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RNA-directed DNA methylation (RdDM) is a biological process in which non-coding RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals. To date, the RdDM pathway is best characterized within angiosperms (flowering plants), and particularly within the model plant Arabidopsis thaliana. However, conserved RdDM pathway components and associated small RNAs (sRNAs) have also been found in other groups of plants, such as gymnosperms and ferns. The RdDM pathway closely resembles other sRNA pathways, particularly the highly conserved RNAi pathway found in fungi, plants, and animals. Both the RdDM and RNAi pathways produce sRNAs and involve conserved Argonaute, Dicer and RNA-dependent RNA polymerase proteins. RdDM has been implicated in a number of regulatory processes in plants. The DNA methylation added by RdDM is generally associated with transcriptional repression of the genetic sequences targeted by the pathway. Since DNA methylation patterns in plants are heritable, these changes can often be stably transmitted to progeny. As a result, one prominent role of RdDM is the stable, transgenerational suppression of transposable element (TE) activity. RdDM has also been linked to pathogen defense, abiotic stress responses, and the regulation of several key developmental transitions. Although the RdDM pathway has a number of important functions, RdDM-defective mutants in Arabidopsis thaliana are viable and can reproduce, which has enabled detailed genetic studies of the pathway. However, RdDM mutants can have a range of defects in different plant species, including lethality, altered reproductive phenotypes, TE upregulation and genome instability, and increased pathogen sensitivity. Overall, RdDM is an important pathway in plants that regulates a number of processes by establishing and reinforcing specific DNA methylation patterns, which can lead to transgenerational epigenetic effects on gene expression and phenotype.

Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds. When animals or plants reproduce sexually, the DNA in a sperm or pollen is combined with that in an egg cell to generate an offspring that inherits two copies of each gene, one from each parent. For a very small number of genes, the copy from one of the parents is consistently turned off. This process—called imprinting—means that the same gene can have different effects depending on if it is inherited from the mother or the father. In plants, imprinting is vital for the production of seeds and typically occurs in the endosperm: the tissue within a seed that provides nourishment to the plant embryo. One way genes can be imprinted is by adding small chemical marks—called methyl groups—on to the DNA that makes up the gene or nearby sequences. These marks can either switch on, or switch off, the expression of the gene. DNA methylation also immobilises stretches of DNA called transposable elements, stopping them from moving from one location to another in the genome. These stretches of DNA are identified and targeted for methylation by small molecules of RNA that match their DNA sequences. Genes that are imprinted in the endosperm of the model plant Arabidopsis are often associated with transposable elements, which can be methylated differently in the naturally occurring varieties, or strains, of Arabidopsis. However it is unclear how many genes are differently imprinted between these different strains. Pignatta et al. looked for differences in gene imprinting, DNA methylation and small RNA production in the seeds, embryos and endosperm tissue from three strains of Arabidopsis. They also examined seeds from crosses between these three strains. While most genes had the same imprinting pattern in all strains and crosses examined, 12 genes were imprinted differently depending on whether they were inherited from the male or female of a given strain. For example, for some genes the copy inherited from the male parent is always turned off, unless it is inherited via the pollen of one specific Arabidopsis strain. Half of this variation could be explained by a transposable element near to each gene that was methylated differently among the strains. By comparing the differentially methylated regions in the genomes of 140 Arabidopsis strains, Pignatta et al. found that differences in methylation may affect 11% of imprinted genes—and went on to confirm variable imprinting in some Arabidopsis strains based on the presence or absence of DNA methylation. Future work is needed to understand how variation in gene imprinting might affect the traits of hybrid seeds, and how it might affect the evolution of new traits in hybrid plants.

5-Hydroxymethylcytosine (5-hmC) is an intermediate in active demethylation in metazoans, as well as a potentially stable epigenetic mark. Previous reports investigating 5-hydroxymethylcytosine in plants have reached conflicting conclusions. We systematically investigated whether 5-hmC is present in plant DNA using a range of methods. Using the model organism Arabidopsis thaliana, in addition to other plant species, we assayed the amount or distribution of 5-hydroxymethylcytosine by thin-layer chromatography, immunoprecipitation-chip, ELISA, enzymatic radiolabeling, and mass spectrometry. The failure to observe 5-hydroxymethylcytosine by thin-layer chromatography established an upper bound for the possible fraction of the nucleotide in plant DNA. Antibody-based methods suggested that there were low levels of 5-hmC in plant DNA, but these experiments were potentially confounded by cross-reactivity with the abundant base 5-methylcytosine. Enzymatic radiolabeling and mass spectrometry, the most sensitive methods for detection that we used, failed to detect 5-hydroxymethylcytosine in A. thaliana genomic DNA isolated from a number of different tissue types and genetic backgrounds. Taken together, our results led us to conclude that 5-hmC is not present in biologically relevant quantities within plant genomic DNA.

Balance between maternal and paternal genomes within the triploid endosperm is necessary for normal seed development. The majority of genes in Arabidopsis endosperm are expressed in a 2:1 maternal:paternal ratio, reflecting endosperm genomic DNA content. Here we find that the 2:1 transcriptional ratio is not, unexpectedly, a passive default but is actively regulated. We describe an inverse relationship between the parent-of-origin of small RNAs and mRNAs in endosperm on a genome-wide scale. Disruption of the Pol IV small RNA pathway causes the entire transcriptome to become more maternally biased. Furthermore, paternal inheritance of a RNA Pol IV mutation is sufficient to rescue seed abortion caused by excess paternal genome dosage. These results indicate that maintenance of the maternal:paternal transcriptome ratio in endosperm is an active process and reveal a function for RNA Pol IV in mediating the global transcriptional balance between maternally and paternally inherited genomes in endosperm.

Evaluating the Use of Peer Instruction in Civil Engineering Courses Interactive teaching strategies have demonstrated the ability to bring about increased learning gains when compared to traditional lecture style approaches (Freeman et al. 2014). One such strategy, Peer Instruction (PI), aims to convert students from passive listeners to active and engaged learners. During PI, conceptual questions are posed to the students and students respond individually via a personal response system (PRS). If a significant proportion of the class is confused, students engage in peer discussion moderated by the instructor, which is then followed by another PRS vote (Vickrey et al. 2014). PI has been evaluated for its efficacy as an instructional strategy in the natural sciences including chemistry, biology and physics, but there are few studies evaluating the use of PI in engineering education. In this study, we evaluated the use of PI in civil engineering courses including a required introductory environmental engineering course (junior level, n=53) and an elective course in structural engineering (senior and graduate students, n=11) and an elective course in transportation engineering (senior and graduate students, n=20-25) taught via synchronous distance education. In each course, one-half of the course topics were covered using a PI approach. A pre- and post-knowledge test was given to all students to evaluate learning gains on both PI and non-PI topics. Classroom activities from topics covered with and without the use of PI were video recorded and evaluated using the Classroom Observation Protocol for Undergraduate STEM (COPUS) as described in Smith et al. (2013). At the end of the course, a student satisfaction survey was administered to students using an instrument adapted from Crossgrove and Curran (2008). From these assessment techniques, we will measure learning gains on topics presented using PI versus traditional lecture styles. We hypothesize that the use of PI will increase the classroom time spent by both the instructor and students in active teaching and learning modes. Finally, we anticipate that the use of PI will increase student satisfaction with the course and that students will recommend the continued use of PI in civil engineering courses. To our knowledge, this study constitutes one of the first to evaluate the efficacy of PI in civil engineering education. Crossgrove, K. and K.L. Curran. (2009). Using Clickers in Nonmajors- and Majors-Level Biology Courses: Student Opinion, Learning, and Long-Term Retention of Course Material. CBE-Life Science Education, 7: 146-154, doi: 10.1187/cbe.07–08–0060 Freeman, S., Eddy S.L., McDonough, M., Smith, M.K., Okorafor, N., Jordt, H., and Wenderoth M.P. (2013). Active learning increases student performance in science, engineering and mathematics. Proceedings of the National Academies of Science, 111: 8410-8415, doi: 10.1073/pnas.1319030111 Smith, M.K., Jones, F.H., Gilbert, S.L., and Wieman, C.E. (2013). The Classroom Observation Protocol for Undergraduate STEM (COPUS): a new instrument to characterize university STEM classroom practices. CBE-Life Science Education, 12: 618-627, doi: 10.1187/cbe.13-08-0154 Vickrey, T., Rosploch, K., Rahmanian, R., Pilarz, M., and Stains, M. (2014). Research-Based Implementation of Peer Instruction: A Literature Review. CBE-Life Science Education, 14: 1-11, doi: 10.1187/cbe.14-11-0198.

Relative abundances of bacterial species in the gut microbiome have been linked to many diseases. Species of gut bacteria are ecologically differentiated by their abilities to metabolize different glycans, making glycan delivery a powerful way to alter the microbiome to promote health. Here, we study the properties and therapeutic potential of chemically diverse synthetic glycans (SGs). Fermentation of SGs by gut microbiome cultures results in compound-specific shifts in taxonomic and metabolite profiles not observed with reference glycans, including prebiotics. Model enteric pathogens grow poorly on most SGs, potentially increasing their safety for at-risk populations. SGs increase survival, reduce weight loss, and improve clinical scores in mouse models of colitis. Synthetic glycans are thus a promising modality to improve health through selective changes to the gut microbiome. Here, the authors characterize the gut microbiome fermentation properties and therapeutic potential of chemically diverse synthetic glycans (SGs), showing they promote specific shifts in taxonomic and metabolite profiles, and exhibit therapeutic benefits in mouse models of colonic inflammation, together implying SGs as a potential avenue to treat disease by modulating the composition and metabolites produced by the gut microbiome.

In a multicenter, controlled trial, patients undergoing transplantation of a liver from a donor after circulatory death were randomly assigned to receive the liver after hypothermic oxygenated machine perfusion or conventional static cold storage. Hypothermic perfusion led to a lower risk of post-transplantation nonanastomotic biliary strictures.

Circulating levels of glycine have previously been associated with lower incidence of coronary heart disease (CHD) and type 2 diabetes (T2D) but it remains uncertain if glycine plays an aetiological role. We present a meta-analysis of genome-wide association studies for glycine in 80,003 participants and investigate the causality and potential mechanisms of the association between glycine and cardio-metabolic diseases using genetic approaches. We identify 27 genetic loci, of which 22 have not previously been reported for glycine. We show that glycine is genetically associated with lower CHD risk and find that this may be partly driven by blood pressure. Evidence for a genetic association of glycine with T2D is weaker, but we find a strong inverse genetic effect of hyperinsulinaemia on glycine. Our findings strengthen evidence for a protective effect of glycine on CHD and show that the glycine-T2D association may be driven by a glycine-lowering effect of insulin resistance. Epidemiological studies have associated circulating levels of the amino acid glycine with cardiometabolic outcomes. Here, in a genome-wide meta-analysis of 80,003 individuals, Wittemans et al. identify 22 novel genetic loci for glycine and find a causal relationship with coronary heart disease using MR.