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Background Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. Results We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. Conclusions Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development., Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by certain bacteria and fungi that establish symbiotic relationships with plants. Here, the authors show that LCOs are produced also by many other, non-symbiotic fungi, and regulate fungal growth and development.

Systemic lupus erythematosus (SLE) is a complex and heterogeneous autoimmune disease involving multiple immune cells. A major hurdle to the elucidation of SLE pathogenesis is our limited understanding of dysregulated gene expression linked to various clinical statuses with a high cellular resolution. Here, we conducted a large-scale transcriptome study with 6,386 RNA sequencing data covering 27 immune cell types from 159 SLE and 89 healthy donors. We first profiled two distinct cell-type-specific transcriptomic signatures: disease-state and disease-activity signatures, reflecting disease establishment and exacerbation, respectively. We next identified candidate biological processes unique to each signature. This study suggested the clinical value of disease-activity signatures, which were associated with organ involvement and responses to therapeutic agents such as belimumab. However, disease-activity signatures were less enriched around SLE risk variants than disease-state signatures, suggesting that the genetic studies to date may not well capture clinically vital biology in SLE. Together, we identified comprehensive gene signatures of SLE, which will provide essential foundations for future genomic, genetic, and clinical studies.

Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 ("Kv137-1036") retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Background Behçet’s syndrome (BS) is an immune-mediated disease characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin symptoms. HLA-B51, as well as other genetic polymorphisms, has been reported to be associated with BS; however, the pathogenesis of BS and its relationship to genetic risk factors still remain unclear. To address these points, we performed immunophenotyping and transcriptome analysis of immune cells from BS patients and healthy donors. Methods ImmuNexUT is a comprehensive database consisting of RNA sequencing data and eQTL database of immune cell subsets from patients with immune-mediated diseases and healthy donors, and flow cytometry data and transcriptome data from 23 BS patients and 28 healthy donors from the ImmuNexUT study were utilized for this study. Differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify genes associated with BS and clinical features of BS. eQTL database was used to assess the relationship between genetic risk factors of BS with those genes. Results The frequency of Th17 cells was increased in BS patients, and transcriptome analysis of Th17 cells suggested the activation of the NFκB pathway in Th17 cells of BS patients. Next, WGCNA was used to group genes into modules with similar expression patterns in each subset. Modules of antigen-presenting cells were associated with BS, and pathway analysis suggested the activation of antigen-presenting cells of BS patients. Further examination of genes in BS-associated modules indicated that the expression of YBX3, a member of a plasmacytoid dendritic cell (pDC) gene module associated with BS, is influenced by a BS risk polymorphism, rs2617170, in pDCs, suggesting that YBX3 may be a key molecule connecting genetic risk factors of BS with disease pathogenesis. Furthermore, pathway analysis of modules associated with HLA-B51 indicated that the association of IL-17-associated pathways in memory CD8+ T cells with HLA-B51; therefore, IL-17-producing CD8+ T cells, Tc17 cells, may play a critical role in BS. Conclusions Various cells including CD4+ T cells, CD8+ T cells, and antigen-presenting cells are important in the pathogenesis of BS. Tc17 cells and YBX3 may be potential therapeutic targets in BS.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple immune cells. To elucidate SLE pathogenesis, it is essential to understand the dysregulated gene expression pattern linked to various clinical statuses with a high cellular resolution. Here, we conducted a large-scale transcriptome study with 6,386 RNA sequencing data covering 27 immune cell types from 136 SLE and 89 healthy donors. We profiled two distinct cell-type-specific transcriptomic signatures: disease-state and disease-activity signatures, reflecting disease establishment and exacerbation, respectively. We then identified candidate biological processes unique to each signature. This study suggested the clinical value of disease-activity signatures, which were associated with organ involvement and therapeutic responses. However, disease-activity signatures were less enriched around SLE risk variants than disease-state signatures, suggesting that current genetic studies may not well capture clinically vital biology. Together, we identified comprehensive gene signatures of SLE, which will provide essential foundations for future genomic and genetic studies.

Summary: Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming

Comparative functional genomics offers a powerful approach to study species evolution. To date, the majority of these studies have focused on the transcriptome in mammalian and yeast phylogenies. Here, we present a novel multi-species proteomic dataset and a computational pipeline to systematically compare the protein levels across multiple plant species. Globally we find that protein levels diverge according to phylogenetic distance but is more constrained than the mRNA level. Module-level comparative analysis of groups of proteins shows that proteins that are more highly expressed tend to be more conserved. To interpret the evolutionary patterns of conservation and divergence, we develop a novel network-based integrative analysis pipeline that combines publicly available transcriptomic datasets to define co-expression modules. Our analysis pipeline can be used to relate the changes in protein levels to different species-specific phenotypic traits. We present a case study with the rhizobia-legume symbiosis process that supports the role of autophagy in this symbiotic association.

Phototherapy using narrowband ultraviolet-B (NB-UVB) has been shown to be more effective than conventional broadband UVB (BB-UVB) in treating a variety of skin diseases. To assess the difference in carcinogenic potential between NB-UVB and BB-UVB, we investigated the cytotoxicity via colony formation assay, genotoxicity via sister chromatid exchange (SCE) assay, mutagenicity via hypoxanthine phosphoribosyltransferase (HPRT) mutation assay, as well as cyclobutane pyrimidine dimer (CPD) formation and reactive oxygen species (ROS) generation in Chinese hamster ovary (CHO) and their NER mutant cells. The radiation dose required to reduce survival to 10% (D10 value) demonstrated BB-UVB was 10 times more cytotoxic than NB-UVB, and revealed that NB-UVB also induces DNA damage repaired by nucleotide excision repair. We also found that BB-UVB more efficiently induced SCEs and HPRT mutations per absorbed energy dosage (J/m2) than NB-UVB. However, SCE and HPRT mutation frequencies were observed to rise in noncytotoxic dosages of NB-UVB exposure. BB-UVB and NB-UVB both produced a significant increase in CPD formation and ROS formation (p &lt, 0.05), however, higher dosages were required for NB-UVB. These results suggest that NB-UVB is less cytotoxic and genotoxic than BB-UVB, but can still produce genotoxic effects even at noncytotoxic doses.

Ascorbic acid 2-glucoside (AA2G), glucosylated ascorbic acid (AA), has superior properties for bioavailability and stability compared to AA. Although AA2G has shown radioprotective properties similar to AA, effects for UV light, especially UVC and UVB, are not studied. AA2G was tested for cytotoxicity and protective effects against ionizing radiation, UVC, and broadband and narrowband UVB in Chinese hamster ovary (CHO) cells and compared to AA and dimethyl sulfoxide (DMSO). Pretreatment with DMSO, AA, and AA2G showed comparative protective effects in CHO wild type and radiosensitive xrs5 cells for cell death against ionizing radiation with reducing the number of radiation-induced DNA damages. Pretreatment with AA and AA2G protected CHO wild type and UV sensitive UV135 cells from UVC and broadband UV, but not from narrowband UVB. DMSO showed no protective effects against tested UV. The UV filtration effects of AA and AA2G were analyzed with a spectrometer and spectroradiometer. AA and AA2G blocked UVC and reduced short wavelengths of UVB, but had no effect on wavelengths above 300 nm. These results suggest that AA2G protects cells from radiation by acting as a radical scavenger to reduce initial DNA damage, as well as protecting cells from certain UVB wavelengths by filtration.

Early rehabilitation for acute stroke patients is widely recommended. We tested the hypothesis that daily intervention by speech therapists promotes safe oral intake of patients with acute stroke.We analyzed hospitalized patients who experienced cerebral infarction and cerebral hemorrhage and who underwent rehabilitation between October 2010 and September 2014 at our hospital. In total, 936 patients were analyzed, and 452 patients underwent daily speech therapy. We examined the association of training frequency and eating status.Multiple linear regression analysis indicated that daily speech therapy was correlated significantly and positively with a reduction in the number of days of hospitalization until oral intake commenced (coefficient, -.998; 95% confidence interval, -1.793 to -.202; P .05), and was not correlated with the cessation of oral intake due to aspiration pneumonia after resuming oral intake.Our retrospective cohort study demonstrated that daily intervention by speech therapists in patients with acute stroke shortens the number of days until oral intake without increasing the incidence of aspiration pneumonia.

International audience; Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor. Compared with the wildtype Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.

Summary Genetic studies have revealed many variant loci that are associated with immune-mediated diseases. To elucidate the disease pathogenesis, it is essential to understand the function of these variants, especially under disease-associated conditions. Here, we performed a large-scale immune cell gene-expression analysis, together with whole-genome sequence analysis. Our dataset consists of 28 distinct immune cell subsets from 337 patients diagnosed with 10 categories of immune-mediated diseases and 79 healthy volunteers. Our dataset captured distinctive gene-expression profiles across immune cell types and diseases. Expression quantitative trait loci (eQTL) analysis revealed dynamic variations of eQTL effects in the context of immunological conditions, as well as cell types. These cell-type-specific and context-dependent eQTLs showed significant enrichment in immune disease-associated genetic variants, and they implicated the disease-relevant cell types, genes, and environment. This atlas deepens our understanding of the immunogenetic functions of disease-associated variants under in vivo disease conditions.

Legumes are essential components of agricultural systems because they enrich the soil in nitrogen and require little environmentally deleterious fertilizers. A complex symbiotic association between legumes and nitrogen-fixing soil bacteria called rhizobia culminates in the development of root nodules, where rhizobia fix atmospheric nitrogen and transfer it to their plant host. Here we describe a quantitative proteomic atlas of the model legume Medicago truncatula and its rhizobial symbiont Sinorhizobium meliloti, which includes more than 23,000 proteins, 20,000 phosphorylation sites, and 700 lysine acetylation sites. Our analysis provides insight into mechanisms regulating symbiosis. We identify a calmodulin-binding protein as a key regulator in the host and assign putative roles and targets to host factors (bioactive peptides) that control gene expression in the symbiont. Further mining of this proteomic resource may enable engineering of crops and their microbial partners to increase agricultural productivity and sustainability.

Heavy ions, characterized by high linear energy transfer (LET) radiation, have advantages compared with low LET protons and photons in their biological effects. The application of heavy ions within veterinary clinics requires additional background information to determine heavy ion efficacy. In the present study, comparison of the cell-killing effects of photons, protons and heavy ions was investigated in canine osteosarcoma (OSA) cells in vitro. A total of four canine OSA cell lines with various radiosensitivities were irradiated with 137Cs gamma-rays, monoenergetic proton beams, 50 keV/µm carbon ion spread out Bragg peak beams and 200 keV/µm iron ion monoenergetic beams. Clonogenic survival was examined using colony-forming as says, and relative biological effectiveness (RBE) values were calculated relative to gamma-rays using the D10 value, which is determined as the dose (Gy) resulting in 10% survival. For proton irradiation, the RBE values for all four cell lines were 1.0–1.1. For all four cell lines, exposure to carbon ions yielded a decreased cell survival compared with gamma-rays, with the RBE values ranging from 1.56–2.10. Iron ions yielded the lowest cell survival among tested radiation types, with RBE values ranging from 3.51–3.69 observed in the three radioresistant cell lines. The radiosensitive cell line investigated demonstrated similar cell survival for carbon and iron ion irradiation. The results of the present study suggest that heavy ions are more effective for killing radioresistant canine OSA cells when compared with gamma-rays and protons. This markedly increased efficiency of cell killing is an attractive reason for utilizing heavy ions for radioresistant canine OSA.

The flavonoids quercetin, and its natural glycosides isoquercetin and rutin, are phytochemicals commonly consumed in plant-derived foods and used as a food beverage additive. Semi-synthetic maltooligosyl isoquercetin, monoglucosyl rutin and maltooligosyl rutin were developed by synthetic glycosylation to improve their water solubility for food and other applications. Using a system of Chinese hamster ovary (CHO) cells, this study examined the differences in cytotoxic responses induced by short and continuous exposure of natural and synthetic flavonoids. By assessing cell viability after short term exposure and clonogenicity with continuous exposure under various flavonoids, quercetin aglycone is confirmed to be the most cytotoxic flavonoids, and heavily glucosylated maltooligosyl rutin was the least cytotoxic. The other heavily glucosylated maltooligosyl isoquercetin showed intermediate cytotoxicity and similar toxicity as isoquercetin. Keywords: Cytotoxicity, Flavonoid, Quercetin, Isoquercetin, Rutin

BACKGROUND In Japan, familial Mediterranean fever (FMF) is a rare cause of fever of unknown origin (FUO). However, we experienced an extraordinary number of FMF cases over 3 years. This suggests that many patients with FMF remain misdiagnosed in Japan. This study examines the clinical picture of FMF to assist Japanese clinicians in daily practice dealing with FUO. PATIENTS AND METHODS Three years of medical records were reviewed, and 38 patients with FMF or suspected FMF were collected from our patient database. We applied the Tel-Hashomer criteria to those patients. RESULTS Of the 38 patients, 30 were classified as having FMF in this investigation. The mean patient age was 27.8 years. MEFV gene mutations were detected in 14 patients. Three cases were colchicine-resistant. CONCLUSION Clinicians should recognize the pattern of short, spontaneously resolving attacks of fever with fever-free intervals, especially when they see patients with recurrent FUO in the outpatient setting.

BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU&lsquo, s effects have not been extensively studied yet. Therefore, we investigated EdU&rsquo, s potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were observed in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concentrations (similar to manufacturers suggested concentration, &gt, 5&ndash, 10 &mu, M) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with additional precautions.

Signals and signaling pathways underlying the symbiosis between legumes and rhizobia have been studied extensively over the past decades. In a previous phosphoproteomic study on the Medicago truncatula–Sinorhizobium meliloti symbiosis, we identified plant proteins that are differentially phosphorylated upon the perception of rhizobial signals, called Nod factors. In this study, we provide experimental evidence that one of these proteins, Early Phosphorylated Protein 1 (EPP1), is required for the initiation of this symbiosis. Upon inoculation with rhizobia, MtEPP1 expression was induced in curled root hairs. Down-regulation of MtEPP1 in M. truncatula roots almost abolished calcium spiking, reduced the expression of essential symbiosis-related genes (MtNIN, MtNF-YB1, MtERN1 and MtENOD40) and strongly decreased nodule development. Phylogenetic analyses revealed that orthologs of MtEPP1 are present in legumes and specifically in plant species able to host arbuscular mycorrhizal fungi, suggesting a possible role in this association too. Short chitin oligomers induced the phosphorylation of MtEPP1 like Nod factors. However, the down-regulation of MtEPP1 affected the colonization of M. truncatula roots by arbuscular mycorrhizal fungi only moderately. Altogether, these findings indicate that MtEPP1 is essential for the establishment of the legume–rhizobia symbiosis but might plays a limited role in the arbuscular mycorrhizal symbiosis.

Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a (64)Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of (64)Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from (64)Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair-deficient xrs5 cells were exposed to (64)Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D(10) values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that (64)Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/μm). γH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from (64)Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after (64)Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that (64)Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of (64)Cu-ATSM as a cancer treatment modality for hypoxic tumors.

The flavonoids quercetin, and its natural glycosides isoquercetin and rutin, are phytochemicals commonly consumed in plant-derived foods. Semi-synthetic water-soluble isoquercetin and rutin glycosides, maltooligosyl isoquercetin, monoglucosyl rutin and maltooligosyl rutin were developed by synthetic glycosylation to overcome solubility challenges for improved incorporation in food and medicinal applications. Quercetin and its natural glycosides are known to induce genetic instability and decrease cell proliferation. Using a system of Chinese hamster ovary (CHO) cells, this study examined the differences in cytotoxic and genotoxic responses induced by natural and synthetic flavonoids. Bioactivity evaluations using poly(ADP-ribose) polymerase (PARP) ELISA showed that the synthetic flavonoids were less effective in inhibiting PARP than the natural flavonoids, where PARP inhibitory effects decreased with glycosylation of flavonoids. In the genotoxic studies, treatments with flavonoids at a concentration range of 0.2 μM-1 mM induced significant frequencies of sister chromatid exchange (SCE) and micronuclei in CHO cells compared to spontaneous occurrences. The synthetic flavonoids monoglucosyl rutin and maltooligosyl rutin induced less genotoxic effects than the natural flavonoids. However, maltooligosyl isoquercetin induced similar responses as isoquercetin and rutin. The growth inhibition studies showed glycosylation dependent cytotoxicity in natural flavonoids. The quercetin aglycone exhibited the highest toxicity out of all the flavonoids studied. Differences in growth inhibition were not observed between the synthetic flavonoids, maltooligosyl isoquercetin and monoglucosyl rutin, and natural isoquercetin and rutin, respectively. Maltooligosyl rutin induced less cytotoxicity than rutin and monoglucosyl rutin. Our in vitro studies demonstrated that the synthetic flavonoids generally induced less genotoxic responses than their natural counterparts.

In the present study, the role of monoglucosyl-rutin as a potential radioprotector was investigated using mammalian cell culture models. Cell survival and DNA damage were assessed using colony formation, sister chromatid exchange and γH2AX assays. It was demonstrated that monoglucosyl-rutin was able to increase cell survival when exposed to ionizing radiation, possibly by decreasing the amount of base damage experienced by the cell. However, the present study also demonstrated that, despite monoglucosyl-rutin exhibiting radioprotective effects at low doses, high doses of monoglucosyl-rutin led to a decrease in plating efficiency and an increased doubling time. This effect may be due to double-strand breaks caused by high concentrations of monoglucosyl-rutin.

Poly(ADP-ribose) polymerase (PARP) inhibitors have been proven to represent superior clinical agents targeting DNA repair mechanisms in cancer therapy. We investigated PARP inhibitory effects of the natural and synthetic flavonoids (quercetin, rutin, monoglucosyl rutin and maltooligosyl rutin) and tested the synthetic lethality in BRCA2 mutated cells. In vitro ELISA assay suggested that the flavonoids have inhibitory effects on PARP activity, but glucosyl modifications reduced the inhibitory effect. Cytotoxicity tests of Chinese hamster cells defective in BRCA2 gene (V-C8) and its parental V79 cells showed BRCA2-dependent synthetic lethality when treated with the flavonoids. BRCA2 mutated cells were three times more sensitive to the flavonoids than the wild-type and gene complemented cells. Reduced toxicity was observed in a glucosyl modification-dependent manner. The present study provides support for the clinical use of new treatment drugs, and is the beginning of the potential application of flavonoids in cancer prevention and the periodic consumption of appropriate flavonoids to reduce cancer risk in individuals carrying a mutant allele of the BRCA2 gene.

Purpose : The impact of the damage distribution to cellular survival and chromosomal aberrations following high Linear Energy Transfer (LET) radiation was investigated. Materials and methods : High LET iron-ions (500 MeV/n, LET 200 keV/ um) were delivered to G1-phase synchronized Chinese Hamster Ovary (CHO) cells located at a vertical or horizontal angle relative to the ion beam in order to give same dose but diff erent fl uence and damage distribution. Results : Horizontal irradiation produced DNA double-strand break (DSB) along each ion track represented as clustered lines, and vertical irradiation produced a greater fl uence. The initial damages measured by premature chromosome condensation were equal per dose in both irradiation types. Horizontal irradiation proved to be less eff ective in cell killing than vertical at doses of more than 3 Gy. Vertical irradiation produced a higher number of metaphase chromosomal aberrations compared to horizontally irradiated samples. In particular, formation of exchange-type aberrations was the same, but that of deletiontype aberrations were signifi cantly higher after vertical irradiation than horizontal irradiation. Conclusions : Therefore, we concluded that high fl uence per dose is more eff ective than low fl uence per dose to produce radiationinduced chromosomal aberrations and to kill exposed cells following high LET heavy-ion exposure.

Flavivirus infections (including Japanese encephalitis, West Nile encephalitis and dengue fever/severe dengue) present a worldwide public health problem. Recent climate change may affect the geographical distribution of the arthropod vectors for these viruses and so the risk of flavivirus epidemics may increase. Many methods have been developed for the serological diagnosis of flavivirus infections, such as haemagglutination inhibition assay, enzyme-linked immunosorbent assay, and immunofluorescence in staining. However, the specificity of these assays varies. The plaque reduction neutralizing test (PRNT) using live viruses is currently the 'gold standard' for the differential serodiagnosis of flaviviruses. The specificity of results obtained with PRNT is better than that for other protocols and many laboratories apply the PRNT protocol to the differential serodiagnosis of flaviviruses. Here, recent refinements to the PRNT protocols with genetically modified recombinant viruses or reporter-harbouring virus-like particles are reviewed. Further, the problems associated with the differential serodiagnosis of flaviviruses using PRNT are discussed.

Plant science is an important, rapidly developing area of study. Within plant science, one area of study that has grown tremendously with recent technological advances, such as mass spectrometry, is the field of plant-omics; however, plant peptidomics is relatively underdeveloped in comparison with proteomics and metabolomics. Endogenous plant peptides can act as signaling molecules and have been shown to affect cell division, development, nodulation, reproduction, symbiotic associations, and defense reactions. There is a growing need to uncover the role of endogenous peptides on a molecular level. Mass spectrometric imaging (MSI) is a valuable tool for biological analyses as it allows for the detection of thousands of analytes in a single experiment and also displays spatial information for the detected analytes. Despite the prediction of a large number of plant peptides, their detection and imaging with spatial localization and chemical specificity is currently lacking. Here we analyzed the endogenous peptides and proteins in Medicago truncatula using matrix-assisted laser desorption/ionization (MALDI)-MSI. Hundreds of endogenous peptides and protein fragments were imaged, with interesting peptide spatial distribution changes observed between plants in different developmental stages.

Charged particle therapy utilizing protons or carbon ions has been rapidly intensifying over recent years. The present study was designed to jointly investigate these two charged particle treatment modalities with respect to modeled anatomical depth-dependent dose and linear energy transfer (LET) deliveries to cells with either normal or compromised DNA repair phenotypes. We compared cellular lethality in response to dose, LET and Bragg peak location for accelerated protons and carbon ions at 70 and 290 MeV/n, respectively. A novel experimental live cell irradiation OptiCell™ in vitro culture system using three different Chinese hamster ovary (CHO) cells as a mammalian model was conducted. A wild-type DNA repair-competent CHO cell line (CHO 10B2) was compared to two other CHO cell lines (51D1 and xrs5), each genetically deficient with respect to one of the two major DNA repair pathways (homologous recombination and non-homologous end joining pathways, respectively) following genotoxic insults. We found that wild-type and homologous recombination-deficient (Rad51D) cellular lethality was dependent on both the dose and LET of the carbon ions, whereas it was only dependent on dose for protons. The non-homologous end joining deficient cell line (Ku80 mutant) showed nearly identical dose-response profiles for both carbon ions and protons. Our results show that the increasingly used modality of carbon ions as charged particle therapy is advantageous to protons in a radiotherapeutic context, primarily for tumor cells proficient in non-homologous end joining DNA repair where cellular lethality is dependent not only on the dose as in the case of more common photon therapeutic modalities, but more importantly on the carbon ion LETs. Genetic characterization of patient tumors would be key to individualize and optimize the selection of radiation modality, clinical outcome and treatment cost.

Sphingomonas sp. NP5 can degrade a wide range of nonylphenol (NP) isomers that have widely contaminated aquatic environments as major endocrine-disrupting chemicals. To understand the biochemical and genetic backgrounds of NP degradation, a gene library of strain NP5 was constructed using a broad-host-range vector pBBR1MCS-2 and introduced into Sphingobium japonicum UT26. Several transformants accumulated reddish brown metabolites on agar plates dispersed with a mixture of NP isomers. Two different DNA fragments (7.6 and 9.3 kb) involved in the phenotype were isolated from the transformants. Sequence analysis revealed that both fragments contained an identical 1593 bp monooxygenase gene (nmoA), the predicted protein sequence of which showed 83 % identity to the octylphenol-4-monooxygenase of Sphingomonas sp. PWE1. The nmoA gene in the 7.6 kb fragment was surrounded by an IS21-type insertion sequence (IS) and IS6100, while another in the 9.3 kb fragment was adjacent to an IS66-type IS, suggesting that they have been acquired through multiple transposition events. A fast-growing recombinant Pseudomonas putida strain harbouring nmoA was constructed and used for degradation of a chemically synthesized NP isomer, 4-(1-ethyl-1-methylhexyl)phenol. This strain converted the isomer into hydroquinone stoichiometrically. 3-Methyl-3-octanol, probably originating from the alkyl side chain, was also detected as the metabolite. These results indicate that these two nmoA genes are involved in the NP degradation ability of strain NP5.

A method for identifying the enantiomers of N,O-di-trifluoroacetylated ephedrine (EP) and norephedrine (NE) and the enantiomers of pseudoephedrine (PEP) and pseudonorephedrine (PNE) in plasma was developed using chiral capillary gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). N,O-Di-trifluoroacethyl (TFA) derivatization was accomplished in a dried hydrochloride extract of plasma (minimum quantity of 0.2 mL). An SIM GC-MS method with a β-cyclodextrin chiral capillary column allowed the successful and simultaneous detection of each TFA-derivatized stereoisomer of EP, NE, PEP, PNE, and an internal standard (IS; S-(+)-ethylamphetamine). Each TFA-drivatized stereoisomer was identified using four mass fragment ions (m/z 140, 154, 168, and 230). The TFA-derivatized stereoisomers of EP, NE, PEP, PNE, and IS were separated completely and were detected with sufficient sensitivity. The assay allowed the stereoisomers to be determined in a linear range of 12.5-1250 ng/mL for the EP stereoisomers and a linear range of 5-1250 ng/mL for the PEP, NE, and PNE stereoisomers. The detection limits were 7.5 ng/mL for the EP stereoisomers and 2.5 ng/mL for the PEP, NE, and PNE stereoisomers. The intra- and interday precisions were less than 5.9% and 8.2%, respectively. This chiral capillary SIM GC-MS method was sufficiently effective in the analysis of plasma from users of over-the-counter cold medicines and was also fully applicable to the plasma analysis of guinea pigs following their treatment with racemic EP.

Depression has been known to reduce the prefrontal activity associated with the execution of certain cognitive tasks, although whether a temporarily depressed or anxious mood in healthy individuals affects the prefrontal blood oxygen level during cognitive tasks is unknown. Combining the measurement of prefrontal activity with near-infrared spectroscopy (NIRS) and the two cognitive tasks, namely the letter version of the verbal fluency test (VFT-l) and the Stroop test, we measured the effect of a depressed or anxious mood and gender on the changes in the prefrontal oxygenated hemoglobin (Oxy-Hb) levels during those cognitive tests in healthy individuals. Depressed mood or anxious mood was assessed by the Hospital Anxiety and Depression Scale (HADS). Thereby we aimed to explore the possibility of NIRS measurement for detecting the early subclinical manifestation of major depression. Moreover, we examined the possible relationships between prefrontal activation and the functional Val66Met polymorphisms of the brain derived neurotropic factor (BDNF) gene and serum BDNF level. As a result, the increased prefrontal Oxy-Hb levels during cognitive tasks were significantly correlated with the severity of depressed mood in males. The course of the prefrontal Oxy-Hb increase was different depending on the cognitive tasks, i.e., the VFT-l or the Stroop test, in both genders. Correlations of BDNF genotype and serum BDNF level with the prefrontal Oxy-Hb levels during those cognitive tasks were negative. Our results suggest that the early subclinical manifestation of depressed mood in males might be detected by the NIRS measurement, which is not correlated with the individual properties of BDNF.

Plants are associated with a complex microbiota that contributes to nutrient acquisition, plant growth, and plant defense. Nitrogen-fixing microbial associations are efficient and well characterized in legumes but are limited in cereals, including maize. We studied an indigenous landrace of maize grown in nitrogen-depleted soils in the Sierra Mixe region of Oaxaca, Mexico. This landrace is characterized by the extensive development of aerial roots that secrete a carbohydrate-rich mucilage. Analysis of the mucilage microbiota indicated that it was enriched in taxa for which many known species are diazotrophic, was enriched for homologs of genes encoding nitrogenase subunits, and harbored active nitrogenase activity as assessed by acetylene reduction and 15N2 incorporation assays. Field experiments in Sierra Mixe using 15N natural abundance or 15N-enrichment assessments over 5 years indicated that atmospheric nitrogen fixation contributed 29%–82% of the nitrogen nutrition of Sierra Mixe maize., Author summary Nitrogen is an essential nutrient for plants, and for many nonlegume crops, the requirement for nitrogen is primarily met by the use of inorganic fertilizers. These fertilizers are produced from fossil fuel by energy-intensive processes that are estimated to use 1% to 2% of the total global energy supply and produce an equivalent share of greenhouse gases. Because maize (Zea mays L.) is a significant recipient of nitrogen fertilization, a research goal for decades has been to identify or engineer mechanisms for biological fixation of atmospheric nitrogen in association with this crop. We hypothesized that isolated indigenous landraces of maize grown using traditional practices with little or no fertilizer might have evolved strategies to improve plant performance under low-nitrogen nutrient conditions. Here, we show that for one such maize landrace grown in nitrogen-depleted fields near Oaxaca, Mexico, 29%–82% of the plant nitrogen is derived from atmospheric nitrogen. High levels of nitrogen fixation are supported, at least in part, by the abundant production of a sugar-rich mucilage associated with aerial roots that provides a home to a complex nitrogen-fixing microbiome.