DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.

Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING 1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion

DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets:Validation with camera-recorded diets

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of fecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free-living animals. This study validates that DNA metabarcoding of feces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify the relative biomass of dietary taxa. Validation was done with the hole-nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size-adjusted counts of food items delivered to nestlings were used as a proxy for provided biomass of prey orders and families, and subsequently, nestling feces were assessed through DNA metabarcoding. To explore potential effects of digestion, gizzard and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera-recorded taxa. Overall, and in each replicate year (N = 3), the relative scaled biomass of prey taxa and COI read numbers correlated at R =.85 (95CI:0.68–0.94) at order level and at R =.75 (CI:0.67–0.82) at family level. Similarity in arthropod community composition between gizzard and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds

Medicare Part D Plan Optimization: The Need for an Annual Check-Up

Background: Since its inception, Medicare Part D requires beneficiaries to choose from a myriad of insurance plans in order to receive prescription drug coverage. Moreover, each year beneficiaries are confronted with plan cancellations, new plan offerings, changes in existing plan formularies and cost-sharing structure. Objective: This study prospectively examined the relationship between stand-alone prescription drug plan (PDP) costs, subsidy status, and the number of plans offered in California from 2009-2012. Methods: Forty-one community outreach events were held throughout Central/Northern California during the Medicare Part D annual election periods from 2008-2011. In total, 1,578 beneficiaries were assisted, 983 (62.2%) of whom had a PDP. During each intervention, beneficiary subsidy status, cost data for the beneficiary\u27s current plan and lowest cost plan for the upcoming year were recorded from the Medicare website. The percent of beneficiaries that did not need to switch plans to reduce their out-of-pocket (OOP) drug costs was compared to the number of available plans in the subsequent year.Results: On average, 14.3-23.7% of beneficiaries would have been enrolled in the lowest cost plan in the upcoming year had they remained in their current plan. Subsidy recipients were significantly more likely to be in the lowest cost plan each year. The chance of being in the lowest cost plan was significantly negatively correlated to the number of drug plans offered in the subsequent year. Conclusion: Annual Part D plan reexamination is essential to ensure that beneficiaries optimize their prescription medication coverage and minimize their OOP costs

N-arachidonylglycine is a caloric state-dependent circulating metabolite which regulates human CD4+T cell responsiveness

Acknowledgments We thank Drs. Zoe Hall and Sonia Liggi of the University of Cambridge Biochemistry Department for their contributions to metabolomics analysis and data processing. Shahin Hassanzadeh of the Laboratory of Mitochondrial Biology and Metabolism for developing the PBMC RNAseq library.Matthew Rodman of the Laboratory of Mitochondrial Biology and Metabolism for preparing lean/obese samples. Dr. Duck-Yeon Lee of the NHLBI Biochemistry Core for NAGly analysis in cell culture. Special thanks to the National Institutes of Health Oxford-Cambridge Scholars Program and the International Biomedical Research Alliance for their sponsorship and support. Funding: NHLBI Division of Intramural Research (MNS – ZIA-HL005199) and the UK MRC (JLG – MR/P011705/2; UKDRI-5002; MAPUK).Peer reviewedPublisher PD

Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism

CRISPR-delivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a browning process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed CRISPR-delivery particles, denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease

Propionate functions as a feeding state-dependent regulatory metabolite to counter proinflammatory signaling linked to nutrient load and obesity.

The authors thank Drs. Antonio Murgia and Ben McNally of the University of Cambridge Biochemistry Department for their contributions to metabolomics and lipidomic data processing. They thank and acknowledge the assistance of the National Heart, Lung, and Blood Institute DNA Sequencing and Genomics Core in performing the RNA library sequencing and the National Heart, Lung, and Blood Institute Flow Cytometry Core for performing the flow cytometry.Peer reviewe

Mechanisms for collaboration: a design and evaluation framework for multi-user interfaces

Multi-user interfaces are said to provide “natural” interaction in supporting collaboration, compared to individual and noncolocated technologies. We identify three mechanisms accounting for the success of such interfaces: high awareness of others' actions and intentions, high control over the interface, and high availability of background information. We challenge the idea that interaction over such interfaces is necessarily “natural” and argue that everyday interaction involves constraints on awareness, control, and availability. These constraints help people interact more smoothly. We draw from social developmental psychology to characterize the design of multi-user interfaces in terms of how constraints on these mechanisms can be best used to promote collaboration. We use this framework of mechanisms and constraints to explain the successes and failures of existing designs, then apply it to three case studies of design, and finally derive from them a set of questions to consider when designing and analysing multi-user interfaces for collaboration

Corticosterone pattern-dependent glucocorticoid receptor binding and transcriptional regulation within the liver

Ultradian glucocorticoid rhythms are highly conserved across mammalian species, however, their functional significance is not yet fully understood. Here we demonstrate that pulsatile corticosterone replacement in adrenalectomised rats induces a dynamic pattern of glucocorticoid receptor (GR) binding at ~3,000 genomic sites in liver at the pulse peak, subsequently not found during the pulse nadir. In contrast, constant corticosterone replacement induced prolonged binding at the majority of these sites. Additionally, each pattern further induced markedly different transcriptional responses. During pulsatile treatment, intragenic occupancy by active RNA polymerase II exhibited pulsatile dynamics with transient changes in enrichment, either decreased or increased depending on the gene, which mostly returned to baseline during the inter-pulse interval. In contrast, constant corticosterone exposure induced prolonged effects on RNA polymerase II occupancy at the majority of gene targets, thus acting as a sustained regulatory signal for both transactivation and repression of glucocorticoid target genes. The nett effect of these differences were consequently seen in the liver transcriptome as RNA-seq analysis indicated that despite the same overall amount of corticosterone infused, twice the number of transcripts were regulated by constant corticosterone infusion, when compared to pulsatile. Target genes that were found to be differentially regulated in a pattern-dependent manner were enriched in functional pathways including carbohydrate, cholesterol, glucose and fat metabolism as well as inflammation, suggesting a functional role for dysregulated glucocorticoid rhythms in the development of metabolic dysfunction

Energy-dependent solar neutrino flux depletion in the Exact Parity Model and implications for SNO, SuperKamiokande and BOREXINO

Energy-dependent solar neutrino flux reduction caused by the Mikheyev-Smirnov-Wolfenstein (MSW) effect is applied to the Exact Parity Model. Several scenarios are possible, depending on the region of parameter space chosen. The interplay between intergenerational MSW transitions and vacuum ``intragenerational'' ordinary-mirror neutrino oscillations is discussed. Expectations for the ratio of charged to neutral current event rates at the Sudbury Neutrino Observatory (SNO) are estimated. The implications of the various scenarios for the Boron neutrino energy spectrum and BOREXINO are briefly discussed. The consequences of MSW-induced solar neutrino depletion within the Exact Parity Model differ in interesting ways from the standard $\nu_e - \nu_{\mu, \tau}$ and $\nu_e - \nu_s$ cases. The physical causes of these differences are determined.Comment: 43 pages, 8 figures, RevTeX; to appear in Phys. Rev. D, accepted versio