oxygens of corilagin.

8-HEPE-Concentrated Materials from Pacific Krill Improve Plasma Cholesterol Levels and Hepatic Steatosis in High Cholesterol Diet-Fed Low-Density Lipoprotein (LDL) Receptor-Deficient Mice.

Eicosapentaenoic acid (EPA), one of the N-3 polyunsaturated fatty acids (n-3 PUFAs), is a major active ingredient of fish that contributes to improve dyslipidemia. Recently, we demonstrated that 8-hydroxyeicosapentaenoic acid (8-HEPE) had a more positive effect on metabolic syndrome than EPA, and that 8-HEPE induced peroxisome proliferator-activated receptor (PPAR)α activation in the liver. We investigated the effects of 8-HEPE-concentrated materials from Pacific krill on dyslipidemia and hepatic steatosis in low-density lipoprotein (LDL) receptor-deficient (LDLR-KO) mice. Eight-week-old male LDLR-KO mice were fed a Western diet (0.15% cholesterol, WD), WD supplemented with 8-HEPE-concentrated materials from Pacific krill (8-HEPE included; WD +8-HEPE), or a standard diet (SD) for eighteen weeks, respectively. Murine J774.1 macrophages were incubated in the absence or presence of 8-HEPE (50 µM) or EPA (50 µM). 8-HEPE-concentrated materials significantly increased the plasma high-density lipoprotein (HDL)-cholesterol level, and decreased the plasma LDL-cholesterol and hepatic triglyceride levels in WD-fed LDLR-KO mice. Moreover, the rate of Oil Red O-positive staining was higher in the liver of WD-fed LDLR-KO mice than in that of 8-HEPE + WD-fed LDLR-KO mice. 8-HEPE but not EPA significantly increased gene expression levels of ABCA1, CD36, and interleukin 6 (IL-6) in murine J774.1 macrophages compared with those in the control. These results suggest that 8-HEPE-concentrated materials improve dyslipidemia and hepatic steatosis increasing ABCA1, CD36, and IL-6 gene expressions in macrophages.

Euphausia pacifica as a source of 8(R)-hydroxy-eicosapentaenoic acid (8R-HEPE), 8(R)-hydroxy-eicosatetraenoic acid (8R-HETE) and 10(R)-hydroxy-docosahexaenoic acid (10R-HDHA).

Although not fully investigated, 8-HEPE, 8-HETE, and 10-HDHA have potentially beneficial effects for human health. Euphausia pacifica (North Pacific krill) is unique in containing several ppm level of 8R-HEPE, and sub-ppm levels of 8R-HETE and 10R-HDHA. Obtaining sufficient quantities of these compounds is a major bottleneck for conducting in vivo experiments to evaluate their biological activities. In this study, we examined an efficient way of obtaining 8R-HEPE, 8R-HETE, and 10R-HDHA by enzymatic production in E. pacifica. We devised a novel method to purify 199.4 mg of 8R-HEPE, 2.1 mg of 8R-HETE and 5.6 mg of 10R-HDHA from 1 kg of E. pacifica. We identified the stereochemistry of the hydroxy group at C-8 of HEPE and HETE and C-10 of HDHA as the R configuration by chiral column chromatography analysis using LC/QTOFMS.Abbreviations: 8-HEPE: 8-hydroxy-eicosapentaenoic acid; 8-HETE: 8-hydroxy-eicosatetraenoic acid; 10-HDHA: 10-hydroxy-docosahexaenoic acid; EPA: eicosapentaenoic acid; TLC-FID, thin layer chromatograph-Flame Ionization Detector; LC/QTOFMS: liquid chromatography/hybrid quadrupole time of flight mass spectrometry.

Effect of fatty acids on melanogenesis and tumor cell growth in melanoma cells.

Fatty acids have various physiological effects on melanoma. For example, palmitic acid (PA) increases melanin levels; linoleic acid and DHA decrease melanin levels; and DHA suppresses tumor growth. In this study, we focused on the relationship between the structure of fatty acids and their physiological effects in melanoma to examine the likely mechanisms of action. We showed that saturated fatty acids and PUFAs display opposing effects on melanin content in melanoma cells. Likewise, PA and EPA have opposing effects in terms of actin polymerization. Our findings suggest that PA and EPA change melanin content in melanoma to alter melanosome trafficking by modulating actin polymerization. Here, we also examined the mechanism of the anti-tumor effect of DHA. We found that DHA interacts with receptor for activated C kinase 1 and represses melanoma cell proliferation by suppressing protein kinase C signaling. Our results suggest a new mechanism to explain the physiological effects of fatty acids.

Changes in Lipids During the Ovary Maturation Process of Balanus rostratus.

Balanus rostratus is a large cold-water acorn barnacle distributed around the northern coast of the Pacific Ocean. In Mutsu Bay, Aomori, Japan, B. rostratus, which adhere naturally to scallop shells, are cultured as food. However, current culture methods do not generate sufficient supplies to satisfy market demand. Knowledge of the physiology of B. rostratus reproduction is important for the development of more efficient aquaculture methods. Previous studies have suggested that fatty acids and their metabolites play an important role in barnacle reproduction and development; however, few studies have analyzed lipids, particularly during ovary maturation. Here we analyzed lipid content, lipid class, and fatty acid composition of B. rostratus ovary throughout the year. The clutch in the present study was observed once per year at the end of November. The lipid content increased as the ovary underwent maturation. The proportion of triacylglycerol increased with increasing lipid content. The proportions of myristic acid, arachidonic acid, EPA and DHA significantly decreased in December. By contrast, the proportion of these fatty acids in lipid extracted from larvae was high relative to lipid extracted from B. rostratus ovary in December. These findings suggest that these fatty acids are transferred from the ovary to the larvae. Our novel findings on lipid metabolism during ovary maturation in B. rostratus indicate the importance of lipids during reproduction. This information may be useful in establishing methods for the aquaculture of B. rostratus.

Gtgen3A, a novel plant GH3 ß-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana.

Gentiobiose, a ß-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian (Gentiana triflora). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a ß-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 ß-glucan exohydrolases, ExoI of Hordeum vulgare, and ExgI from Zea mays, which preferentially hydrolyzed ß-1,3- and ß-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from ß-1,2-, ß-1,3-, ß-1,4-, and ß-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.

Geminin is an indispensable inhibitor of Cdt1 in mouse embryonic stem cells.

Geminin is implicated in regulation of the cell cycle and differentiation. Although loss of Geminin triggers unscheduled DNA rereplication as a result of interruption of its interaction with Cdt1 in some somatic cancer cells, whether such cell cycle regulation also operates in embryonic stem cells (ESCs) has remained unclear. To characterize the Geminin-Cdt1 axis in ESCs and compare it with that in somatic cells, we established conditional knockout (KO) of Geminin in mouse ESCs and mouse embryonic fibroblasts (MEFs). Geminin KO ESCs manifest a large flattened morphology, develop polyploidy accompanied by DNA damage and G2 -M checkpoint activation, and subsequently undergo apoptosis. Rereplication in Geminin KO ESCs was attenuated by inhibition of G2 -M checkpoint signaling or by expression of wild-type Geminin, but not by expression of a Geminin mutant that does not bind to Cdt1, indicating the importance of sequestration of Cdt1 by Geminin in G2 phase. In contrast, Geminin KO MEFs did not manifest disturbance of the cell cycle unless they were treated to force abnormal accumulation of Cdt1. Together, our results indicate that Geminin is a key inhibitor of Cdt1 in mouse ESCs, but that it plays a backup role in MEFs to compensate for accidental up-regulation of Cdt1.

Structural Revisions in Natural Ellagitannins.

Ellagitannins are literally a class of tannins. Triggered by the oxidation of the phenolic parts on ß-pentagalloyl-d-glucose, ellagitannins are generated through various structural conversions, such as the coupling of the phenolic parts, oxidation to highly complex structures, and the formation of dimer and lager analogs, which expand the structural diversity. To date, more than 1000 natural ellagitannins have been identified. Since these phenolic compounds exhibit a variety of biological activities, ellagitannins have potential applications in medicine and health enhancement. Within the context of identifying suitable applications, considerations need to be based on correct structural features. This review describes the structural revisions of 32 natural ellagitannins, namely alnusiin; alnusnin A and B; castalagin; castalin; casuarinin; cercidinin A and B; chebulagic acid; chebulinic acid; corilagin; geraniin; isoterchebin; nobotanin B, C, E, G, H, I, J, and K; punicalagin; punicalin; punigluconin; roxbin B; sanguiin H-2, H-3, and H-6; stachyurin; terchebin; vescalagin; and vescalin. The major focus is on the outline of the initial structural determination, on the processes to find the errors in the structure, and on the methods for the revision of the structure.

Non-Enzymatic Oxidation of a Pentagalloylglucose Analogue into Members of the Ellagitannin Family.

The occurrence of more than 1000 structurally diverse ellagitannins has been hypothesized to begin with the oxidation of penta-O-galloyl-ß-d-glucose (ß-PGG) for the coupling of the galloyl groups. However, the non-enzymatic behavior of ß-PGG in the oxidation is unknown. Disclosed herein is which galloyl groups tended to couple and which axial chirality was predominant in the derived hexahydroxydiphenoyl groups when an analogue of ß-PGG was subjected to oxidation. The galloyl groups coupled in the following order: at the 4,6-, 1,6-, 1,2-, 2,3-, and 3,6-positions with respective S-, S-, R-, S-, and R-axial chirality. Among them, the most preferred 4,6-coupling reflected the what was observed for natural ellagitannins. A new finding was that the second best coupling occured at the 1,6-positions. With the detection of a 3,6-coupled product, this work demonstrated that even ellagitannin skeletons with an axial-rich glucose core may be generated non-enzymatically.

Hydroxyeicosapentaenoic acids from the Pacific krill show high ligand activities for PPARs.

PPARs regulate the expression of genes for energy metabolism in a ligand-dependent manner. PPARs can influence fatty acid oxidation, the level of circulating triglycerides, glucose uptake and insulin sensitivity. Here, we demonstrate that 5-hydroxyeicosapentaenoic acid (HEPE), 8-HEPE, 9-HEPE, 12-HEPE and 18-HEPE (hydroxylation products of EPA) obtained from methanol extracts of Pacific krill (Euphausia pacifica) can act as PPAR ligands. Two of these products, 8-HEPE and 9-HEPE, enhanced the transcription levels of GAL4-PPARs to a significantly greater extent than 5-HEPE, 12-HEPE, 18-HEPE, EPA, and EPA ethyl-ester. 8-HEPE also activated significantly higher transcription of GAL4-PPARα, GAL4-PPARγ, and GAL4-PPARδ than EPA at concentrations greater than 4, 64, and 64 µM, respectively. We also demonstrated that 8-HEPE increased the expression levels of genes regulated by PPARs in FaO, 3T3-F442A, and C2C12 cells. Furthermore, 8-HEPE enhanced adipogenesis and glucose uptake. By contrast, at the same concentrations, EPA showed weak or little effect, indicating that 8-HEPE was the more potent inducer of physiological effects.