Germ cell-specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens).

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Understanding buckwheat allergies for the management of allergic reactions in humans and animals.

Buckwheat allergy is an immediate hypersensitivity reaction that includes anaphylaxis mediated by specific IgE antibodies. Several IgE-binding proteins in common buckwheat have been reported to be possible clinically relevant buckwheat allergens. Although common buckwheat is popularly consumed in Asia, buckwheat allergy is becoming a serious problem not only in Asia but also in Europe. In addition, common buckwheat has also been found to be a causative agent of allergic symptoms in animals. In recent years, in addition to conventional food allergy testing methods, the development of component-resolved diagnosis (CRD) has improved the diagnostic accuracy of food allergy. The identification of allergens is essential for the construction of CRD. In this review, we introduce the different types of buckwheat allergens and discuss how each buckwheat allergen contributes to the diagnosis of buckwheat allergy. We also present the analysis of buckwheat allergen that will help reduce the allergenicity of common buckwheat and reduce buckwheat allergen molecules. These findings may be beneficial in overcoming buckwheat allergies in humans and animals.

Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko Gnathopogon caerulescens.

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.

[Concentration of Radioactive Cesium in Domestic Foods Collected from the Japanese Market in Fiscal Years 2014-2016].

Between fiscal years 2014 and 2016, we surveyed the concentration of radioactive cesium in commercial foods produced in areas where there is a risk of radiation contamination due to the Fukushima Daiichi nuclear disaster. The number of samples with a concentration of radioactive cesium that exceeded the regulatory limit （100 Bq/kg for general foods） was 9 out of 1,516 （0.6％） in fiscal 2014, 12 out of 900 （1.3％） in fiscal 2015, and 10 out of 654 （1.5％） in fiscal 2016. Even though some samples were expected to be contaminated with radioactive cesium, because wild mushrooms and edible wild plants were intentionally included in this survey, the percentage of samples that exceeded the regulatory limit was only around 1％. The surveillance results confirmed that the pre-shipment food monitoring conducted by local governments was properly and efficiently performed, although continuous monitoring of the concentration of radioactive cesium in cultivated and wild mushrooms, edible wild plants, and wild animal meats is still required.

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

Multiresidue determination of pesticides in tea by gas chromatography-tandem mass spectrometry.

An efficient and reliable GC-MS/MS method for the multiresidue determination of pesticides in tea was developed by modifying the Japanese official multiresidue method. Sample preparation was carefully optimized for the efficient removal of coextracted matrix components. The optimal sample preparation procedure involved swelling of the sample in water; extraction with acetonitrile; removal of water by salting-out; and sequential cleanup by ODS, graphitized carbon black/primary secondary amine (GCB/PSA) and silica gel cartridges prior to GC-MS/MS analysis. The recoveries of 162 pesticides from fortified (at 0.01 mg kg(-1)) green tea, oolong tea, black tea and matcha (powdered green tea) were mostly (95-98% of the tested pesticides) within the range of 70-120%, with relative standard deviations of <20%. Poor recovery of triazole pesticides was considered to be due to low recovery from the silica gel cartridges. The test solutions obtained by the modified method contained relatively small amounts of pigments, caffeine and other matrix components and were cleaner than those obtained by the original Japanese official multiresidue method. No interfering peaks were observed in the blank chromatograms, indicating the high selectivity of the modified method. The overall results suggest that the developed method is suitable for the quantitative analysis of GC-amenable pesticide residues in tea.

Differential analysis of protein expression in RNA-binding-protein transgenic and parental rice seeds cultivated under salt stress.

Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.

Identification and detection of genetically modified papaya resistant to papaya ringspot virus strains in Thailand.

Many lines of genetically modified (GM) papaya (Carica papaya Linnaeus) have been developed worldwide to resist infection from various strains of papaya ringspot virus (PRSV). We found an unidentified and unauthorized GM papaya in imported processed papaya food. Transgenic vector construct that provides resistance to the PRSV strains isolated in Thailand was detected. An original and specific real-time polymerase chain reaction method was generated to qualitatively detect the PRSV-Thailand-resistant GM papaya.

RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds.

KEY MESSAGE: RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.

Characterization of natural aryl hydrocarbon receptor agonists from cassia seed and rosemary.

Many recent studies have suggested that activation of the aryl hydrocarbon receptor (AhR) reduces immune responses, thus suppressing allergies and autoimmune diseases. In our continuing study on natural AhR agonists in foods, we examined the influence of 37 health food materials on the AhR using a reporter gene assay, and found that aqueous ethanol extracts of cassia seed and rosemary had particularly high AhR activity. To characterize the AhR-activating substances in these samples, the chemical constituents of the respective extracts were identified. From an active ethyl acetate fraction of the cassia seed extract, eight aromatic compounds were isolated. Among these compounds, aurantio-obtusin, an anthraquinone, elicited marked AhR activation. Chromatographic separation of an active ethyl acetate fraction of the rosemary extract gave nine compounds. Among these compounds, cirsimaritin induced AhR activity at 10-10² µM, and nepitrin and homoplantagenin, which are flavone glucosides, showed marked AhR activation at 10-10³ µM.

A new reliable method for detecting specific IgE antibodies in the patients with immediate type wheat allergy due to hydrolyzed wheat protein: correlation of its titer and clinical severity.

BACKGROUND: Immediate-type wheat allergy caused by a specific hydrolyzed wheat protein (HWP-IWA), Glupearl 19S (GP19S), typically develops food-dependent exercise-induced anaphylaxis (FDEIA), but is different from conventional FDEIA, or simple wheat allergy in many aspects. The skin prick test (SPT) is considered to be the most effective method for diagnosis of HWP-IWA. As SPT is a relatively qualitative method, we developed quantitative and high-throughput test method for HWP-IWA. METHODS: An enzyme-linked immunosorbent assay (ELISA)-based GP19S-specific IgE assay was tested using sera from 14 HWP-IWA and five conventional wheat-dependent exercise-induced anaphylaxis (CO-WDEIA) patients, as well as five healthy subjects. Then a validation study at five different institutions was carried out using sera from 10 HWP-IWA and five CO-WDEIA patients, as well as five healthy subjects different from the previous studies. RESULTS: The mean unit values converted from measured absorbance of ELISA were 68.3, 1.3 and 1.1 respectively. Furthermore, the validation study revealed reproducible results across all five institutions, with the standard deviation (SD) being 0.3-0.4 for the healthy group, 0.2-0.6 for the CO-WDEIA group, and 3.8-9.6 for HWP-IWA group except for one case. One case of HWP-IWA was excluded from analysis due to the high SD of 53.3 units, indicating that samples with a unit value > 100.0 will affect inter-laboratory reproducibility. CONCLUSIONS: Our findings suggest that the ELISA-based GP19S-specific IgE assay can be used to test HWP-IWA using venous blood samples, except for those with a unit value > 100.0.

[Immunotoxicity and environmental substances].

A well functioning immune system is essential in maintaining integrity of the organism, and malfunction may have severe health consequences. Environmental substances may pose direct toxicity to components of the immune system, often leading to immunosuppression and resulting reduced resistance to infections and tumors. Alternatively, such substances may be recognized by the immune system in a specific fashion, which may result in allergy and autoimmunity. A proper risk assessment of environmental substances in terms of immunotoxicity is necessary. In this manuscript, I reviewed recent three topics about immunotoxicity: (1) IPCS/WHO Guidance for immunotoxicity risk assessment for chemicals, (2) Intestinal immunotoxicity, and (3) Epicutaneous sensitization of food proteins.

Establishment of monoclonal antibodies of BW10kDa distinguish it from Fag e 2 related to anaphylaxis.

BW10kDa, which is a buckwheat (BW) allergen, belongs to the 2S-albumin protein family, akin to Fag e 2. Detailed analyses of BW10kDa were lacking until this study. Herein, we conducted these analyses using monoclonal antibodies (mAbs) to recombinant BW10kDa (rBW10kDa). We successfully generated anti-rBW10kDa mAbs capable of distinguishing between Fag e 2 and BW10kDa. These mAbs were categorised into two types (type 1 and type 2) based on their reactivity to BW plant seed extracts in western blot analyses. Type 1 mAbs revealed two bands (15 kDa and 10 kDa), while type 2 mAbs showed a single band (15 kDa). Spot analyses using these mAbs confirmed that type 1 mAbs recognised epitopes near the C-terminal region, with the 10 kDa band representing the C-terminal subunit cleaved by protease. The mAbs targeting rBW10kDa enabled to assess the concentration of BW10kDa in wild type and also in diagnostic buckwheat extracts.

Effect of Left Ventricular Diastolic Dysfunction on the Cardiopulmonary Exercise Test in Patients With Cardiovascular Disease.

Left ventricular diastolic dysfunction exists in patients with heart failure with reduced ejection fraction and causes activity restriction and a poor prognosis, but there have been few reports about exercise tolerance in patients with diastolic dysfunction, regardless of left ventricular ejection fraction (LVEF). In this study, 294 cardiovascular disease patients who performed a cardiopulmonary exercise test (CPX) with an adequate examination by echocardiography at Fukuoka University Hospital from 2011 to 2020 were investigated. Patients were divided into groups with grade I and grade II or III diastolic dysfunction according to diagnostic criteria, regardless of LVEF, by echocardiography. After adjusting for age, gender, body mass index, smoking, and LVEF by propensity score matching, we compared the results of CPX between the grade I and grade II/III groups. There were no significant differences in hemodynamic parameters, or in the respiratory exchange ratio, oxygen uptake per body weight, oxygen uptake per heart rate, or parameters of ventilatory volume. Ventilatory equivalents per oxygen uptake and per carbon dioxide output were significantly worse in the grade II/III group from the rest to peak periods during CPX. In conclusion, left ventricular diastolic dysfunction worsens ventilatory efficacy during CPX. This effect potentially contributes to a poor prognosis in left ventricular diastolic dysfunction.

MucoRice-cholera toxin B-subunit, a rice-based oral cholera vaccine, down-regulates the expression of α-amylase/trypsin inhibitor-like protein family as major rice allergens.

To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application.

Evaluation of allergenicity of acid-hydrolyzed wheat protein using an in vitro elicitation test.

BACKGROUND: We performed an in vitro elicitation test to determine the ability of different types of wheat-allergic patients' IgE to induce humanized mast cell activation after the addition of various time-treated acid-hydrolyzed wheat proteins (HWPs). METHODS: The reactivity of heat- and various time-treated acid-hydrolyzed glutens (acid-HGs) and commercial acid-HWP (HWP1), using serum IgE from wheat allergy accompanied by skin and rhinoconjunctival sensitization to HWP1 in the facial soap, pediatric subjects with food allergy to native wheat, adult wheat-dependent exercise-induced anaphylaxis subjects, and nonatopic healthy subjects, was elucidated by dot blot and a luciferase assay-based in vitro elicitation test (EXiLE test). RESULTS: Serum from subjects sensitized with HWP1 reacted only to acid-HGs (acid-HGs treated for 0.5-3 or 6 h), but not native gluten, in the results of the dot blot. In contrast, sera from pediatric subjects sensitized with native wheat reacted to native gluten more strongly and showed only slight reactions to 0.5- to 1-hour-treated acid-HGs. The results of the in vitro elicitation test showed that acid hydrolyzation of the gluten attenuated antigen-induced luciferase expression in a time-dependent manner for sera from native-wheat-sensitized pediatric subjects. On the other hand, in the sera from HWP1-sensitized subjects, acid hydrolyzation of the gluten for 0.5 h dramatically increased luciferase expression. CONCLUSIONS: Even after prolonged hydrolyzation, acid-HGs still retained the ability to activate mast cells in the case of HWP1-sensitized subjects.