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Bioelectrocatalytic methanol production from formaldehyde with alcohol dehydrogenase from Saccharomyces cerevisiae was developed toward establishing a method for synthesizing CO2 to methanol.

Visible-light-driven molecular conversion using the NADH regeneration system of triethanol amine, water-soluble zinc porphyrin, colloidal Rh nanoparticles and NAD+ in the presence of various dehydrogenases was developed.

We discovered the catalytic activity of Rh nanoparticles dispersed by polyvinylpyrrolidone (Rh–PVP) for NADH regeneration. The selective reduction of NAD+ to enzyme active NADH has successfully been achieved in the presence of an electron donor, ZnTPPS as a photosensitizer, and Rh–PVP. Only 1,4-NADH was produced as the reduction product of NAD+, which was validated by an enzymatic assay and HPLC.

Unlike most mammalian cell lines, fish cell lines are immortal and resistant to cellular senescence. Elevated expression of H-Ras contributes to the induction of senescence in a fish cell line, EPC, but is not sufficient to induce full senescence. Here, we focused on the absence of a p16

Malic enzyme (ME) from chicken liver (EC 1.1.1.40) is an enzyme that catalyzes the decarboxylation of malate into pyruvate and CO2 and the reverse reaction that introduces CO2 as a carboxy-group to pyruvate to form malate via oxaloacetate in the presence of natural co-enzyme NADP+/NADPH. Thus, ME is an attractive biocatalyst for building carbon–carbon bonds through the carboxylation of pyruvate with CO2. Since ME mainly catalyzes the decarboxylation of malate to produce pyruvate via oxaloacetate, it is necessary to devise ways to promote the carboxylation of pyruvate with CO2 to produce oxaloacetate based on the building of carbon–carbon bonds. Enhancing the carboxylation of pyruvate by the addition of metal ions with CO2 and using ME as a catalyst will lead to new insight into biocatalytic CO2 utilization research. The effect of adding divalent and trivalent metal ions to promote the ME-catalyzed building of carbon–carbon bonds through the carboxylation of pyruvate with CO2 was investigated. Specifically, it was found for the first time that the addition of trivalent aluminium and iron ions accelerates ME-catalyzed carboxylation of pyruvate with CO2. Moreover, it was found that a high concentration of aluminum ions (>100 μM) and a low concentration of iron ions (

CO2 is a representative greenhouse gas (GHG), and the Paris Agreement adopted by consensus on 12 December 2015 mandates worldwide reduction of CO2 emissions by 80% until the end of 2050 relative to that of 2013. In order to achieve a significant reduction in CO2, it is necessary to establish not only CO2 capture and storage (CCS), but also a wide range of technologies such as CO2 conversion to alcohol fuels such as methanol, utilization as chemical raw materials, and so on (CO2 capture and utilization CCU). The establishment of utilization technology aimed at reducing CO2 will contribute to “7. Use of renewable feedstocks” and “9. Catalyst” from the 12 principles of green chemistry. CO2 utilization technology can be divided into two categories: multi-electron reduction with multiple protons and binding to organic molecules. In the former case, CO, formic acid, and methanol are produced, so no extension of the carbon number of the target product is observed. In the latter case, CO2 is bonded to the organic molecule as a carboxyl group, so that a C–C bond can be newly built. In other words, with the latter technology, it becomes possible to fix CO2 to organic molecules and create new functional materials. For example, natural photosynthesis skilfully utilizes sunlight as a renewable energy and enzyme catalysts to synthesize glucose from CO2 and water as a raw material. In other words, CCU technology based on mimicking natural photosynthesis contributes to the above principles of green chemistry. To establish this technology, a catalyst that promotes the binding of CO2 to organic molecules is an essential factor. In other words, it is necessary to develop a catalyst for activating C–H bonds in organic molecules and promoting C–C bond formation with CO2. In this review, molecular and enzyme-based catalysts for C–H bond activation and building C–C bonds with CO2 applicable to light-driven CCU using a photoredox system are introduced. Moreover, light-driven C–H bond activation and building C–C bonds with CO2 using various catalysts and photofunctional materials are also introduced. The reaction yield and selectivity of both systems for C–H bond activation and building C–C bonds with CO2 are also discussed. Finally, we discussed the problems of the light-driven C–H bond activation and building C–C bonds with CO2 and mentioned some areas for improvement in the future. We also discussed the outline of the life cycle assessment (LCA) of a typical system for light-driven C–H bond activation and building C–C bonds with CO2.

Immersion vaccination with a biomimetic mucoadhesive nanovaccine has been shown to induce a strong mucosal immune response against columnaris disease, a serious bacterial disease in farmed red tilapia caused by Flavobacterium columnare. However, the induction of a systemic immune response by the vaccine is yet to be investigated. Here, we examine if a specific humoral immune response is stimulated in tilapia by a biomimetic-mucoadhesive nanovaccine against Flavobacterium columnare using an indirect-enzyme-linked immunosorbent assay (ELISA), serum bactericidal activity (SBA) and the expression of immune-related genes within the head-kidney and spleen, together with assessing the relative percent survival of vaccinated fish after experimentally infecting them with F. columnare. The anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, supporting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after challenging fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation of IgM, IgT, TNF α, and IL1-β genes in the spleen and kidney of vaccinated fish. Significant upregulation of IgM and IgT genes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish.

Summary The suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling required to prevent excessive cellular responses. In particular, SOCS3 is involved in the regulation of metabolic syndromes, such as obesity and diabetes, by suppressing leptin and insulin signals. SOCS3 also suppresses the inflammatory response associated with metabolic stress, but this specific role remains undefined. Wild-type mice on a high-fat diet (HFD) exhibited only fatty liver, whereas systemic deletion of SOCS3 resulted in excessive myeloid hematopoiesis and hepatic inflammation. In addition, depletion of the gut microbiota resulted in considerable improvement in excess granulopoiesis and splenomegaly, halting the progression of systemic inflammation in SOCS3KO mice on the HFD. This result suggests that intestinal dysbiosis is involved in inflammation associated with SOCS3KO. Although contributing to diet-induced obesity and fatty liver, SOCS3 is nevertheless critical to suppress excess myeloid hematopoiesis and severe systemic inflammation associated with intestinal dysbiosis on HFD., Graphical abstract, Highlights • SOCS3 suppresses severe systemic inflammation associated with high-fat diet • SOCS3 deficiency on high-fat diet accelerates excess myeloid hematopoiesis • SOCS3 controls gut dysbiosis on high-fat diet, Immunology; Microbiome

Recently, our lab reported that CUBIC, a tissue-clearing technique, could help reveal the initial route of infection, its spread, and the localization of pathogens in infected goldfish Carassius auratus. ​However, this technique, in its original form, failed to clear melanin pigmented parts of the fish. ​We show here that bleaching with H2O2 clears melanin pigments of Black Moor goldfish without causing severe histological damage. ​Furthermore, nuclear staining with PI helps visualize the internal structures of cleared fish in a dark field. ​Since bleaching does not significantly quench the fluorescence of GFP, it may be applied to the in vivo imaging of GFP.

Although allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is a curative therapy for high‐risk acute leukemia (AL), some patients still relapse. Since patients simultaneously have many prognostic factors, difficulties are associated with the construction of a patient‐based prediction algorithm of relapse. The alternating decision tree (ADTree) is a successful classification method that combines decision trees with the predictive accuracy of boosting. It is a component of machine learning (ML) and has the capacity to simultaneously analyze multiple factors. Using ADTree, we attempted to construct a prediction model of leukemia relapse within 1 year of transplantation. With the model of training data (n = 148), prediction accuracy, the AUC of ROC, and the κ‐statistic value were 78.4%, 0.746, and 0.508, respectively. The false positive rate (FPR) of the relapse prediction was as low as 0.134. In an evaluation of the model with validation data (n = 69), prediction accuracy, AUC, and FPR of the relapse prediction were similar at 71.0%, 0.667, and 0.216, respectively. These results suggest that the model is generalized and highly accurate. Furthermore, the output of ADTree may visualize the branch point of treatment. For example, the selection of donor types resulted in different relapse predictions. Therefore, clinicians may change treatment options by referring to the model, thereby improving outcomes. The present results indicate that ML, such as ADTree, will contribute to the decision‐making process in the diversified allo‐HSCT field and be useful for preventing the relapse of leukemia.

A feeding trial was conducted to evaluate the effect of nucleotides supplementation to low‐fish meal feed on growth and fatty acid composition of rainbow trout. Six isonitrogenous (42% crude protein) and isolipidic (18% crude lipid) diets were formulated containing fish meal and plant ingredients as main protein sources. The control diet was a basal diet without supplementation of nucleotides, and five experimental diets were prepared by supplementing one of the five different nucleotides in the form of 5′‐monophosphate (0.15%), that is inosine (IMP), adenosine (AMP), guanosine (GMP), uridine (UMP) and cytidine (CMP) onto basal diet. Two hundred forty juvenile rainbow trout with an initial average body weight 9.8 g were randomly distributed into twelve aquaria. After 15 weeks of feeding period, growth performance and feed utilization of rainbow trout were not significantly different among dietary treatments. Dietary GMP, UMP and CMP tended to accumulate crude lipid in the muscle and whole fish body. Moreover, dietary GMP, UMP and CMP significantly increased hepatic 18:3n‐3 and long‐chain homologue 18:4n‐3 and 20:4n‐3 contents. Hepatic 18:2n‐6 content showed also increase in fish fed GMP, UMP and CMP diets, but decreased in long‐chain homologue 20:3n‐6 and 20:4n‐6 contents. Decrease in 20:4n‐6, 20:5n‐3 and 22:6n‐3 contents was also found in the muscle of fish fed IMP, GMP and CMP diets. The present study clearly showed that there was no positive effect of dietary nucleotides on growth of fish, but dietary nucleotides particularly GMP, UMP and CMP altered polyunsaturated fatty acid composition of rainbow trout.

Fish cell lines are known to be immortal and do not show the signs of cellular senescence despite the absence of transformation. Furthermore, high telomerase activities responsible for maintenance of telomere length are detected in many organs in live fish, irrespective of fish age. On the other hand, although it is reported that cytosine methylation at CpG island shores decreases as zebrafish age, the relationship between DNA methylation and cellular senescence in fish has not been explored. In this study, we investigated the induction of cellular senescence and senescence-associated secretory phenotype (SASP) in a fathead minnow Pimephales promelas immortal cell line, Epithelioma papulosum cyprini (EPC) treated with the DNA demethylating agent 5-Aza-2′-deoxycytidine (5-Aza-dC). DNA demethylation by 10 μM of 5-Aza-dC caused cell growth arrest, morphological senescence-like phenotypes and induction of senescence-associated β-galactosidase (SA-β-gal) activity, likely due to a mitotic catastrophe caused by disruption of chromosome segregation. Furthermore, RT-qPCR analyses revealed significant up-regulation of senescence markers such as p53-p21 and p16-Rb pathways as well as several SASP factors in 5-Aza-dC treated cells. Meanwhile, although DNA demethylation suppressed the transcription of myc and its downstream target, telomerase reverse transcriptase (tert), telomerase activity was no more than modestly decreased. These results suggest that although DNA methylation may be involved in the suppression of cellular senescence, it not critical for the immortalization of the fish cell line.

Malate dehydrogenase (oxaloacetate-decarboxylating) commonly named malic enzyme (ME) from chicken liver (EC 1.1.1.40) catalyzes a reaction introducing CO2 as a carboxy-group to pyruvate to form mal...

Background The Rh complex contributes to cell membrane structural integrity of erythrocytes. Rhnull syndrome is characterized by the absence of the Rh antigen on the erythrocyte membrane, resulting in chronic hemolytic anemia. We recently came across 3 Rhnull phenotype probands within two families with the same novel RHAG mutation in the Japanese population. Materials and methods Detailed Rh phenotyping by hemagglutination was performed using monoclonal and polyclonal anti-D, -C, -c, -E, and -e; monoclonal and polyclonal anti-Rh17 antibodies; and polyclonal anti-Rh29 antibodies. RHAG mRNA transcripts were analyzed by reverse transcription-polymerase chain reaction, and the mutation was verified by genomic sequencing. Results The genomic region spanning exon 6 contained a G > A transition in the invariant GT motif of the 5' donor splice-site of Intron 6 (c.945+1G>A). The Rhnull phenotype was caused by an autosomal recessive mutation in Probands 1 and 2, determined by family history. Regarding clinical features, the degree of hemolysis varied slightly between these individuals, with Proband 3 displaying acute hemolytic anemia with an infection. While no standard therapy has been established, the condition of the patient in this study improved with conservative treatment, including hydration and antibiotics. Conclusion The mechanisms of hemolysis due to the Rhnull phenotype can vary, but our findings indicate that acute hemolytic crisis caused by the Rhnull syndrome could be associated with infection.

Columnaris, a highly contagious bacterial disease caused by Flavobacterium columnare, is recognized as one of the most important infectious diseases in farmed tilapia, especially during the fry and fingerling stages of production. The disease is associated with characteristic lesions in the mucosa of affected fish, particularly their skin and gills. Vaccines delivered via the mucosa are therefore of great interest to scientists developing vaccines for this disease. In the present study, we characterized field isolates of F. columnare obtained from clinical columnaris outbreaks in red tilapia to select an isolate to use as a candidate for our vaccine study. This included characterizing its colony morphology, genotype and virulence status. The isolate was incorporated into a mucoadhesive polymer chitosan-complexed nanovaccine (CS-NE), the efficacy of which was determined by experimentally infecting red tilapia that had been vaccinated with the nanoparticles by immersion. The experimental infection was performed 30-days post-vaccination (dpv), which resulted in 89% of the unvaccinated control fish dying, while the relative percentage survival (RPS) of the CS-NE vaccinated group was 78%. Histology of the mucosal associated lymphoid tissue (MALT) showed a significantly higher presence of leucocytes and a greater antigen uptake by the mucosal epithelium in CS-NE vaccinated fish compared to control fish and whole cell vaccinated fish, respectively, and there was statistically significant up-regulation of IgT, IgM, TNF α, IL1-β and MHC-1 genes in the gill of the CS-NE vaccinated group. Overall, the results of our study confirmed that the CS-NE particles achieved better adsorption onto the mucosal surfaces of the fish, elicited great vaccine efficacy and modulated the MALT immune response better than the conventional whole cell-killed vaccine, demonstrating the feasibility of the mucoadhesive nano-immersion vaccine as an effective delivery system for the induction of a mucosal immune response against columnaris disease in tilapia.

Wilms’ tumor 1 (WT1) is a tumor-associated antigen and immunotherapy target in myelodysplastic syndrome (MDS). Further information is needed on the characteristics of WT1-specific CD8 + T cells to develop immunotherapeutic strategies for MDS. To clarify the frequency, distribution, and phenotype of WT1-specific CD8 + T cells, which occur innately in MDS patients, we analyzed paired peripheral blood (PB) and bone marrow (BM) samples from 39 patients with MDS or acute myeloid leukemia with myelodysplasia-related changes. The median frequency of WT1 tetramer-binding CD8 + T cells in the CD8 + T cell population was 0.11% in PB and 0.18% in BM. A further tetramer assay combined with mixed lymphocyte peptide culture (MLPC assay) was used to detect functional WT1-specific CD8 + T cells that could respond to the WT1 peptide. Functional WT1-specific CD8 + T cells were detected in BM in 61% of patients, which was significantly higher than in PB (23%, p = 0.001). The frequency of these cells estimated by the MLPC assay was tenfold higher in BM than in PB. The majority of WT1 tetramer-binding CD8 + T cells in BM had a unique phenotype with co-expression of CD39 and CXCR4. These findings will facilitate the development of novel immunotherapeutic strategies for MDS.

The bone marrow (BM) microenvironment, known as the BM niche, regulates hematopoiesis but is also affected by interactions with hematopoietic cells. Recent evidence indicates that extracellular matrix components are involved in these interactions. Chondroitin sulfate (CS), a glycosaminoglycan, is a major component of the extracellular matrix; however, it is not known whether CS has a physiological role in hematopoiesis. Here, we analyzed the functions of CS in hematopoietic and niche cells. CSGalNAcT1, which encodes CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, was highly expressed in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs), but not in mesenchymal stromal cells (MSCs) in BM. In T1 knockout (T1KO) mice, a greater number of HSPCs existed compared with the wild-type (WT), but HSPCs from T1KO mice showed significantly impaired repopulation in WT recipient mice on serial transplantation. RNA sequence analysis revealed the activation of IFN-α/β signaling and endoplasmic reticulum stress in T1KO HSPCs. In contrast, the number of WT HSPCs repopulated in T1KO recipient mice was larger than that in WT recipient mice after serial transplantation, indicating that the T1KO niche supports repopulation of HSPCs better than the WT niche. There was no obvious difference in the distribution of vasculature and MSCs between WT and T1KO BM, suggesting that CS loss alters vascular niche functions without affecting its structure. Our results revealed distinct roles of CS in hematopoietic cells and BM niche, indicating that crosstalk between these components is important to maintain homeostasis in BM.

Erdheim-Chester disease (ECD), a rare form of non-Langerhans cell histiocytosis, is characterized by the infiltration of foamy CD68+ and CD1a- histiocytes into multiple organ systems. Central nervous system (CNS) involvement has recently been reported to be a poor prognostic factor when treating ECD with interferon alpha. We report the case of a 66-year-old Japanese patient with ECD involving the CNS who harbored the BRAF V600E mutation and also concomitantly developed polycythemia vera with the JAK2 V617F mutation. We confirmed 2-chlorodeoxyadenosine (cladribine) therapy to be effective for the patient in this case.

Rosemary (Rosmarinus officinalis) is a new therapeutic agent against streptococcosis and monogenean infections in fish. However, the toxicity of rosemary and the kinetics of one of its major component (1,8-Cineole) are unclear. In the present study, liver, kidney and intestine histopathology, plasma chemistry, kinetics of 1,8-Cineole in blood and mucus were examined in common carp fed a diet containing rosemary extract. Feeding the fish ≥20 ml aqueous extract/100 g feed caused nuclear pyknosis and cell atrophy in the liver. Fish fed ≥40 ml aqueous extract/100 g developed pyknotic cells with cytoplasmic vacuoles in the kidney, leading to tubular necrosis. Feeding the fish the rosemary diet for 10 and 20 days increased plasma aspartate aminotransferase levels, a sign of liver damage. Following oral administration of 80 ml aqueous extract/100 g feed, the 1,8-Cineole blood level peaked at 60 min at 117.9 ± 3.5 ng/ml. The elimination half-life (T1/2) of 1,8-Cineole in the blood was 248 min. In mucus, the levels of 1,8-Cineole at the end of 5, 10 and 20 days were 1.5 ± 0.8 ng, 5 ± 4.1 ng and 6.1 ± 3.8 ng per mg crude dried mucus, respectively. These results suggest that at high doses, rosemary aqueous extract diets can cause liver and kidney damage in common carp. Further studies are needed to establish a suitable dosage for oral treatment of parasitic diseases.

From the view point of green chemistry, CO2 utilization technologies with solar energy including the photoredox system have been received a lot of attention. As one of them, photoredox system containing a photosensitizer and a catalyst catalyzing a reaction of a carbon–carbon bond formation from CO2 as a feed stock were constructed. In a recent study, we reported the visible light-induced malate (C4 compound) production from pyruvate (C3 compound) and CO2 due to carbon–carbon bond formation with the system consisting an electron donor, a photosensitizer, diphenylviologen (PV2+) derivative as an electron mediator in the presence of malic enzyme (ME). However, the interaction between a photosensitizer and PV2+ derivative has not been clarified yet. In this study, water-soluble PV2+ derivative, 1,1′-bis(p-sulfonatophenyl)-4,4′-bipyridinium salt (PSV2+) was synthesized, and its electro-, photochemical properties were evaluated. Moreover, the photoredox properties of PSV2+ with water-soluble Zn porphyrin were studied using fluorescence spectroscopy and steady state irradiation. The fluorescence of Zn porphyrin was quenched by PSV2+ and the two-electron reduced form of PSV2+ were produced with Zn porphyrin with steady state irradiation. In addition, reaction solution containing triethanolamine, tetraphenylporphyrin tetrasulfonate, pyruvate, ME, Mg2+ and PSV2+ in CO2 saturated bis-tris buffer (pH 7.4) was irradiated with visible light, the oxaloacetate and malate were produced. This result indicates that PSV2+ is an efficient electron mediator in the visible light-induced redox system for carbon–carbon bond formation with ME from CO2 as a feedstock.

The Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) is a widely used tool for pre-transplant risk assessment. Allogeneic hematopoietic cell transplantation (HCT) is performed on patients with diverse backgrounds, highlighting the need for other predictors to complement the HCT-CI and support bedside decision-making. There is a strong body of evidence supporting the use of pre-transplant serum ferritin (SF) in risk assessments of allogeneic HCT. We additionally found that the Glasgow Prognostic Score (GPS), which assesses inflammatory biomarkers and predicts survival of patients with solid organ malignancies, is a useful predictive marker for overall survival (OS) and non-relapse mortality (NRM) in allogeneic HCT, independent of HCT-CI and SF. In this study, we refined the GPS by adding pre-transplant SF to improve its prognostic ability and enable better stratification; we call this revised index the HCT-specific revised Glasgow Prognostic Score (HCT-GPS). We observed that the HCT-GPS more accurately predicted NRM and early-term OS than the GPS. Moreover, the HCT-GPS provides an independent prognostic factor adjusted for the HCT-CI and disease status, and stratifies patients into four risk groups by OS and NRM. Thus, the HCT-GPS is a useful index for predicting early-term complications after allogeneic HCT in patients with hematopoietic diseases.

Formate dehydrogenase (FDH) is an attractive biocatalyst for converting CO 2 to formic acid in ambient conditions. FDH is applied to the catalyst for the visible-light induced CO 2 – formic acid conversion system consisting of an electron donor, a photosensitizaer and an electron carrier. For improvement of the formic acid production efficiency with this system, the enhancement of electron relay processes among a photosensitizer, an electron carrier and FDH has been needed. We aimed to solve this problem by developing novel electron carrier based on the viologen derivative instead of methylviologen (MV), widely used as an electron carreir. In this study, viologen derivatives with carbamoyl group, 1,1′-dicarbamoylmethyl-4,4′-bipyridinium diiodide (CV) and 1-carbamoylmethyl-1′-methyl-4,4′-bipyridinium diiodide (CMV) were synthesized and applied to the electron carrier for photoinduced CO 2 -formic acid conversion system consisting of triethanolamine, water-soluble zinc porphyrin and FDH. By using CV and CMV, the effective formic acid production in the system of water-soluble zinc porphyrin and FDH was improved compared with that of MV.

Molecular conversion reaction system containing a dye and a biocatalyst catalyzing a reaction of a carbon–carbon bond formation from CO2 as a feedstock were constructed. For example, NADP-malic enzyme (ME, EC1.1.1.40) catalyzes the reaction of introducing CO2 as a carboxy group to pyruvate (C3 compound) to form malate (C4 compound) via oxaloacetate in the presence of natural co-enzyme NADPH. In a recent study, visible light-induced oxaloacetate production from pyruvate and CO2 with the system consisting an electron donor, a photosensitizer, diphenylviologen (PV2+) derivative as an electron mediator and ME was reported. However, the visible light-induced reduction properties of PV2+ with water-soluble Zn porphyrin have not been clarified. In this study, chemical and photo-redox properties of PV2+ were studied. In addition, to produce malate, CO2 saturated reaction solution containing triethanolamine, tetraphenylporphine tetrasulfonate (H2TPPS), pyruvate, PV2+ and ME in the presence of Mg2+, co-factor for ME was irradiated with visible-light, the oxaloacetate and malate were produced.

Introduction Late non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation(allo-HCT) is a problem that is yet to be solved. Moreover, no efficient markers exist to predict late NRM. Vascular endothelial damage is known to be a cause of late NRM after allo-HCT. The transplant endothelial activation and stress index (EASIX) was initially established for the diagnosis of thrombotic microangiopathy. Pre-transplant EASIX (EASIX-pre) quartiles were reported to be predictive markers for early NRM after allo-HCT, but EASIX could also potentially help in the evaluation of late NRM. Since late NRM may be affected by time dependent factors that cannot be evaluated before allo-HCT, the timing of EASIX evaluation may be important. Therefore, we focused on EASIX 1 year after allo-HCT (EASIX-1year). Aims This study aimed to clarify the usefulness of EASIX-pre and EASIX-1year as predictive markers of late NRM and overall survival (OS) after allo-HCT. Methods Among 210 patients with hematological disease who underwent a first allo-HCT between 2006 and 2019at our facility, we evaluated EASIX-1year in 102(53 males and 49 females) patients who were alive after 1 year without relapse and/or withdrawal. EASIX was calculated as LDH level (U/I) × Cre level (mg/dL) / Plt level (nL). EASIX-pre was evaluated 7-10 days before conditioning. EASIX-1year was evaluated within 1 month of 1 year after allo-HCT. Landmark analysis was used to perform statistical analysis of late NRM and OS starting 1 year after allo-HCT. Late NRM and OS assessments using EASIX-pre and EASIX-1year were performed with two risk groups based on the cutoff values from the receiver operating characteristic curve. Forty-four patients had acute myeloid leukemia, 24 had acute lymphoblastic leukemia, 14 had myelodysplastic syndrome, 8 had malignant lymphoma, and 12 had other diseases. The median age of the patients was 40 years (range: 16-66 years). Fifty-seven patients received myeloablative conditioning and the others received reduced intensity conditioning regimens. The number of patients in each HCT-comorbidity index (HCT-CI) risk group was as follows: low risk: 51, intermediate risk: 28, and high risk: 23. This study was performed in accordance with the Japanese Ethical Guidelines for Medical and Health Research Involving Humans and approved by the Ethical Committee of our facility. Results Median EASIX-pre was0.98 (0.12-24.1). The C-statistic of EASIX-pre for late NRM and OS were 0.561 (cutoff value: 0.595) and 0.591 (cutoff value: 0.766), respectively. Univariate analysis revealed that a high EASIX-pre value was not significantly associated with late NRM (5-year NRM 3.4% vs. 0%, p=0.24) and OS (5-year OS 91.3% vs. 93.5%, p=0.22). Moreover, the HCT-CI at pre-transplantation was not an indicator of late NRM (5-year NRM 10.2 % vs. 0%, p=0.29, 5-year OS 79.8% vs. 96.2%, P=0.19). Median EASIX-1year was 0.98 (0.15-21.8). The C-statistic of EASIX-1year for late NRM and OS were 0.63 (cutoff value 2.396) and 0.663 (cutoff value 1.159), respectively. Univariate analysis revealed that a high EASIX-1year value was significantly associated with late NRM (5-year NRM 10.1% vs. 1.3%, p By adjusting age, donor source, HCT-CI, chronic graft versus host disease, and conditioning in multivariate analysis, a high of EASIX-1year was extracted as the risk factor for late NRM (Hazard ratio 4.26, p Conclusion The present study indicated that EASIX-1year may be useful as a prognostic index for late NRM. Otherwise, pre-transplant conditions, such as EASIX-pre and pre-transplant HCT-CI, may have limited effects on late NRM. Disclosures No relevant conflicts of interest to declare.

Introduction Despite advances in the genetic analysis of acute myeloid leukemia (AML), Wilms' tumor oncogene 1 (WT1) mRNA remains an important pan-marker of AML. A log reduction in WT1 mRNA expression after chemotherapy is a predictor of prognosis, and WT1 mRNA can be used as a marker of minimal residual disease or relapse. In the 1990s, a high expression level of WT1 mRNA at diagnosis was considered a poor prognostic factor. However, recent analyses have found that WT1 mRNA expression varies with AML type. Since the prognosis is affected by cytogenetic abnormalities and therapeutic intensity, we re-evaluated the clinical significance of WT1 mRNA expression at diagnosis in the context of the European Leukemia Net (ELN) risk classification and treatment. Method We retrospectively analyzed 216 patients at five institutions between 2011 and 2020. The expression level of WT1 mRNA was measured for all patients at diagnosis using bone marrow (BM) samples from 191 patients and peripheral blood (PB) samples from 25. WT-1 mRNA expression was measured using real-time quantitative polymerase chain reaction and the measured values were converted in normal logarithm. The median age at diagnosis was 62 (range: 23-93) years. The cytogenetic risk of ELN was classified as favorable (n = 41), intermediate (n = 123), and adverse risk (n = 41). Selected therapeutics were standard chemotherapy (n = 182, 84.3%, including CAG regimen; cytarabine, aclarubicin and G-CSF), hypomethylating agents or low-dose cytarabine (n = 19, 8.8%), and best supportive care (n = 15, 6.9%). Also, 143 patients (66.2%) received one or more courses of standard consolidation chemotherapy and 61 (28.3%) underwent allogeneic hematopoietic stem cell transplantation (allo-HCT). The median observation period was 518 [1-3418] days (Table 1). The overall survival (OS) and event-free survival (EFS) rates were assessed. Relapse or death was defined as an event, and OS was evaluated on the date of death. Result The median expression level of WT1 mRNA was 4.68 (range: 1.0-5.72) [log copies/µg RNA, units are omitted thereafter] in BM and 3.66 (range: 1.34-5.20) in PB (Table 1). Favorable-risk AML had the highest WT1 mRNA expression level in BM (4.95, p = 0.0054), whereas there was no difference between the expression levels in intermediate- and adverse-risk AML in BM (4.63 and 4.47, p = 0.711, Fig. 1A). WT1 mRNA expression in PB were 4.04, 3.84, and 3.05 for favorable-, intermediate-, and adverse-risk AML, respectively, and were higher for those with a favorable-risk AML (vs. adverse risk, p = 0.048, Fig. 1B). When WT1 mRNA expression level in BM was compared between the two groups, i.e., Since AML prognosis is affected by ELN risk and selected therapeutics, we evaluated 109 intermediate-risk patients who had received at least one course of standard chemotherapy. Fifty of them (45.9%) underwent subsequent allo-HCT. The 2-year EFS rates of PB or BM-WT1 low (n = 25, PB = 2 and BM = 23) vs. PB or BM-WT1 high (n = 84, PB = 23 and BM = 61) were 23.9% and 50.4%, respectively (p = 0.023, Fig. 2A) and the 2-year OS rates were 51.2% and 64.6%, respectively (p = 0.03, Fig. 2B). PB or BM-WT1 low of intermediate-risk AML had a poor prognosis even with standard chemotherapy. Multivariate analysis adjusted for ages, the reduction rate of WT1 mRNA after induction chemotherapy, duration of receiving consolidation chemotherapy, allo-HCT, and PB or BM-WT1 low at diagnosis were independent poor prognosis factors for EFS in intermediate-risk AML (HR: 3.32, 95%CI: 1.29-8.53, p = 0.013). The OS rate of PB or BM-WT1 low also worsened (HR: 2.33, 95%CI: 0.88-6.18, p = 0.089) (Table 2). Whereas, the outcome of adverse-risk AML was not affected by PB or BM-WT1 low/high. Conclusion The expression level of WT1 mRNA at diagnosis was negatively correlated with prognosis. Favorable-risk AML had higher expressions of WT1 mRNA. PB or BM-WT1 low in intermediate-risk AML was associated with a poor prognosis. In standard-risk AML, WT1 mRNA may be a useful pan-marker to predict prognosis at diagnosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

[Introduction] Remarkable advances have been made in cancer immunotherapy, including the development of peptide-based cancer vaccines. Wilms' tumor 1 (WT1) is one of the cancer-testis antigens, and the WT1 gene is overexpressed in hematologic malignancies. Several clinical trials of WT1 peptide vaccines showed promising efficacy and high safety of the vaccines for hematologic malignancies. In these trials, immunological responses were assessed within 2 years after vaccination, and the transient WT1-specific immune response observed in many cases early after vaccination was confirmed. However, the long-term durability of the response of WT1-specific CD8+ cytotoxic T lymphocytes (CTLs) after peptide vaccine therapy and the T-cell receptor (TCR) diversity in those CTLs has not been clarified. More than 10 years ago, a patient with chronic myeloid leukemia (CML) received WT1 peptide vaccination after the failure of tyrosine kinase inhibitor therapy. After vaccination, WT1-specific CD8+ CTLs were observed. We continued the immunological assessment of the patient for more than 10 years after the WT1 peptide vaccination. Herein, we report our findings from the long-term monitoring of WT1-specific CTLs in the patient with CML and describe the results of our detailed analysis, including the functionality and clonality of the CTLs. [Methods] After obtaining written consent from a patient whose CML was difficult to control by imatinib, HLA-A*24:02-restricted modified-type WT1 peptide (WT1 peptide; 9 mer peptide of CYTWNGMNL) was administered to the patient. Post-vaccination, we followed up with the patient. Immune monitoring was performed using a WT1 tetramer assay after mixed lymphocyte peptide culture (MLPC assay). The limiting-dilution (mononuclear cells divided into 20 wells or more, equally containing 3 × 10 5 cells), 2-week cultures with WT1 peptide stimulation and counting of "positive wells" containing expanded WT1 tetramer+ CD8+ T cells were performed for the MLPC assay. The MLPC assay was used to detect functional WT1-specific CD8+ T cells that can expand in response to the WT1 peptide and estimate the frequency of these WT1-specific CD8+ T cells among all CD8+ T cells. For the functionality of WT1-specific CD8+ T cells, we evaluated WT1-specific cytotoxicity and cytokine production in the presence and absence of WT1 peptide pulse to T2A24 cells transfected with the GFP gene (T2A24-GFP). The phenotype and TCR of the WT1-specific CD8+ T cells expanded by MLPC were analyzed using flow cytometry and next-generation sequencing, respectively. [Results] After the WT1 peptide vaccination, the copy numbers of major bcr-abl transcripts gradually decreased, and a therapy free remission was achieved in the patient. No severe adverse effects were observed. The estimated frequency of WT1-specific CD8+ T cells peaked in the third year after vaccination (27 cells per 10 6 CD8+ T cells, 0.00027%) and then declined to 1 - 5 per 10 6 CD8+ T cells at 13 years after vaccination. The WT1-specific CD8+ T cells showed that WT1 peptide-specific cytotoxicity and WT1 peptide-specific IFN-γ release in vitro. These WT1-specific CTLs had different TCRs in each MLPC well. This result was confirmed by three independent analyses, and no common TCRs were detected. Twelve different TCRs were detected in the three analyses. [Conclusion] The WT1 peptide vaccine successfully generated long-lasting and diverse WT1-specific immune responses in a patient with CML. The WT1 peptide vaccine may be a efficient immune therapy for CML patients. Disclosures No relevant conflicts of interest to declare.

Monogenean parasites are important ectoparasites of fish, and are responsible for severe economic impacts in the aquaculture industry. They are usually treated with chemicals, but the chemicals can have harmful side effects in the fish and may pose threats to human health. Rosemary (Rosmarinus officinalis) is a common medicinal herb, with antimicrobial and antitumor properties. Here, we examined the anthelmintic activity of rosemary extract against the monogenean (Dactylogyrus minutus) in vitro and in vivo using bath treatment and oral administration. The in vitro experiments showed that parasite survival was affected by both rosemary extract concentration and the solvent (water and ethanol). Parasites were dead at 61.8±5.6 and 7.8±1.4min when exposed to 100 and 200g aqueous rosemary extract solution/L of water respectively. It took 166.7±48.2 and 5.4±1.01min to kill the parasites when exposed to 1 and 32g ethanol rosemary extract solution/L of water respectively. Moreover, pure component of rosemary extract obtained commercially used in in vitro experiments showed that 1,8-Cineole was the most toxic component of the main components tested. Parasite intensity and prevalence in fish exposed to 50 and 100g aqueous rosemary solution/L water for 30min were significantly lower than they were in controls (p

Introduction: Severe acute graft versus host disease (GVHD) represents a major risk associated with allogeneic hematopoietic cell transplantation (HSCT). Both acute and progressive GVHD following HSCT are in large part attributable to responses of donor T cells to host allo-antigens. Alloreactive T cell activation can be modulated by signals from the tissue microenvironment (TME). Glycosaminoglycans (GAGs) in the TME or modifying T cell surface proteoglycans are known to regulate T cell function. However, the roles of GAGs in acute GVHD following allogeneic HSCT remain to be elucidated, since a suitable murine model has not been available. We previously established a unique mouse model (T1KO), in which knockout of the chondroitin sulfate (CS) N-acetylgalactosaminyltransferase-1 (T1) gene - encoding the rate-limiting CS-synthesizing enzyme - decreases CS production. We also reported a role for CS in murine hematopoietic stem cells. In the present study, we focus on donor T cell CS levels to assess the role of T cell CS in acute GVHD following allogeneic HSCT. We were able to mitigate the clinical features of GVHD in a murine model using T1KO donors. Methods: Eight- to 12-week-old T1KO mice were generated from a C57BL/6N (WT) strain as donors (H-2b) for allogeneic bone marrow transplantation (BMT). Donor BM cells were then transplanted into eight- to 12-week-old BALB/c recipients (H-2d). Prior to transplantation, recipients were irradiated at a dose of 7 Gy. They then received 5 x 106 BM cells and splenocytes of identical genotype, as well as low (5 x 105), intermediate (1 x 106), or high (4 x 106) doses of CD90.2+ cells from either WT or T1KO donors. For five weeks following BMT, survival was monitored daily and clinical GVHD scores (incorporating body weight, activity, fur condition, alopecia, and tortoiseshell-like mucosae) were calculated three times per week. Results: The peripheral blood CD4+/CD8+ T cell ratio of T1KO mice was equivalent to that of WT mice. Following allogeneic BMT, histopathologic analysis confirmed onset of acute recipient GVHD on day 49 when donors were WT mice. As a result, significant lymphocyte infiltration was observed in target organs (liver, colon, and skin). High-dose transplantation of splenocytes from T1KO rather than WT mice significantly prolonged recipient median survival (27.0 versus 20.5 days, p = 0.02). A similar trend was observed for low- and intermediate-dose transplantation (low: 53.0 versus 29.0 days, p = 0.09; intermediate: 50.5 versus 30.5 days, p = 0.13. Similarly, a significant improvement in day 11 GVHD score was observed following high-dose splenocyte transplantation when donors were T1KO mice (mean scores: 1.00 versus 2.75, p < 0.01). However, no significant differences in GVHD score were observed following low- and intermediate-dose transplantation. Taken together, our results suggest that donor T cells producing lower CS levels alleviate acute GVHD following allogeneic HSCT in a murine model. Conclusion: Alloreactive T cell activation following allogeneic HSCT may be down-regulated by lower CS levels, thereby mitigating GVHD severity. Figure 1 Disclosures No relevant conflicts of interest to declare.

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70-90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.

Recently, CO2 utilization technology, including artificial photosynthesis, has received much attention. In this field, CO2 is used as a feedstock for fuels, polymers and in other chemical processes. Of note are malic enzymes (MEs) which catalyze the reaction of malic acid to pyruvic acid and CO2 with the co-enzyme NADP+, and catalyze the reverse reaction of pyruvic acid and CO2 to malic acid with the co-enzyme NADPH. Thus, MEs are also an attractive biocatalyst for carbon–carbon bond formation from CO2. Studies of the visible light-induced malic acid production from pyruvic acid and CO2 using an electron donor, a photosensitizer, an electron mediator, ferredoxin-NADP+ reductase, NADP+, and ME have been reported. However, modification of these systems is required, as they are very complicated. In this study, the visible light-induced carbon–carbon bond formation from pyruvic acid and CO2 with ME using the photoreduction of 1,1′-diphenyl-4,4′-bipyridinium salt derivatives as a novel electron mediator with water-soluble tetraphenylporphyrin tetrasulfonate (H2TPPS) in the presence of triethanolamine (TEOA) as an electron donor was developed. When a sample solution containing TEOA, H2TPPS, 1,1′-diphenyl-4,4′-bipyridinium salt derivative, pyruvic acid, and ME in CO2-saturated bis–tris buffer was irradiated, the major product was oxaloacetic acid. Thus, a visible light-induced photoredox system for carbon–carbon bond formation from CO2 with ME using 1,1′-diphenyl-4,4′-bipyridinium salt derivative as an electron mediator was developed.

The host-bacterium interaction between striped pangasius catfish (Pangasianodon hypophthalmus) and Edwardsiella ictaluri was examined during experimentally induced bacillary necrosis of pangasius (BNP). After infection by immersion challenge, fish samples were taken over the course of the infection. Necrotic dermatitis, associated with vasculitis-perivasculitis changes, was the most notable gross and histopathological change observed, similar to that described during channel catfish enteric septicemia. Typical histopathological change associated with BNP were initially observed by 96 h post-infection (hpi) and these lasted until 14 days post infection (dpi) with no granulomatous formation evident. The presence of the bacterium was observed in various organs using immunohistochemistry (IHC), except for the brain, with positive staining observed within differing cell types, including phagocytic cells, myocardia, gill epithelia and capillary endothelia. No extracellular deposition of bacterial antigen was observed. Bacterial antigen persisted up to 1 month in the independent phagocytic cells, necrotic-associated phagocytic cells and melano-macrophage centers, confirming the intracellular nature of E. ictaluri disseminated in the tissues during septicemia. The bacterium was evident in the gills early in the infection (6–12 hpi) and in necrotic skin at 72 hpi. With the histopathological changes observed, this suggests that skin is another possible natural route for disseminating E. ictaluri in pangasius catfish during infection. Statement of relevance This information will improve our understanding of the pathogenesis of BNP, and establish more effective methods of control for this important disease, including methods for quantifying sub-lethal infections and assessing response against the invading pathogen.

Introduction: Suppressors of cytokine signaling (SOCS) are negative regulators of cytokine signaling; SOCS3 controls myeloid hematopoiesis by suppressing G-CSF. SOCS3 haploinsufficiency attenuates diet-induced obesity by enhanced leptin sensitivity while improving insulin resistance. We established SOCS3 conditional knock-out (KO) mice to elucidate how high fat diets (HFD) affect inflammation and obesity in complete SOCS3 deficiency. Methods: Embryonic lethality of complete SOCS3 deficiency was overcome using a conditional Socs3 allele inactivated by tamoxifen (TAM)-inducible cre-recombinase with SOCS3fl/flRosa26-CreERT2T/+ mice. They were fed HFD (60 kcal% fat) or control chaw (10 kcal% fat) starting at 4 weeks of age and TAM or vehicle administration was done at 8 weeks. Results: Although insulin resistance improved and obesity and fatty liver were not confirmed in SOCS3-KO with HFD (S3HFD), the S3HFD group looked moribund around day 30 post-TAM by systemic inflammation; their mean survival was 62.5 days after TAM. Other groups did not look moribund. Significant granulopoiesis was also observed in S3HFD (peripheral blood neutrophil counts 29301 ± 20581/mm3). This myeloid hematopoiesis occurred in the spleen and not the bone marrow. S3HFD exhibited the progression of various granulocytes from progenitor cells (c-kitint∼highLy6G-) to mature granulocytes (c-kit-Ly6G+).Hematopoietic infiltration was confirmed in the liver in S3HFD with granulocytes, monocytes, and macrophages. Inflammasome-associated genes (IL-1β and Caspase1) were elevated in the liver at day 65 post-TAM in S3HFD. Conclusion: SOCS3 deficiency showed myeloid proliferation under HFD without obesity and adiposity. SOCS3-KO may help in the clarification of the specific mechanism of myeloid proliferation and invasion in HFD-induced inflammation. Disclosure K. Cho: None. T. Ushiki: None. H. Ishiguro: None. T. Katagiri: None. T. Suwabe: None. H. Hirai: None. Y. Nakagawa: None. M. Masuko: None. W. Alexander: None. H. Shimano: None. H. Sone: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited.

The Rh complex contributes to cell membrane structural integrity of erythrocytes. RhDetailed Rh phenotyping by hemagglutination was performed using monoclonal and polyclonal anti-D, -C, -c, -E, and -e; monoclonal and polyclonal anti-Rh17 antibodies; and polyclonal anti-Rh29 antibodies. RHAG mRNA transcripts were analyzed by reverse transcription-polymerase chain reaction, and the mutation was verified by genomic sequencing.The genomic region spanning exon 6 contained a GA transition in the invariant GT motif of the 5' donor splice-site of Intron 6 (c.945+1GA). The RhThe mechanisms of hemolysis due to the Rh

In contrast to most mammals including human, fish cell lines have long been known to be immortal, with little sign of cellular senescence, despite the absence of transformation. Recently, our laboratory reported that DNA demethylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induces telomere-independent cellular senescence and senescence-associated secretory phenotype (SASP) in an immortal fish cell line, EPC (Epithelioma papulosum cyprini). However, it is not known how fish derived cultured cells are usually resistant to aging in vitro. In this study, we focused on Ras, which carries out the main role of Ras-induced senescence (RIS), and investigated the role of Ras in the regulation of senescence in EPC cells. Our results show that 5-Aza-dC induced the expression of the ras (hras, kras, nras) gene in EPC cells. EPC cells overexpressing HRas or its constitutively active form (HRasV12) showed p53-dependent senescence-like growth arrest and senescence-associated β-galactosidase (SA-β-gal) activity with a large and/or flat morphology characteristic of cell senescence. On the other hand, the SASP was not induced. These results imply that the increased expression of HRas contributes to early senescence in EPC cells, but it alone may not be sufficient for the full senescence, even if HRas is aberrantly activated. Thus, the limited mechanism of RIS may play a role in the senescence-resistance of fish cell lines.

Hypoxia is known as a potential immunomodulator in fish. This study therefore assesses the impact of chronic, moderate hypoxia on vaccine efficacy in Oreochromis niloticus. Serum antibody titer was used as a surrogate marker to detect vaccine efficacy. The fish were acclimatized to either moderate hypoxia (55 ± 5% DO) or normoxia (85 ± 5%DO) and immunized with formalin inactivated Vibrio anguillarum. Significantly, a higher antibody titer was found in normoxic fish than in moderate hypoxia. The normoxic group titer peaked at 14th dpv (days post vaccination) while the moderate hypoxic group peaked at 21st or 28th dpv. The absolute blood lymphocyte counts and serum bactericidal activities against V. anguillarum were significantly higher in normoxic fish. Serum killing of V. anguillarum appeared to be mainly via antibody-dependent classical complement pathway. Furthermore, the first week following vaccination appears critical for antibody production. This view was further supported by results obtained from gene expression assay, where the transcription level of all the detected immune related genes (IgM, IL-1 β, TCR-β, MHC-II β), except B cell activating factor, were significantly suppressed following exposure to moderate hypoxia. The overall results highlight that even though moderate hypoxia is not easily detectable in Oreochromis niloticus, it negatively affects antibody production by suppressing and delaying antibody response, ultimately affecting vaccine efficacy.

Interleukin-1 receptor-associated kinase 4 (IRAK-4) is an important component of the mammalian innate and adaptive defense system against infectious disease. In this study, ayu Plecoglossus altivelis altivelis IRAK-4 cDNA and genomic DNA were cloned and analyzed. The open reading frame of ayu IRAK-4 cDNA encodes 463 amino acids, including a death domain and a serine/threonine protein kinase domain of the mammalian IRAK-4 motif. The ayu IRAK-4 gene comprises 11 exons, and was found to be very similar to vertebrate IRAK-4 genes described thus far. Ayu IRAK-4 mRNA was expressed in all tissues, and predominantly in the gill, heart, intestine, whole kidney, and spleen. At 12 h after immersion challenge with Flavobacterium psychrophilum, ayu IRAK-4 mRNA expression in the whole blood was up-regulated (21.6-fold) compared to the control. The present study suggests that ayu IRAK-4 may be involved in the immune response.

Saccharomyces cerevisiae JCM 7255 was tested as a probiotic candidate in tilapia after encapsulating and freeze drying. Viability and morphology during storage and during transit through simulated gut and bile conditions were determined. Growth performance, anti-streptococcal activity and gut mucosal immune parameters were also tested. The viability of encapsulated yeasts was significantly high in simulated gastric and bile conditions and remained high after storage at room temperature for 14 days. The morphology of free S. cerevisiae revealed rough, bumpy, ruptured surface during incubation in gut and bile conditions. Agar spot anti-streptococcal activity showed inhibition of 20 out of 30 strains of Streptococcus agalactiae. Supplementation improved the intestinal structure and growth performance in tilapias. Intraepithelial lymphocytes in the proximal intestine were significantly observed. Lower cumulative mortality after the oral streptococcal challenge was also seen. The results suggest that encapsulated S. cerevisiae JCM 2755 could be a potential probiotic strain in tilapia culture.