Pollen Grain Counting Using a Cell Counter

The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum)

Genome-wide association study of aphid abundance highlights a locus affecting plant growth and flowering in Arabidopsis thaliana

Plant life-history traits, such as size and flowering, contribute to shaping variation in herbivore abundance. Although plant genes involved in physical and chemical traits have been well studied, less is known about the loci linking plant life-history traits and herbivore abundance. Here, we conducted a genome-wide association study (GWAS) of aphid abundance in a field population of Arabidopsis thaliana. This GWAS of aphid abundance detected a relatively rare but significant variant on the third chromosome of A. thaliana, which was also suggestively but non-significantly associated with the presence or absence of inflorescence. Out of candidate genes near this significant variant, a mutant of a ribosomal gene (AT3G13882) exhibited slower growth and later flowering than a wild type under laboratory conditions. A no-choice assay with the turnip aphid, Lipaphis erysimi, found that aphids were unable to successfully establish on the mutant. Our GWAS of aphid abundance unexpectedly found a locus affecting plant growth and flowering

Delineating Genetic Alterations for Tumor Progression in the MCF10A Series of Breast Cancer Cell Lines

To gain insight into the role of genomic alterations in breast cancer progression, we conducted a comprehensive genetic characterization of a series of four cell lines derived from MCF10A. MCF10A is an immortalized mammary epithelial cell line (MEC); MCF10AT is a premalignant cell line generated from MCF10A by transformation with an activated HRAS gene; MCF10CA1h and MCF10CA1a, both derived from MCF10AT xenografts, form well-differentiated and poorly-differentiated malignant tumors in the xenograft models, respectively. We analyzed DNA copy number variation using the Affymetrix 500 K SNP arrays with the goal of identifying gene-specific amplification and deletion events. In addition to a previously noted deletion in the CDKN2A locus, our studies identified MYC amplification in all four cell lines. Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a. We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line. Furthermore, we found that RUNX1 expression was reduced in high-grade primary breast tumors compared to low/mid-grade tumors. Mutational analysis identified an activating PIK3CA mutation, H1047R, in MCF10CA1h and MCF10CA1a, which correlates with an increase of AKT1 phosphorylation at Ser473 and Thr308. Furthermore, we showed increased expression levels for genes located in the genomic regions with copy number gain. Thus, our genetic analyses have uncovered sequential molecular events that delineate breast tumor progression. These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors

Plant trichomes and a single gene GLABRA1 contribute to insect community composition on field-grown Arabidopsis thaliana

BACKGROUND: Genetic variation in plants alters insect abundance and community structure in the field; however, little is known about the importance of a single gene among diverse plant genotypes. In this context, Arabidopsis trichomes provide an excellent system to discern the roles of natural variation and a key gene, GLABRA1, in shaping insect communities. In this study, we transplanted two independent glabrous mutants (gl1-1 and gl1-2) and 17 natural accessions of Arabidopsis thaliana to two localities in Switzerland and Japan. RESULTS: Fifteen insect species inhabited the plant accessions, with the insect community composition significantly attributed to variations among plant accessions. The total abundance of leaf-chewing herbivores was negatively correlated with trichome density at both field sites, while glucosinolates had variable effects on leaf chewers between the sites. Interestingly, there was a parallel tendency for the abundance of leaf chewers to be higher on gl1-1 and gl1-2 than on their different parental accessions, Ler-1 and Col-0, respectively. Furthermore, the loss of function in the GLABRA1 gene significantly decreased the resistance of plants to the two predominant chewers; flea beetles and turnip sawflies. CONCLUSIONS: Overall, our results indicate that insect community composition significantly varies among A. thaliana accessions across two distant field sites, with GLABRA1 playing a key role in altering the abundance of leaf-chewing herbivores. Given that such a trichome variation is widely observed in Brassicaceae plants, the present study exemplifies the community-wide effect of a single plant gene on crucifer-feeding insects in the field

The UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME 1 For Plant Survival Under Sunlight In Field Conditions

As sessile, photoautotrophic organisms, plants are subjected to fluctuating sunlight that includes potentially detrimental ultraviolet-B radiation (UV-B). Experiments under controlled conditions have shown that the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) controls acclimation and tolerance to UV-B in Arabidopsis thaliana; however, its long-term impacts on plant fitness remain poorly understood in naturally fluctuating environments. Here we quantified the survival and reproduction of different Arabidopsis mutant genotypes in diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type when grown in growth chambers under artificial low UV-B conditions but not in natural field conditions, indicating a fitness cost in absence of UV-B stress. Importantly, independent double mutants of UVR8 and the blue-light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. Experiments with UV-B attenuation experiments in field and with supplemental UV-B in growth chambers demonstrated that UV-B caused the conditional cry1 uvr8 lethality phenotype. Using RNA-seq data of field-grown single and double mutants, we explicitly identified genes showing statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. They were enriched in Gene Ontology categories related to oxidative stress, photoprotection, and DNA damage repair in addition to UV-B response. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with that of cry1. Moreover, these data provide an integral picture of gene expression associated with plant responses under field conditions

Point defects and clustering in uranium dioxide by LSDA+U calculations

A comprehensive investigation on point defects and their clustering behavior in nonstoichiometric uranium dioxide UO2+x is carried out using LSDA+U method based on density functional theory. Accurate energetic information and charge transfers available so far are obtained. With these energies that have improved more than 50% over that of pure GGA and LDA, we show the density functional theory predicts the predominance of oxygen defects over uranium ones at any compositions, which is possible only after treated the localized 5f electrons properly. Calculations also suggest an upper bound of x~0.03 for oxygen clusters to start off. The volume change induced by point uranium defects is monotonic but nonlinear, whereas for oxygen defects, increase x always reduces the system volume linearly, except dimers that require extra space for accommodation, which has been identified as meta-stable ionic molecule. Though oxygen dimers usually occupy Willis O'' sites and mimic a single oxygen in energetics and charge state, they are rare at ambient conditions. Its decomposition process and vibrational properties have been studied carefully. To obtain a general clustering mechanism in anion-excess fluorites systematically, we also analyze the local stabilities of possible basic clustering modes of oxygen defects. The result shows an unified way to understand the structure of Willis type and cuboctahedral clusters in UO2+x and beta-U4O9. Finally we generalize the point defect model to the independent clusters approximation to include clustering effects, the impact on defect populations is discussed.Comment: 20 pages, 12 figure

N-myc downstream regulated gene 1 modulates Wnt-β-catenin signalling and pleiotropically suppresses metastasis

Wnt signalling has pivotal roles in tumour progression and metastasis; however, the exact molecular mechanism of Wnt signalling in the metastatic process is as yet poorly defined. Here we demonstrate that the tumour metastasis suppressor gene, NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signalling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumour cells. Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumour cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG−/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a novel negative master regulator of Wnt signalling during the metastatic progression, which opens an opportunity to define a potential therapeutic target for metastatic disease

Systematic inclusion of mandatory interprofessional education in health professions curricula at Gunma University: a report of student self-assessment in a nine-year implementation

<p>Abstract</p> <p>Background</p> <p>The mandatory interprofessional education programme at Gunma University was initiated in 1999. This paper is a statistical evaluation of the programme from 1999 to 2007.</p> <p>Methods</p> <p>A questionnaire of 10 items to assess the achievement levels of the programme, which was developed independently of other assessment systems published previously, was distributed, as well as two or three open-ended questions to be answered at the end of each annual module. A multivariate analysis of variance model was used, and the factor analysis of the responses was performed with varimax rotation.</p> <p>Results</p> <p>Over all, 1418 respondents of a possible 1629 students completed the survey, for a total response rate of 87.1%. Cronbach's alpha of 10 items was 0.793, revealing high internal consistency. Our original questionnaire was categorized into four subscales as follows: "Role and responsibilities", "Teamwork and collaboration", "Structure and function of training facilities", and "Professional identity". Students in the Department of Occupational Therapy reached a relatively lower level of achievement. In the replies to the open-ended questions, requests for the participation of the medical students were repeated throughout the evaluation period.</p> <p>Conclusion</p> <p>The present four subscales measure "understanding", and may take into account the development of interprofessional education programmes with clinical training in various facilities. The content and quality of clinical training subjects may be remarkably dependent on training facilities, suggesting the importance of full consultation mechanisms in the local network with the relevant educational institutes for medicine, health care and welfare.</p

Tumoricidal efficacy coincides with CD11c up-regulation in antigen-specific CD8+ T cells during vaccine immunotherapy

Background: Dendritic cells (DCs) mount tumor-associated antigens (TAAs), and the double-stranded RNA adjuvant Poly(I:C) stimulates Toll-like receptor 3 (TLR3) signal in DC, which in turn induces type I interferon (IFN) and interleukin-12 (IL-12), then cross-primes cytotoxic T lymphocytes (CTLs). Proliferation of CTLs correlates with tumor regression. How these potent cells expand with high quality is crucial to the outcome of CTL therapy. However, good markers reflecting the efficacy of DC-target immunotherapy have not been addressed. Methods: Using an EG7 (ovalbumin, OVA-positive) tumor-implant mouse model, we examined what is a good marker for active CTL induction in treatment with Poly(I:C)/OVA. Results: Simultaneous administration of Poly(I:C) and antigen (Ag) OVA significantly increased a minor population of CD8+ T cells, that express CD11c in lymphoid and tumor sites. The numbers of the CD11c+ CD8+ T cells correlated with those of induced Ag-specific CD8+ T cells and tumor regression. The CD11c+ CD8+ T cell moiety was characterized by its high killing activity and IFN-γ-producing ability, which represent an active phenotype of the effector CTLs. Not only a TLR3-specific (TICAM-1-dependent) signal but also TLR2 (MyD88) signal in DC triggered the expansion of CD11c+ CD8+ T cells in tumor-bearing mice. Notably, human CD11c+ CD8+ T cells also proliferated in peripheral blood mononuclear cells (PBMC) stimulated with cytomegalovirus (CMV) Ag. Conclusions: CD11c expression in CD8+ T cells reflects anti-tumor CTL activity and would be a marker for immunotherapeutic efficacy in mouse models and probably cancer patients as well

Phosphoinositide 3-Kinaseγ Controls the Intracellular Localization of CpG to Limit DNA-PKcs-Dependent IL-10 Production in Macrophages

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ−/−). By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ−/− cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ−/− cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ−/− cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ−/− cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages