Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

Site-specific glycosylation and single amino acid substitution dramatically reduced the immunogenicity of ß-lactoglobulin.

To reduce the immunogenicity of ß-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the D28N/P126A was conjugated with a ∼4 kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.

Simulation of the optical system in the GroundBIRD telescope.

GroundBIRD is a ground-based telescope for measuring the polarization of cosmic microwave background radiation, and it is soon to be operational at the Teide Observatory. The GroundBIRD telescope employs Mizuguchi-Dragone dual reflectors and 161 kinetic inductance detectors coupled with single polarization antennas as photon detectors. We present the results of our optical simulation on the pointing direction, stray light response, and influence of the blackbody radiation from the baffle. We also find that the power of the baffle radiation incident on the detectors is reduced by 99.95% when corrugated feed horns are coupled to the detectors.

Aggravation of Food Allergy by Skin Sensitization via Systemic Th2 Enhancement.

BACKGROUND: Recently, the relationship between antigen contact via skin (skin sensitization) and the development of food allergies has gained increasing attention. However, few studies have examined the effects of skin sensitization on healthy skin. OBJECTIVE: To examine the effect of sensitization in healthy skin on IgE and cytokine production during food allergy development. METHODS: The effect of skin sensitization on food allergy was evaluated using DO11.10 mice whose T cells express ovalbumin (OVA)-specific T-cell receptors. OVA was applied to the back skin of mice dehaired by various methods, and then food allergy was induced by providing them with an OVA-containing diet. OVA-specific IgE production in the sera and decreases in body temperature due to anaphylactic reaction were measured as indicators of food allergy. In addition, IL-4 production and proliferation of splenocytes were measured in mice with food allergy after skin sensitization. RESULTS: Skin sensitization in healthy skin increased IgE production and exacerbated anaphylactic symptoms induced by ingesting the antigen. Moreover, skin sensitization enhanced IL-4 production from splenocytes during the onset of food allergy. In contrast, oral tolerance was induced even after establishing skin sensitization. CONCLUSION: Skin sensitization temporarily exacerbated food allergy by enhancing systemic Th2 responses. These findings will help identify the mechanisms involved in food allergy and help develop treatments.

Preparation of hypoallergenic ovalbumin by high-temperature water treatment.

The high-temperature water treatment is one of the methods used to reduce the molecular weight of proteins. In this study, in order to establish a practical method for preparing hypoallergenic materials using the high-temperature water treatment, we investigated the effects of processing temperature on the antigenicity and allergenicity of a food allergen. Additionally, the foaming ability of the samples was also evaluated as a function desired in the food industry. We used ovalbumin as a model allergen. As a result, although there was no significant difference among the samples treated with different processing temperatures, all the antigens treated with high-temperature water showed a decrease in antigenicity and allergenicity. In addition, when ovalbumin was treated at a temperature of 130 °C or higher, there was a significant improvement in foaming properties. These findings indicate that high-temperature water treatment is a potential strategy for preparing practical hypoallergenic materials.

The Simons Observatory: metamaterial microwave absorber and its cryogenic applications.

Controlling stray light at millimeter wavelengths requires special optical design and selection of absorptive materials that should be compatible with cryogenic operating environments. While a wide selection of absorptive materials exists, these typically exhibit high indices of refraction and reflect/scatter a significant fraction of light before absorption. For many lower index materials such as commercial microwave absorbers, their applications in cryogenic environments are challenging. In this paper, we present a new tool to control stray light: metamaterial microwave absorber tiles. These tiles comprise an outer metamaterial layer that approximates a lossy gradient index anti-reflection coating. They are fabricated via injection molding commercially available carbon-loaded polyurethane (25% by mass). The injection molding technology enables mass production at low cost. The design of these tiles is presented, along with thermal tests to 1 K. Room temperature optical measurements verify their control of reflectance to less than 1% up to 65∘ angles of incidence, and control of wide angle scattering below 0.01%. The dielectric properties of the bulk carbon-loaded material used in the tiles is also measured at different temperatures, confirming that the material maintains similar dielectric properties down to 3 K.

Characterization of multicopper oxidase genes in the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), with focus on salivary gland-specific genes.

Multicopper oxidase (MCO) enzymes are present ubiquitously and act on diverse substrates. Recently, the presence of multiple MCO genes has been described in many insects. Based on sialotranscriptome data, we identified and comprehensively characterized six MCO genes: NcLac1S, 1G, and 2-5 in the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae). NcLac1S and NcLac1G belong to the MCO1 ortholog of other insects. NcLac2 forms a clade with MCO2s involved in the sclerotization and pigmentation of the cuticle. NcLac3 and NcLac4 form a clade with NlMCO3 -5 of the hemipteran Nilaparvata luges. NcLac5 forms a clade with MCORPs (MCO-related proteins) that lack amino acid residues normally highly conserved in copper-coordinated MCOs. NcLac1S and NcLac3 were specifically expressed in the salivary glands; whereas NcLac5 was primarily expressed in the salivary glands. Only NcLac3 protein is considered to have laccase activity in the salivary glands and salivary sheaths ejected by the insect. NcLac1G expression was relatively high in the testis. NcLac2 and NcLac4 were specifically expressed in the integument and in Malpighian tubules, respectively. Knockdown by RNA interference (RNAi) of either NcLac2 and NcLac5 in nymphs caused high mortality. All NcLac2-knockdown nymphs showed depigmentation and soft cuticle, and eventually died, as did other MCO2-knockdown insects. DsNcLac5-injected nymphs (third, fourth, and fifth-instar) showed high mortality, but injection into adults had no effect on survival or number of eggs deposited, suggesting that NcLac5 is not essential for survival after molting (eclosion). NcLac5 could be a promising target gene for control of N. cincticeps.

Lactobacillus helveticus SBT2171 alleviates allergic symptoms in a murine model for pollen allergy.

Lactic acid bacteria are known to have various health-promoting effects and are highly expected to find applications in anti-allergic food materials. In this study, we focused on Lactobacillus helveticus SBT2171 (LH2171), which reportedly modifies some unique immune responses and ameliorated symptoms of patients allergic to mites and house dust in the previous studies. We examined the effect of LH2171 on cytokine production by antigen-stimulated murine naïve splenocytes in vitro and demonstrated that it inhibited IL-4 and IL-13 production while enhancing IFN-γ and IL-10 production. Then, we examined the anti-allergic effect of LH2171 in vivo using a murine model of pollen allergy and found that LH2171 reduced the sneezing frequency when orally administered to mice. We successfully confirmed the immune modulatory activity of LH2171 and its anti-allergic activity against inhaled antigens. These evidences would contribute to identifying the anti-allergic mechanism of LH2171.Abbreviations: ALDH: aldehyde dehydrogenase; EGCG: epigallocatechin gallate; LAB: lactic acid bacteria; LH2171: Lactobacillus helveticus SBT2171; NALT: nasal-associated lymphoid tissue; OVA: ovalbumin.

Cyclopia Extracts Enhance Th1-, Th2-, and Th17-type T Cell Responses and Induce Foxp3+ Cells in Murine Cell Culture.

Cyclopia genistoides, one of the traditional South African medicinal plants, and other species of the same genus offer noteworthy phenolic profiles, in particular high levels of the anti-allergic xanthone mangiferin. Hot water and 40% ethanol-water (v/v) extracts, prepared from C. genistoides, Cyclopia subternata, and Cyclopia maculata, were tested for immune-regulating activity in vitro using murine splenocytes and mesenteric lymph node cells. The 40% ethanol-water extracts of C. genistoides and C. subternata significantly enhanced production of several types of cytokines, including IL-4, IL-17, and IFN-γ, by antigen-stimulated splenocytes. A concentration-dependent response was observed, noticeably for IFN-γ production. The activity of the extracts did not correlate with the content of any of the major phenolic compounds, indicative that other extract constituents also play a role in immunomodulation. Additionally, the increased ratio of CD4+CD25+Foxp3+ Treg cells to total CD4+ cells indicated induction of Foxp3+ cells when mesenteric lymph node cells were cultured in the presence of these two extracts. This study is the first reporting immunostimulatory activity for Cyclopia, which are widely consumed as the herbal tea known as honeybush, underpinning further investigations into the potential use of its extracts as adjuvants for mucosal immunotherapy.

Comprehensive analysis of epigenetically regulated genes in anergic T cells.

T cell anergy is one of the important mechanisms for immune tolerance. The results of many studies investigating the mechanism for T cell anergy induction have revealed that the expression of several genes was up-regulated in anergic T cells. It has also been demonstrated that the molecules encoded on those genes played a critical role in anergy induction. However, the mechanism for their up-regulation has not previously been clarified. We examined in this study the changes in gene expression and DNA methylation status caused by anergy induction. Our results demonstrate that the expression of many genes was changed by anergy induction, and that the DNA methylation status of some of these genes was also changed. We show here by a GO analysis that the extent of the change in methylation status caused by anergy induction was distinct between the groups of genes that were categorized.

Early detection of speech and voice disorders in Parkinson's disease patients treated with subthalamic nucleus deep brain stimulation: a 1-year follow-up study.

We previously reported that Parkinson's disease (PD) patients treated with subthalamic nucleus deep brain stimulation (STN-DBS) had distinct phenotypes of speech and voice disorders: hypokinetic dysarthria, stuttering, breathy voice, strained voice, and spastic dysarthria. However, changes over time remain unclear. In the present study, 32 consecutive PD patients were assessed before and up to 1 year after surgery (PD-DBS). Eleven medically treated PD patients were also assessed (PD-Med). Speech, voice, motor, and cognitive functions were evaluated. At baseline, the incidence of hypokinetic dysarthria (63% of PD-DBS vs. 82% of PD-Med), stuttering (50% vs. 45%), breathy voice (66% vs. 73%), and strained voice (3% vs. 9%) was similar between groups. At 1 year, a slight but significant deterioration in speech intelligibility (p < 0.001) and grade of dysphonia (p = 0.001) were observed only in PD-DBS group compared with baseline. During the follow-up, stuttering (9% vs. 18%) and breathy voice (13% vs. 9%) emerged in PD-DBS and PD-Med, but strained voice (28%) and spastic dysarthria (44%) emerged only in PD-DBS. After the stimulation was stopped, strained voice and spastic dysarthria improved in most patients, while stuttering and breathy voice improved in a minority of patients. These findings indicate that the most common DBS-induced speech and voice disorders are strained voice and spastic dysarthria and that STN-DBS potentially aggravates stuttering and breathy voice. An improved understanding of these types of disorders may help detect speech and voice deteriorations during the early phase and lead to appropriate treatments.

GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE).

RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species.

Functional improvements in ß-lactoglobulin by conjugating with soybean soluble polysaccharide.

Soybean soluble polysaccharide (SSPS) was hydrolyzed by autoclaving for 6 and 12 h to obtain SSPS (6 h) and SSPS (12 h). Bovine ß-lactoglobulin (BLG) was conjugated with each SSPS by the Maillard reaction to improve its function. Conjugation between BLG and each SSPS was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. BLG-SSPS (6 h) and BLG-SSPS (12 h), respectively, retained approximately 56 and 43% of the retinol-binding activity of BLG. Structural analyses by intrinsic fluorescence and enzyme-linked immunosorbent assay with monoclonal antibodies indicated that the native structure of BLG had almost been maintained in each conjugate and that the surface structure was covered by conjugated SSPS. The emulsifying properties of BLG were improved in each conjugate at pH 3, 5, and 7 in the presence of 0.2 M NaCl. The antibody responses to BLG-SSPS (6 h) and BLG-SSPS (12 h) were considerably reduced in BALB/c mice. We conclude that conjugation with SSPS was very effective for improving the function of BLG and this study would contribute to greater utilization of SSPS.

Establishment and characterization of a novel murine model for pollen allergy.

Although there have been many studies revealing the mechanism and establishing the therapeutical method for allergic rhinitis, no suitable animal models for allergic rhinitis, especially for pollen allergy, are currently available. We therefore aimed in this study to develop a murine model producing IgE in response to an inhaled antigen without using any adjuvants. Ovalbumin (OVA)-specific T cell receptor transgenic mice (DO11.10) inhaled an OVA solution for one h, twice a week, for six weeks. The resulting increase of OVA-specific IgE in the serum was observed depending on the times of inhalation. Spleen cells from mice that had inhaled the antigen produced more IL-4 and less IFN-γ than those from the control mice in vitro. These results indicate that inhaled antigen enhanced the Th2-type responses and induced IgE production in a T cell-mediated manner. Our findings would contribute to studies on prevention and treatment of pollen allergy.

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens.

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F2 progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.

Anti-allergic effect of Cyclopia (honeybush) extracts via anti-degranulation activity in a murine allergy model for inhaled antigen.

The anti-allergic effects of extracts prepared from two species of honeybush, Cyclopia genistoides and Cyclopia subternata, were demonstrated in vivo in a murine allergy model for inhaled antigen induced with ovalbumin (OVA) inhalation to mimic pollen allergy. Intake of the extracts increased the production of OVA-specific immunoglobulin (Ig) E (IgE), IgG1, and IgG2a antibodies in serum and significantly suppressed anaphylactic reaction-induced body temperature decline. Moreover, the extracts significantly inhibited antigen-antibody-induced degranulation in RBL-2H3 cells. They also inhibited body temperature decline when the allergic mice were given them after antigen sensitization, indicating that anti-degranulation activity is the major mechanism underlying the anti-allergic effect of Cyclopia extracts. Despite their qualitative and quantitative differences in phenolic composition, the two extracts exhibited similar effects, suggesting that several active compounds might be involved in the activity. Therefore, oral administration of either Cyclopia extract potentially exerts a systemic anti-allergic effect, supporting the increased consumption of honeybush tea for general wellness and improved quality of life.

An increased number of CD4+CD25+ cells induced by an oral administration of Lactobacillus plantarum NRIC0380 are involved in antiallergic activity.

BACKGROUND: Our previous study showed that an oral administration of Lactobacillus plantarum NRIC0380 inhibited immunoglobulin E (IgE) production in a murine model, and that orally administered NRIC0380 induced CD4+CD25+ Foxp3+ T, i.e. regulatory T (Treg), cells in the spleen and Peyer's patch of mice. Although it has been reported that Treg cells might suppress the allergic symptoms, the involvement of the cells in the antiallergic activity of lactic acid bacteria has not been clearly demonstrated. We therefore examined in detail the antiallergic activity of Treg cells obtained from mice that had been fed NRIC0380. METHODS: Treg cells were obtained from mice that had been fed NRIC0380. The T cell-suppressive effect of the cells was analyzed by coculturing the cells with splenocytes of ß-lactoglobulin-immunized mice and ß-lactoglobulin. The effects of the Treg cells on the IgE production and cutaneous anaphylaxis reaction were then analyzed by transferring the cells into another mouse. RESULTS: The Treg cells obtained from the mice that had been fed NRIC0380 showed similar T cell-suppressive activity to those cells obtained from the control mice. The Treg cells obtained from the mice fed NRIC0380 significantly inhibited the IgE production and active cutaneous anaphylaxis reaction when transferred into another mouse that was subsequently immunized with the antigen. Furthermore, the Treg cells also significantly suppressed the passive cutaneous anaphylaxis reaction when cotransferred with the IgE antibody into another mouse. CONCLUSIONS: The induction of Treg cells by the oral administration of NRIC0380 would be involved in the antiallergic activity of NRIC0380.

Induction of ALDH activity in intestinal dendritic cells by Lactobacillus plantarum NRIC0380.

Lactic acid bacteria have been reported to have various immune-regulating activities. We also found in the previous study that the oral administration of heat-killed Lactobacillus plantarum NRIC0380 induced CD4(+)CD25(+)Foxp3(+) cells (Treg cells). We examine in this present study the influence of NRIC0380 on the function of intestinal dendritic cells (DCs) in vitro and in vivo. The aldehyde dehydrogenase (ALDH) activity was significantly induced in DCs obtained from the mesenteric lymph node (MLN) by culturing with NRIC0380. The oral administration of NRIC0380 also significantly increased ALDH-positive DCs in MLN. NRIC0380 significantly enhanced the production of TGF-ß from MLN cells in vitro. These effects were not apparent in cells from the Peyer's patch (PP) and spleen (SPL). NRIC0380 also significantly enhanced the expression of B7-H1 on DCs of all organs in vitro. The effects of NRIC0380 on DCs, especially those located in MLN, might be involved in its function to induce Treg cells.

Functional improvements in ß-conglycinin by edible bioconjugation with carboxymethyl dextran.

ß-Conglycinin was conjugated with carboxymethyl dextran (CMD) by the Maillard reaction to improve its function. The ß-conglycinin-CMD conjugate was purified by dialysis. Conjugation was confirmed by SDS-PAGE with CBB and PAS staining. Composition of the ß-conglycinin-CMD was ß-conglycinin:CMD = 1:2.7 (molar ratio) which was confirmed by BCA method and phenol sulfuric acid method. Solubility of ß-conglycinin in the range of pH 2.0-7.0 was much improved by conjugation with CMD. Emulsifying property of ß-conglycinin at pH 7 and in presence of salt was improved by conjugation with CMD. Immunogenicity of ß-conglycinin was reduced by conjugation with CMD. Conjugation method performed in this study was considered to be valuable in that it can be used in food processing.

Reduced immunogenicity and endowed antimicrobial activity in ß-lactoglobulin by preparing edible bioconjugate.

ß-lactoglobulin (BLG) and ε-polylysine (PL) were bound by using microbial transglutaminase. Dextran (Dex) was further conjugated to the BLG-PL conjugate by the Maillard reaction. Confirmation of conjugation was carried out by SDS-PAGE. From the results of isoelectric focusing, it was revealed that the isoelectric point of the BLG-PL conjugate was shifted to the basic side as compared with native BLG. Immunogenicity of BLG in BALB/c mice was lowered by conjugation with PL and further lowered by conjugation with Dex. By conjugation with PL and PL-Dex, antibacterial activity against Staphylococcus aureus was endowed to BLG. Because the conjugation method in this study is a safe method, it is valuable in that it can be applicable to food processing.