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Supplementary Figure 1: Assessment of kidney cancer cell lines A498, 786-O, and A704 via flow cytometry revealed surface expression of P-gp in all lines; Supplementary Figure 2: 786-O and A704 cells were transduced with lentiviral FR1, and flow cytometric analysis shows increased surface FR1 expression; Supplementary Figure 3: NCI/ADR-RES or NCI/ADR-RES-FR cells with known FR levels were treated with increasing concentrations (10-9 M to 10-5 M) of vintafolide for 2 h; Supplementary Figure 4: Ovarian tumors positive for P-gp tend to express lower levels of FR; Supplementary Table 1: P-gp inhibitor elacridar reverses P-gp-mediated DAVLBH and vintafolide resistance; Supplementary Table 2: Functional multi-drug transporter efflux assay.

Legends for Supplementary Figures 1-4 and Supplementary Tables 1 & 2

Silencing of transposable elements (TEs) drives the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here, we show, via single-nucleus RNA-seq of developing male gametophytes, that these repressors are critical for TE silencing in the pollen vegetative cell, a companion cell important for fertilization that undergoes chromatin decompaction. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We show that TEs repressed by MBD5/6 gain chromatin accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system particularly important when chromatin compaction is compromised.

SUMMARYSilencing of transposable elements (TEs) drove the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here we show via single-nucleus RNA-seq of developing male gametophytes that these repressors are critical for TE silencing in the pollen vegetative cell, which undergoes epigenetic reprogramming causing chromatin decompaction to support fertilization by sperm cells. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We found that TEs repressed by MBD5/6 gain accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system especially important when chromatin compaction is compromised.

To compare the mortality of monochorionic-monoamniotic (MCMA) and monochorionic-biamniotic (MCBA) twin pregnancies, from 14weeks of gestation to 28th day of life, before and after exclusion of major congenital malformations.We conducted a retrospective cohort study in two level 3 maternity units of the Hospices civils de Lyon (France) including all patients with a monochorionic twin pregnancy between January 2013 and December 2020. We excluded TRAP sequences and conjoined twins.A total of 38 MCMA and 658 MCBA pregnancies were included. We showed an increase in overall mortality in the MCMA group compared to the MCBA group (31.6% versus 16.4%, P=0.03) even after exclusion of major congenital malformations (20.3% versus 9.5%, P=0.01). The main cause of mortality found in the MCMA group was the occurrence of unexpected IUGR.MCMA pregnancies have a higher foetal and neonatal mortality rate than MCBA pregnancies even after exclusion of congenital malformations related to the occurrence of unexpected MFIU.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B , or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component

Resistance to traditional synthetic compounds by Varroa destructor Anderson and Trueman and shortcomings of the organic acid class of acaracides commonly used in varroa management requires continual development of new controls. V. destructor, however, are difficult to obtain for use in control bioassays because they are obligate parasites that cannot be easily reared outside of a honey bee colony. We conducted bioassays using other, more easily obtainable species to find organisms that could be used as surrogates for V. destructor when testing new potential controls. We compared the toxicities of acetic acid, lactic acid, formic acid, and oxalic acid at 0.005%, 0.05%, 0.5%, 5%, and 50% (20% oxalic acid only) concentrations based on natural volatility (nonheated) for the control of two beetle species, Oryzaephilus surinamensis L. and Alphitobius diaperinus Panzer, greater wax moth larvae, Galleria mellonella L., and V. destructor. The assay results were consistent across all species with formic acid and acetic acid showing 100% mortality of all four test species at 50% concentration. The assays also provided insight into the method of application (vaporization or contact) needed to cause mortality. Our results show that other organisms can be used in place of V. destructor for initial testing of acids and possibly other chemicals for control of the ectoparasite.

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. To analyze the in vivo function of H2A.W, we created a h2a.w null mutant via CRISPR-Cas9, h2a.w-2. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

Significance The plant-specific H3K27me1 methyltransferases ATXR5 and ATXR6 play integral roles connecting epigenetic silencing with genomic stability. However, how H3K27me1 relates to these processes is poorly understood. In this study, we performed a comprehensive transcriptome analysis of tissue- and ploidy-specific expression in a hypomorphic atxr5/6 mutant and revealed that the tissue-specific defects correlate with residual ATXR6 expression. We also determined that ATXR5/6 function is essential for female germline development. Furthermore, we provide a comprehensive analysis of H3K27me1 changes in relation to other epigenetic marks. We also determined that some previously reported suppressors of atxr5/6 may act by restoring the levels of H3K27me1, such as through up-regulation of the ATXR6 transcript in the atxr6 hypomorphic promoter allele., ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms., MORC ATPases are required for transposable element silencing and heterochromatin condensation in plants and animals. Here the authors show that Arabidopsis MORCs colocalize with sites of RNA-directed DNA methylation and provide evidence that they act as molecular tethers to efficiently establish DNA methylation.

Author(s): Martin, Elizabeth Allene | Advisor(s): Panning, Barbara | Abstract: Female (XX) mouse embryonic stem cells (mESCs) differ from their male (XY) counterparts because they have lower levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). This difference in DNA modifications is a result of having two X chromosomes (Xs), both of which are active at this developmental stage. To test whether OGT is one of the X-linked proteins that regulate 5mC and 5hmC in mESCs, we manipulated OGT dose in XX and XY mESCs. We found that OGT abundance controls cytosine modifications, implicating OGT targets in 5mC and 5hmC regulation. Our quantitative comparison of the O-GlcNAcylated proteome in XX and XY mESCs revealed that O-GlcNAc modified TET3 peptides were more abundant in XX mESCs, which reflected an increase in TET3 amount in these cells. In addition to differing in abundance, TET3 and OGT distribution were also different in XX and XY mESCs. In XX cells, TET3 and OGT were enriched in the nucleus, while they were predominantly cytoplasmic in XY cells. TET3 and OGT occur in different high molecular weight complexes in XX and XY mESCs. When OGT is expressed from one X in XX mESCs, OGT and TET3 are predominantly cytoplasmic and 5mC/5hmC levels increase, indicating that OGT is one X-linked regulator of DNA cytosine modifications. To directly query whether TET3 is necessary for the female-specific 5mC and 5hmC in mESCs we generated homozygous TET3 mutant XX mESCs. In these cells, 5mC and 5hmC levels were decreased relative to wildtype XX mESCs, without dramatic gene expression changes. To investigate the developmental significance of TET3, examined the effects of this mutation on the ability of cells to undergo X chromosome inactivation (XCI) an epigenetic change that occurs when mESCs are differentiated into the next developmental stage, epiblast-like (mEpiL) cells. The establishment of XCI is characterized by the up-regulation of a non-coding RNA, Xist RNA, which remains in the nucleus and ‘coats’ the X concomitant with silencing. In TET3 mutant XX mEpiLCs Xist RNA exhibits abnormal distribution and silencing defects. These results link the activity of a dose-sensitive complex containing X and autosomal proteins to regulation of cytosine DNA modifications and XCI.

Significance Site-specific modification of epigenetic marks such as DNA methylation to regulate gene expression is a unique approach to enhance economically important crop traits. This approach allows for the maintenance of the introduced modifications in the absence of the initial transgene inducer in subsequent generations and relies largely on methylation of cytosines in the CG-specific sequence context. We have developed a targeted DNA methylation tool based on a bacterial methyltransferase and the CRISPR-Cas9 platform to directly methylate cytosines at CG sites in Arabidopsis. These tools expand the currently available CRISPR-based targeted DNA methylation tools and provide an approach for the establishment of heritable targeted DNA methylation in plants., CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning., Plant cell wall formation is directed by cortical microtubules, which produce complex patterns needed to support xylem vessels. Here, the authors perform live-cell imaging and simulations of Arabidopsis cells during proto-xylem differentiation to show how local microtubule dynamics control pattern formation.

Introduction Depuis 2015, les recommandations europeennes (ESC) et nord-americaine (IDSA) proposent l’association Amoxicilline-Ceftriaxone (A-C) comme alternative au traitement de reference Amoxicilline-Gentamicine (A-G) dans le traitement de l’endocardite infectieuse a Enterococcus faecalis (EIEF). L’objectif de cette etude est de decrire au niveau regional les pratiques selon les centres et leur evolution depuis la publication de ces recommandations. Materiels et methodes Etude retrospective multicentrique portant sur 7 centres hospitaliers universitaires (CHU) et 6 centres hospitaliers non universitaires (CH). Tous les patients pris en charge entre 2015 et 2019 pour une EIEF certaine selon les criteres modifies de Duke etaient inclus. Un patient etait considere comme ayant ete traite par A-C et/ou A-G s’il avait recu au moins 21 ou 7 jours de bitherapie, respectivement, c’est-a-dire plus de 50% de la duree recommandee. Resultats 272 patients ont ete inclus, dont 210 (77 %) ont ete pris en charge, initialement ou secondairement, dans un CHU et 62 exclusivement en CH (23 %). La population etait composee de 216 hommes (79 %). L’endocardite survenait sur valve native chez 160 patients (59 %). Une ETO etait realisee chez 208 patients (77 %) dont 42/62 patients en CH (68 %) et 166/202 (79 %) patients en CHU, le taux de realisation par centre variant entre 28 % (4/14) et 88% (36/41). La chirurgie etait realisee pendant le traitement pour 89 patients (33 %), le taux de realisation dans les CHU allant de 35 % (5/14) a 53 % (21/40). L’âge median des patients operes etait de 67,5 ans (interquartile [IQR] 60-72) contre 79 ans (IQR 71-85) pour les non operes (p Conclusion Les patients presentant une EIEF sont essentiellement pris en charge en CHU ou la chirurgie cardiaque est accessible. Malgre un âge eleve, le recours a la chirurgie reste frequent. Depuis les recommandations de 2015, on observe une diminution progressive de la bitherapie A-G et un large recours a la bitherapie A-C.

Seeds are a key life cycle stage for many plants. Seeds are also the basis of agriculture and the primary source of calories consumed by humans1. Here, we employ single-nucleus RNA-sequencing to generate a transcriptional atlas of developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the primary site of gene imprinting in flowering plants, mediates the relationship between the maternal parent and the embryo2. We identify transcriptionally uncharacterized nuclei types in the chalazal endosperm, which interfaces with maternal tissue for nutrient unloading3,4. We demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell-cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. Thus, imprinting is spatially and temporally heterogeneous. Increased paternal expression in the chalazal region suggests that parental conflict, which is proposed to drive imprinting evolution, is fiercest at the boundary between filial and maternal tissues. This study generated a transcriptional atlas of developing Arabidopsis seeds with single-nucleus RNA-sequencing, reporting transcriptionally uncharacterized nuclei types in the chalazal endosperm and spatially and temporally heterogenous imprinting in the seeds.

Cellulose synthesis is essential for plant morphology, water transport and defense, and provides raw material for biomaterials and fuels. Cellulose is produced at the plasma membrane by Cellulose Synthase (CESA) protein complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked from the Golgi apparatus and trans-Golgi Network (TGN) to the plasma membrane. Since CESA enzymes are only active in the plasma membrane, control of CSC secretion is a critical step in the regulation of cellulose synthesis. However, the regulatory framework for CSC secretion is not clarified. In this study, we identify members of a family of seven transmembrane domain-containing proteins (7TMs) as important for cellulose production during cell wall integrity stress. 7TM proteins are often associated with guanine nucleotide-binding protein (G) protein signalling and mutants in several of the canonical G protein complex components phenocopied the 7tm mutant plants. Unexpectedly, the 7TM proteins localized to the Golgi apparatus/TGN where they interacted with the G protein complex. Here, the 7TMs and G proteins regulated CESA trafficking, but did not affect general protein secretion. Furthermore, during cell wall stress, 7TMs’ localization was biased towards small CESA-containing vesicles, specifically associated with CSC trafficking. Our results thus outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses upon exposure to cell wall integrity stress.

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.

Seeds are the basis of agriculture, yet their full transcriptional complexity has remained unknown. Here, we employ single-nucleus RNA-sequencing to characterize developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the site of gene imprinting in plants, mediates the relationship between the maternal parent and embryo. We identify new cell types in the chalazal endosperm region, which interfaces with maternal tissue for nutrient unloading. We further demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. These data indicate imprinting in endosperm is heterogeneous and suggest that parental conflict, which is proposed to drive the evolution of imprinting, is fiercest at the boundary between filial and maternal tissues.

Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.Author summaryThe discovery of CRISPR-CAS9 and its non-catalytic variants have provided enormous capacity for crop improvement and basic research by modifying the genome and the epigenome. The standard methods for delivering genome and epigenome editing reagents to plants consist of generating stable transgenic lines through tissue culture processes, which have several drawbacks including the need for plant regeneration and crossing. To overcome some of these challenges, plant virus-based platforms are being developed for genome editing. Although viruses have a limited cargo capacity, limiting the use of viruses to encode entire editing systems, guide RNAs have been successfully delivered to transgenic CAS9 expressing plants for genome editing. However, the use of viruses for CRISPR-based epigenome editing and transcriptional activation have not yet been explored. In this study we show that viral delivery of guide RNAs using a modified tobacco rattle virus can be used for transcriptional activation and heritable epigenome editing. This study advances the use of plant RNA viruses as delivery agents for epigenome editing.

BackgroundModel organisms are at the core of life science research. Notable examples include the mouse as a model for humans, baker’s yeast for eukaryotic unicellular life and simple genetics, or the enterobacteria phage λ in virology. Plant research was an exception to this rule, with researchers relying on a variety of non-model plants until the eventual adoption of Arabidopsis thaliana as primary plant model in the 1980s. This proved to be an unprecedented success, and several secondary plant models have since been established. Currently, we are experiencing another wave of expansion in the set of plant models.ScopeSince the 2000s, new model plants have been established to study numerous aspects of plant biology, such as the evolution of land plants, grasses, invasive and parasitic plant life, adaptation to environmental challenges, and the development of morphological diversity. Concurrent with the establishment of new plant models, the advent of the ‘omics’ era in biology has led to a resurgence of the more complex non-model plants. With this review, we introduce some of the new and fascinating plant models, outline why they are interesting subjects to study, the questions they will help to answer, and the molecular tools that have been established and are available to researchers.ConclusionsUnderstanding the molecular mechanisms underlying all aspects of plant biology can only be achieved with the adoption of a comprehensive set of models, each of which allows the assessment of at least one aspect of plant life. The model plants described here represent a step forward towards our goal to explore and comprehend the diversity of plant form and function. Still, several questions remain unanswered, but the constant development of novel technologies in molecular biology and bioinformatics is already paving the way for the next generation of plant models.

Genomic imprinting is a phenomenon that occurs in flowering plants and mammals, whereby a gene is expressed in a parent-of-origin-specific manner. Although imprinting has now been examined genome-wide in a number of species using RNA-seq, the analyses used to assess imprinting vary between studies, making consistent comparisons between species difficult. Here we present a simple, easy-to-use bioinformatic pipeline for imprinting analyses suitable for any tissue, including plant endosperm. All relevant scripts can be downloaded. As an illustrative example, we reanalyze published data from A. thaliana and Z. mays endosperm using the pipeline and then demonstrate how to use the results to assess the conservation of imprinting between these species. We also introduce the Plant Imprinting Database, a repository for published imprinting datasets in plants that can be used to view, compare, and download data.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. We established long-term live-cell imaging of singleArabidopsiscells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to investigate the microtubule re-organization. The initial disperse microtubule array rapidly readjusted into well-defined microtubule bands, which required local de-stabilization of individual microtubules in band-interspersing gap regions. Using extensive microtubule simulations, we could recapitulate the processin silicoand found that local recruitment of microtubule-bound nucleation is critical for pattern formation, which we confirmedin vivo. Our simulations further indicated that the initial microtubule alignment impact microtubule band patterning. We confirmed this prediction usingkataninmutants, which have microtubule organization defects, and uncovered active KATANIN recruitment to the forming microtubule bands. Our combination of quantitative microscopy and modelling outlines a framework towards a comprehensive understanding of microtubule re-organization during wall pattern formation.

Genomic imprinting is a phenomenon that occurs in flowering plants and mammals, whereby a gene is expressed in a parent-of-origin-specific manner. Although imprinting has now been examined genome-wide in a number of species using RNA-seq, the analyses used to assess imprinting vary between studies, making consistent comparisons between species difficult. Here we present a simple, easy-to-use bioinformatic pipeline for imprinting analyses suitable for any tissue, including plant endosperm. All relevant scripts can be downloaded. As an illustrative example, we reanalyze published data from A. thaliana and Z. mays endosperm using the pipeline and then demonstrate how to use the results to assess the conservation of imprinting between these species. We also introduce the Plant Imprinting Database, a repository for published imprinting datasets in plants that can be used to view, compare, and download data.

Introduction La prevalence, et le mecanisme des symptomes persistants a distance de l’infection SARS-CoV2 restent mal connus. Notre objectif est de mieux les caracteriser a travers une application smartphone de recueil prospectif mis en place des le debut de l’epidemie, et d’evaluer l’apport d’explorations complementaires standardisees. Materiels et methodes Etude observationnelle, descriptive, transversale, monocentrique, a partir de 309 patients ayant ete suivis en ambulatoire pour une Covid19 confirmee virologiquement ou avec forte presomption clinique/radiologique. Un questionnaire internet etait envoye a 6 semaines d’evolution; il evaluait anonymement l’etat respiratoire, nutritionnel, psychologique, la reprise d’activite physique, les troubles du gout et de l’odorat. Un bilan complementaire par TDM thoracique non injecte et EFR, une reeducation a l’effort ou une prise en charge orthophonique etait systematiquement proposes aux patients volontaires et symptomatiques. Resultats Trois cent neuf patients ont ete suivis dans l’application, avec un âge median de 39 ans (IQR 28–49), un sexe ratio H/F de 0,42, et 20 % de patients a risque de complication. Le taux de reponses au questionnaire est de 68 % (186/274): 13 % des repondeurs sont fumeurs (24/186), 44 % presentent encore des symptomes (81/186): perte de poids (n = 73, 43 %), sensation de perte de capacites respiratoires (n = 70, 38 %), dyspnee d’effort (n = 61, 34 %), toux (n = 33, 18 %), douleurs thoraciques (n = 18, 10 %), et dyspnee de repos (n = 7, 4 %). Parmi les 132 patients (71 %) avec une activite physique anterieure, 76 (58 %) ont pu la reprendre. Parmi les 103 patients (57 %) ayant presente une anosmie, et les 98 patients (55 %) ayant presente une agueusie, ces symptomes etaient resolutifs a 3 semaines chez respectivement 64 % (66/103) et 71 % (68/96) des patients. A ce jour, 17 patients ont ete investigues par un TDM thoracique non injecte et des epreuves fonctionnelles respiratoires (EFR) pour explorer les symptomes respiratoires invalidants persistants: Aucune complication n’a ete detectee par les TDM, tandis que les EFR ont mis en evidence une baisse isolee de la DLCO chez 6 patients (35 %), et une hyperreactivite bronchique chez 5 patients (29 %), aboutissant a un traitement par beta-2 mimetiques et corticoides inhales chez 6 patients (35 %). Une reeducation a l’effort et/ou orthophonique a ete prescrite chez 16 patients (9 % des 186 patients suivis a 6 semaines) Conclusion A 6 semaines d’evolution d’une Covid19, seuls 56 % des patients se disent totalement asymptomatiques. Pour les plus symptomatiques, les examens complementaires semblent peu informatifs. L’information du patient, la reassurance, une reeducation et un traitement d’epreuve d’hyperreactivite bronchique sont les principales reponses medicales.

International audience; Le test de Kleihauer (TK) vise à mettre en évidence la présence de cellules fœtales dans le sang maternel afin d’objectiver une hémorragie foeto-maternelle (HFM). Les hématies fœtales sont distinguées des hématies maternelles grâce à leur contenu en hémoglobine fœtale (HbF) ce qui les rend acido-résistantes. De ce fait, elles ne se lysent pas en présence d’un réactif acidophile et apparaissent en rose foncé après coloration à l’éosine tandis que les hématies adultes sont hémolysées et apparaissent incolores [1]. Chez l’adulte, le taux d’HbF est normalement inférieur à 1% [2]. Le syndrome héréditaire de persistance de l’hémoglobine fœtale (SHPHF) est caractérisé par la production à l’âge adulte d’HbF à l’origine d’un TK « faussement » positif. Nous rapportons le cas d’une patiente dont le TK particulièrement élevé en l’absence de signes d’HFM a conduit à identifier un SHPHF, dont l’origine génétique est un réarrangement non décrit.

Cellulose is produced at the plasma membrane of plant cells by cellulose synthase (CESA) complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked to the plasma membrane. Because CESAs are only active in the plasma membrane, control of CSC secretion regulates cellulose synthesis. We identified members of a family of seven transmembrane domain-containing proteins (7TMs) that are important for cellulose production during cell wall integrity stress. 7TMs are often associated with guanine nucleotide-binding (G) protein signaling and we found that mutants affecting the Gβγ dimer phenocopied the 7tm mutants. Unexpectedly, the 7TMs localized to the Golgi/trans-Golgi network where they interacted with G protein components. Here, the 7TMs and Gβγ regulated CESA trafficking but did not affect general protein secretion. Our results outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses to cell wall integrity stress.

Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp–mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998–2008. ©2016 AACR.

Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis.

Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing., Author summary The discovery of CRISPR-CAS9 and its non-catalytic variants have provided enormous capacity for crop improvement and basic research by modifying the genome and the epigenome. The standard methods for delivering genome and epigenome editing reagents to plants consist of generating stable transgenic lines through tissue culture processes, which have several drawbacks including the need for plant regeneration and crossing. To overcome some of these challenges, plant virus-based platforms are being developed for genome editing. Although viruses have a limited cargo capacity, limiting the use of viruses to encode entire editing systems, guide RNAs have been successfully delivered to transgenic CAS9 expressing plants for genome editing. However, the use of viruses for CRISPR-based epigenome editing and transcriptional activation have not yet been explored. In this study we show that viral delivery of guide RNAs using a modified tobacco rattle virus can be used for transcriptional activation and heritable epigenome editing. This study advances the use of plant RNA viruses as delivery agents for epigenome editing.

Introduction La pandemie d’infections a SARS-CoV-2 a conduit a une reorganisation des services de soins en « filieres COVID-19 » et « non COVID-19 » au sein de nombreux hopitaux avec pour objectifs l’orientation precoce vers une prise en charge specifique des patients COVID-19 et la limitation des risques de transmission au sein de ces etablissements. En complement de ces mesures, une surveillance active de la survenue de cas dans les filieres « non COVID-19 » a ete indispensable pour optimiser la gestion de l’epidemie au sein de notre etablissement. Le deploiement d’une equipe mobile (EM) de prelevement, basee sur une organisation pluridisciplinaire, avait pour but de realiser ces depistages dans des conditions optimales pour les patients et les soignants. Materiels et methodes Les demandes de prelevements rhinopharynges etaient systematiquement soumises aux cliniciens du service de Maladies Infectieuses pour validation des indications : prelevement de cas possibles sur la base d’arguments cliniques et d’imagerie ou prelevements a visee systematique. L’EM etait composee d’un binome d’internes, rapidement mobilisable sur les 5 sites du CHU. Au sein des services cliniques concernes, l’EM etait chargee de la realisation du prelevement ainsi que de l’accompagnement de l’equipe soignante autour des precautions d’hygiene specifiques a mettre en place dans l’attente des resultats biologiques. Le prelevement etait achemine aussitot au laboratoire de virologie pour recherche de SARS-CoV-2 par biologie moleculaire. Resultats Sur les 158 prelevements pris en charge par l’EM, 66 % l’ont ete pour des suspicions cliniques et 34 % a visee systematique. Toute indication confondue, la tranche d’âge majoritaire etait celle des plus de 80 ans (32 %), suivie par les 65–79 ans (23 %) et 50–64 ans (23 %), la population pediatrique etait minoritaire (1 %). Pour les patients symptomatiques, les signes cliniques predominants etaient la fievre (66 %), la toux (21 %), les troubles respiratoires a type de dyspnee et desaturation (21 %), la presence de criteres d’imagerie evocateurs (17 %) et les diarrhees (16 %). Le delai moyen entre l’apparition des symptomes et la suspicion clinique ayant conduit a la realisation du prelevement etait de 3 jours. Parmi les suspicions cliniques, 4 cas positifs ont ete detectes par PCR et aucun parmi les depistages systematiques. Conclusion En complement de la reorganisation des services de soins « COVID-19 », la mise en place d’une equipe mobilisable a permis une surveillance de terrain en temps reel. L’EM a ainsi contribue au diagnostic de COVID-19 pour 4 patients initialement hospitalises dans des filieres non dediees. Le faible taux de positivite constate est en accord avec l’epidemiologie locale correspondant a une zone a faible circulation virale. L’EM a egalement participe au maintien du lien avec les services et a grandement facilite la diffusion de l’information autour des bonnes pratiques d’hygiene.

Introduction/Objectif Determiner la duree des differents symptomes lors de la phase initiale d’une infection a SARS-Cov-2. Materiels et methodes Les patients diagnostiques « COVID-19 » et suivis en ambulatoire du 4 mars au 15 mai 2020 ont ete telesurveilles via une application telephonique (ou internet) avec un questionnaire biquotidien standardise recensant leurs symptomes. Une etude prospective observationnelle monocentrique a ete organisee chez ces patients dont l’infection a SARS-Cov-2 etait confirmee par RT-PCR ou avec forte presomption clinique/radiologique. Ceux qui remplissaient moins de 15 % des questionnaires etaient exclus. La duree de suivi prevue a l’inclusion etait de 20 jours, mais pouvait etre plus breve si le patient se considerait gueri. Resultats Parmi les 387 patients suivis sur l’application, 213 (55 %) ont ete inclus. L’âge median etait de 39 ans (IQR : 28–49) et le sex-ratio H/F de 0,42. Un facteur de risque de complication etait rapporte chez 14 % (29/213). Aucun patient de cette cohorte ambulatoire n’a recu d’hydroxychloroquine, lopinavir/ritonavir ou remdesivir. Cinq patients ont secondairement ete hospitalises. Aucun n’est decede. Le suivi median etait de 18 jours (IQR : 16–21). Les durees medianes des symptomes ont ete respectivement (mediane [IQR] en jours) : alteration de l’etat general (11 [7–15]), fievre (9 [5–14]), frissons (9 [6–14]), cephalees (14 [11–18]), courbatures (13 [8–17]), toux (16 [11–19]), rhinorrhee (13 [8–17]), expectorations (13 [8–19]), douleurs thoraciques (12 [7–17]), dyspnee de repos (9 [6–15]), dyspnee d’effort (16 [12–20]), gene respiratoire (10 [7–14]), diarrhee (11 [7–15]), vomissements (6 [4–10]), difficultes alimentaires (11 [7–16]). La duree mediane de prise de paracetamol etait de 12 jours [9–17]. Lors du dernier suivi, aucun patient n’est febrile, 14 % gardaient une rhinite (19/134), 50 % toussaient (87/173) et 50 % restaient dyspneique a l’effort (73/147). Conclusion La duree des symptomes de COVID-19 est souvent plus longue que deux semaines. L’information du patient lors du diagnostic doit le preparer a cette situation.

Background: Gene body methylation at CG dinucleotides is a widely conserved feature of methylated genomes but remains poorly understood. The Arabidopsis thaliana strain Cvi has depleted gene body methylation relative to the reference strain Col. Here, we leverage this natural epigenetic difference to investigate gene body methylation stability. Results: Recombinant inbred lines derived from Col and Cvi were used to examine the transmission of distinct gene body methylation states. The vast majority of genic CG methylation patterns are faithfully transmitted over nine generations according to parental genotype, with only 1-4% of CGs either losing or gaining methylation relative to the parent. Genic CGs that fail to maintain the parental methylation state are shared among independent lines, suggesting that these are not random occurrences. We use a logistic regression framework to identify features that best predict sites that fail to maintain parental methylation state. Intermediate levels of CG methylation around a dynamic CG site and high methylation variability across many A. thaliana strains at that site are the strongest predictors. These data suggest that the dynamic CGs we identify are not specific to the Col-Cvi recombinant inbred lines but have an epigenetic state that is inherently less stable within the A. thaliana species. Extending this, variably methylated genic CGs in maize and Brachypodium distachyon are also associated with intermediate local CG methylation. Conclusions: These results provide new insights into the features determining the inheritance of gene body methylation and demonstrate that two different methylation equilibria can be maintained within single individuals., National Institutes of Health (U.S.) (Grant T32 GM087237)

Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element–binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3β (GSK3β) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K+ (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K+ channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3β inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3β mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3β inhibitors Li+ and/or CHIR-99021, Li+ increased IKACh, and Li+ and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3β activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3β as a new therapeutic target for diabetic autonomic neuropathy.

In plants, imprinted gene expression occurs in endosperm seed tissue and is sometimes associated with differential DNA methylation between maternal and paternal alleles1. Imprinting is theorized to have been selected for because of conflict between parental genomes in offspring2, but most studies of imprinting have been conducted in Arabidopsis thaliana, an inbred primarily self-fertilizing species that should have limited parental conflict. We examined embryo and endosperm allele-specific expression and DNA methylation genome-wide in the wild outcrossing species Arabidopsis lyrata. Here we show that the majority of A. lyrata imprinted genes also exhibit parentally biased expression in A. thaliana, suggesting that there is evolutionary conservation in gene imprinting. Surprisingly, we discovered substantial interspecies differences in methylation features associated with paternally expressed imprinted genes (PEGs). Unlike in A. thaliana, the maternal allele of many A. lyrata PEGs was hypermethylated in the CHG context. Increased maternal allele CHG methylation was associated with increased expression bias in favour of the paternal allele. We propose that CHG methylation maintains or reinforces repression of maternal alleles of PEGs. These data suggest that the genes subject to imprinting are largely conserved, but there is flexibility in the epigenetic mechanisms employed between closely related species to maintain monoallelic expression. This supports the idea that imprinting of specific genes is a functional phenomenon, and not simply a byproduct of seed epigenomic reprogramming. Genomic imprinting is a form of epigenetic gene regulation in flowering plants and mammals in which alleles of genes are expressed in a parent-of-origin dependent manner. Allele-specific gene expression profiling has identified hundreds of imprinted genes in A. thaliana, maize and rice endosperm, the functions of which are largely unknown. Allelic differences in DNA methylation and chromatin modification between maternal and paternal alleles are important for establishing and maintaining imprinted expression. The emerging picture from multiple species is that the paternal allele of PEGs is associated with DNA methylation, and the silent maternal allele is hypomethylated and bears the Polycomb Repressive Complex 2 (PRC2) mark H3K27me3. Several evolutionary theories have been proposed to describe processes that would select for fixation of this unusual pattern of gene expression13. The kinship or parental conflict theory posits that imprinting is selected for because of asymmetric relatedness among kin. In species where the maternal parent directly provisions growing progeny and has offspring by multiple males, maternally and paternally inherited genomes are predicted to have conflicting interests with regard to the extent of maternal investment. Paternally inherited alleles are expected to favour maternal investment at the expense of half-siblings. Low conservation of imprinting between A. thaliana and monocots, limited conservation between rice and maize, evidence for intraspecific variation in imprinting and lack of strong phenotypes for some imprinted gene mutants has cast doubt on whether imprinting of particular genes is functionally important. Additionally, although some imprinted genes are associated with differential methylation, it has been suggested that imprinted expression is simply a byproduct of endosperm DNA methylation changes—changes that could have a primary function outside imprinting regulation. We were motivated by these considerations and by predictions of the parental conflict theory to compare imprinting and seed DNA methylation between two closely related species that differ in breeding strategy. A. lyrata and A. thaliana diverged approximately 13 Myr ago. Although A. thaliana outcrosses to some extent in the wild, as an obligate outcrosser A. lyrata should be subject to a higher degree of parental conflict than A. thaliana and should therefore be under greater pressure to maintain imprinting. To identify A. lyrata imprinted genes, we performed mRNA-seq on parental strains and F1 hybrid embryo and endosperm tissue derived from crosses between the sequenced A. lyrata strain MN47 (MN) and a strain from Karhumäki (Kar) (Supplementary Fig. 1, Supplementary Fig. 2 and Supplementary Tables 1 and 2). After reannotating A. lyrata genes based on our extensive RNA-seq data (see Supplementary Methods), sequence polymorphisms between MN and Kar were used to quantify the contributions of each parental genome to gene expression. All possible pairwise comparisons (n = 12) of parent-of-origin bias among three MN × Kar and four Kar × MN reciprocal cross-replicates were performed to identify imprinted genes using the same criteria we previously applied to A. thaliana. Only genes that were defined as imprinted in at least 40% of comparisons were included in the final set (Fig. 1 and Supplementary Tables 3 and 4, see Supplementary Methods for details of imprinting criteria). This analysis yielded 49 PEGs and 35 maternally expressed imprinted genes (MEGs) in endosperm (Fig. 1a). Allele assignment calls for 13 genes, including both imprinted and non-imprinted genes, were validated by pyrosequencing (Supplementary Fig. 3). As expected3,5, there was little evidence for imprinting in embryos (Fig. 1a). We compared A. lyrata and A. thaliana endosperm imprinted genes (Fig. 1, Supplementary Fig. 4 and Supplementary Table 4). Of the A. lyrata PEGs for which there were sufficient data available in A. thaliana, 72% (26/36) were also paternally biased in A. thaliana, with 50% (18/36) meeting all stringent criteria for being designated as a PEG in both species (Fig. 1b). Conserved PEGs encoded DNA binding proteins and genes related to chromatin modification, among others (Supplementary Table 4). Of the A. lyrata MEGs for which there were sufficient data in A. thaliana, 70% (12/17) were also significantly maternally biased in A. thaliana, with 35% (6/17) meeting all criteria for being called a MEG in both datasets (Fig. 1b). The conserved MEGs included the Polycomb group gene FIS2, the F-box gene SDC, another F-box gene and three genes encoding DNA binding proteins. Although previous research has identified somewhat more imprinted genes in A. thaliana than what we describe in A. lyrata, these studies involved multiple accessions and assessed imprinting for a greater total number of genes. The majority of genes that were imprinted in A. thaliana but not in A. lyrata lacked sufficient data to make an imprinting designation in A. lyrata (Supplementary Fig. 4). Thus, it is presently unclear whether the number of imprinted genes differs significantly between the species. All of the genes that are commonly imprinted among A. thaliana and cereals were also imprinted in A. lyrata. Many mammalian imprinted genes are clearly involved in growth regulation, including genes for nutrient uptake and feeding behaviour. By contrast, we found that proteins encoded by conserved plant imprinted genes were predicted to regulate or affect the expression of many other genes (chromatin proteins and transcription factors) or protein abundance (F-boxes). We also found that some pathways, rather than orthologous genes, were imprinted in both species, as has been previously noted for imprinting of different subunits of the PRC2 complex among Arabidopsis and cereals. In A. thaliana, the large subunit of RNA Polymerase IV, NRPD1, which functions in RNA-directed DNA methylation (RdDM), is a PEG5,6. Although we did not find evidence for imprinting of the NRPD1 gene in A. lyrata, homologues of two other genes involved in RdDM were PEGs (Supplementary Table 4): NRPD4/NRPE4/RDM2 (AL946699), which encodes a common subunit of Pol IV and Pol V, and RRP6L1 (AL337734), which encodes an exosomal protein that impacts RdDM. Thus, in both species the function of RdDM in the endosperm is under paternal influence, but this is achieved through different genes. The kinship theory is essentially an argument about optimal total gene expression levels in offspring. We therefore evaluated the expression levels and patterns of imprinted genes. MEGs appear to be primarily endosperm-specific genes; they have much lower than average expression in embryos and flower buds, and much higher than average expression in the endosperm (Fig. 1c). Conversely, PEGs were more highly expressed in all tissues than genes on average, and showed more modest expression increases in endosperm, suggesting that the expression of MEGs and PEGs is regulated differently. We also compared the percentage of maternal transcripts for homologous imprinted A. lyrata and A. thaliana genes (Fig. 1d). Conserved MEGs and PEGs exhibited similar degrees of parental bias in the two species (Fig. 1d). However, comparison of the A. thaliana and A. lyrata gene expression level for individual imprinted genes indicated that the overall expression level of PEGs was higher in A. lyrata than in A. thaliana (Fig. 1e). These findings are consistent with selection for higher expression of PEGs in species with greater parental conflict, such as obligate outcrossers. In A. thaliana, active DNA demethylation by the 5-methylcytosine DNA glycosylase DME in the central cell (the female gamete that is the progenitor of the endosperm) before fertilization is essential for establishing gene imprinting at many loci1. Imprinting of many A. thaliana genes, particularly PEGs, is correlated with maternal allele demethylation of proximal sequences corresponding to fragments of transposable elements (TEs). A. lyrata PEGs were somewhat enriched for the presence of TEs in 5′ regions compared with all genes, with 30 out of 49 PEGs (61%) associated with at least one TE within 2 kb upstream, compared with 51% of all genes (Supplementary Table 4). To test if the relationship between methylation and imprinting was conserved in A. lyrata, we profiled genome-wide methylation in MN × MN flower bud, embryo and endosperm tissue by whole-genome bisulfite sequencing. Shared and novel endosperm methylation features were observed compared with A. thaliana (Figs 2 and 3, Supplementary Fig. 5 and Supplementary Table 5). In plants, DNA methylation is found in CG, CHG and CHH sequence contexts. CG methylation was strongly decreased in TEs and in the 5′ and 3′ regions of genes in endosperm relative to other tissues (Fig. 2a and Supplementary Fig. 5). By profiling allele-specific DNA methylation in the F1 embryo and endosperm from Kar females crossed with MN males, we determined that maternally inherited DNA was primarily responsible for endosperm CG hypomethylation (Fig. 2b). These data suggest that, as in A. thaliana, A. lyrata maternally inherited genomes are actively demethylated before fertilization.By contrast, we were surprised to discover that A. lyrata endosperm had a non-CG DNA methylation profile distinct from A. thaliana. This was unexpected because DNA methylation patterns in A. lyrata vegetative tissues display similar features to A. thaliana, although overall methylation levels are higher (Supplementary Fig. 5). We found that average CHG methylation in gene bodies was increased in endosperm compared with embryo (Fig. 2a), a phenotype not observed in wild-type A. thaliana endosperm profiled at similar developmental stages (Supplementary Fig. 5). To determine whether differences in aggregate methylation profiles represented small changes in many regions or larger changes in specific regions of the genome, we compared embryo and endosperm methylation profiles to identify differentially methylated regions (DMRs). As in A. thaliana, the most abundant class of DMRs were less CG methylated in the endosperm than the embryo, with 38% of these falling within 2 kb upstream of genes and 34% within 2 kb downstream of genes (Supplementary Table 6). Regions that gained CHG methylation in MN × MN endosperm displayed markedly different characteristics; 84% fell within gene bodies, corresponding to 1,606 genes (Fig. 2c and Supplementary Table 6). CHG endosperm hypermethylated DMRs were also longer than all other DMR types (mean length = 564 bp with 400 bp s.d.) (Supplementary Table 6). CHG gene body hypermethylation was also observed in Kar × MN endosperm, although on fewer genes (n = 194). Allele-specific analysis of methylation indicated that endosperm CHG hypermethylation was specific to maternally inherited alleles (Fig. 2d). Methylation within gene bodies is usually restricted to the CG context, which is maintained after DNA replication by the maintenance methyltransferase MET1. CHG methylation, normally not found in genes, is maintained by the DNA methyltransferase CMT3, which directly binds to the repressive histone modification H3K9me2 (ref. 23). When accompanied by H3K9me2, CHG gene body methylation is associated with transcriptional repression24. We found that gain of gene body CHG methylation in A. lyrata endosperm was associated with reduced gene expression (Supplementary Fig. 6). Of the CHG hypermethylated genes with enough coverage to evaluate differential expression (n = 1,225), 338 were significantly less expressed in endosperm than in embryo, compared with 159 significantly more highly expressed in endosperm. This represents a significant enrichment of CHG hypermethylated genes among genes less expressed in endosperm than embryo (P = 1.766 × 10–21, hypergeometric test) and a significant depletion among genes upregulated in endosperm (P = 2.04 × 10–10, see Supplementary Methods). The mechanism responsible for CHG gene body hypermethylation in A. lyrata endosperm remains unclear. We found significant overlap between A. thaliana genes that gain CHG methylation or H3K9me2 in ibm1 mutants and CHG hypermethylation of orthologous genes in A. lyrata endosperm (Supplementary Fig. 7). IBM1 encodes a histone lysine demethylase that prevents accumulation of H3K9me2, and thus accumulation of CHG methylation, in genes24. IBM1 transcript abundance was lower in the endosperm than embryo (Supplementary Fig. 7). In A. thaliana, methylation in the long intron of IBM1 is required for proper transcript splicing and production of an enzymatically active protein25. We found that A. lyrata IBM1 exhibited decreased CG and non-CG methylation and increased accumulation of RNA-seq reads in the long intron in endosperm relative to embryo (Supplementary Fig. 7). However, A. thaliana endosperm also had reduced methylation in the long intron and decreased IBM1 transcript abundance than the embryo (Supplementary Fig. 7). Thus, differences in IBM1 expression alone are not sufficient to explain CHG hypermethylation in A. lyrata endosperm compared with A. thaliana, although reduced IBM1 activity is likely to be part of the mechanism. Several of the observed endosperm methylation features were correlated with gene imprinting. More than half of the A. lyrata MEGs and approximately one-third of PEGs were associated with endosperm CG hypomethylated DMRs in the 2 kb region upstream of the transcriptional start site, whereas only 11% of non-imprinted genes were similarly associated with these DMRs (Fig. 3, Supplementary Table 4, Supplementary Fig. 8 and Supplementary Fig. 9). CG hypomethylation occurred specifically on the maternally inherited allele (Supplementary Fig. 8). Thus, reduction of CG methylation by active demethylation is also likely to be an important component of the A. lyrata imprinting mechanism. We found a striking and non-mutually exclusive association between PEGs and endosperm CHG hypermethylation. Almost 60% of PEG gene bodies (n = 27) were CHG hypermethylated, and about one-third were also associated with a 5′ or 3′ CG hypomethylated DMR (Supplementary Table 4). The average methylation profile of PEGs containing a CHG endosperm hypermethylated DMR indicated a very strong increase in CHG methylation across the entire gene body, which was specific to the maternally inherited allele (Figs 3 and 4). Results were validated for two PEGs, homologues of AT5G10950 and AT5G26210, by locus-specific bisulfite-PCR (Fig. 4 and Supplementary Fig. 10). In both A. thaliana and A. lyrata these genes were associated with CG or CHH endosperm hypomethylated DMRs in 5′ To determine if there was a quantitative relationship between gain of CHG methylation and allelic expression bias, we plotted the difference in CHG methylation between maternal alleles in the embryo and endosperm relative to the ratio of maternal to paternal allele transcripts (Fig. 3c). The degree to which CHG methylation was gained on the maternal allele in endosperm relative to embryo was positively correlated with the extent of paternal allele expression bias in endosperm. In addition, PEGs were clearly distinct from other genes that gained CHG gene body methylation; they tended to exhibit greater gain of CHG methylation (Fig. 3c) and were also hypermethylated along more of their length than all CHG hypermethylated genes (56 versus 29%) (Supplementary Tables 4 and 6). Thus, a greater extent and amount of maternal allele CHG hypermethylation is correlated with more paternally biased transcription. These data suggest that CHG methylation, perhaps accompanied by gain of H3K9me2, represses the maternal alleles of PEGs. It is unknown whether gene body CHG methylation is established on maternal alleles before or after fertilization. Demethylation of the IBM1 regulatory intron (Supplementary Fig. 7) could be initiated before fertilization in the central cell, leading to its downregulation and an increase in CHG methylation specifically on maternal alleles, which would then be maintained after fertilization. Alternatively, if maternal allele CHG methylation occurs after fertilization, then CMT3 must be able to distinguish maternally and paternally inherited alleles. Retention of CG gene body methylation on the paternal alleles of PEGs (Fig. 4) could possibly protect them from gain of CHG methylation. Interestingly, gain of gene body CHG methylation was also recently shown to occur in both A. thaliana endosperm and embryos when wild-type plants were pollinated by diploid hypomethylated pollen. Diploid pollen creates triploid seeds with tetraploid endosperm that usually abort, but seed abortion is suppressed when the pollen is hypomethylated owing to mutations in met. Many of the genes that gain CHG methylation and have reduced expression in triploid rescued seeds are PEGs. However, this phenotype appears to be distinct from what we observed; the CHG methylation gain is much more modest than what we have described in wild-type A. lyrata endosperm, and only one conserved PEG was affected. Our data further suggest that gene body CHG hypermethylation is not a state restricted to mutant tissues, but can occur in a developmentally regulated manner that could be important for maintaining gene expression programmes. This is the first study to compare imprinting between two closely related plant species that differ in breeding strategy. A. lyrata and A. thaliana homologous imprinted genes are epigenetically modified in a distinct manner despite the close relatedness of the species (Fig. 4). Allele-specific maintenance of gene repression by the PRC2 complex is an important component of the imprinting mechanism in A. thaliana and other species. The PRC2 complex silences the hypomethylated maternal allele of PEGs, and the methylated paternal allele is expressed. Several studies have suggested that H3K9me2 and H3K27me3 are repressive marks that can substitute for one another in mutant contexts. We suggest that this substitution can also occur in wild-type tissues, and favour the hypothesis that in A. lyrata endosperm the maternal allele of at least a subset of PEGs is repressed by CHG methylation/H3K9me2. Overall, our results point to high conservation of imprinting accompanied by a distinct epigenetic signature, at least for PEGs. If the mechanism of imprinting is different but the genes that are imprinted are the same, this argues that imprinting is not simply a byproduct of endosperm methylation dynamics, but that imprinted expression of specific genes is under selection. Thus, the means by which monoallelic expression can be achieved are plastic, but the genes subject to this regulation are conserved.regions, but were additionally associated with gene body CHG hypermethylated DMRs in A. lyrata. Interestingly, gain of CHG methylation on the maternal allele was often accompanied by loss of CG gene body methylation, whereas paternally inherited alleles retained CG gene body methylation and had a similar methylation profile to embryo alleles (Fig. 4 and Supplementary Table 4). For the 22 PEGs lacking a gene body CHG hypermethylated DMR, half had a CG hypomethylated DMR in the flanking regions 2 kb 5′ or 3′, more like typical A. thaliana PEGs (Supplementary Table 4). Interestingly, these genes largely lacked CG gene body methylation in all tissues (Fig. 3a). Thus, there appear to be at least two classes of PEGs in terms of methylation features (Fig. 3a), which may correspond to different modes of epigenetic regulation. PEGs conserved with A. thaliana are found in both classes, although the majority (12/18) are CHG hypermethylated (Supplementary Table 4).To determine if there was a quantitative relationship between gain of CHG methylation and allelic expression bias, we plotted the difference in CHG methylation between maternal alleles in the embryo and endosperm relative to the ratio of maternal to paternal allele transcripts (Fig. 3c). The degree to which CHG methylation was gained on the maternal allele in endosperm relative to embryo was positively correlated with the extent of paternal allele expression bias in endosperm. In addition, PEGs were clearly distinct from other genes that gained CHG gene body methylation; they tended to exhibit greater gain of CHG methylation (Fig. 3c) and were also hypermethylated along more of their length than all CHG hypermethylated genes (56 versus 29%) (Supplementary Tables 4 and 6). Thus, a greater extent and amount of maternal allele CHG hypermethylation is correlated with more paternally biased transcription. These data suggest that CHG methylation, perhaps accompanied by gain of H3K9me2, represses the maternal alleles of PEGs. It is unknown whether gene body CHG methylation is established on maternal alleles before or after fertilization. Demethylation of the IBM1 regulatory intron (Supplementary Fig. 7) could be initiated before fertilization in the central cell, leading to its downregulation and an increase in CHG methylation specifically on maternal alleles, which would then be maintained after fertilization. Alternatively, if maternal allele CHG methylation occurs after fertilization, then CMT3 must be able to distinguish maternally and paternally inherited alleles. Retention of CG gene body methylation on the paternal alleles of PEGs (Fig. 4) could possibly protect them from gain of CHG methylation. Interestingly, gain of gene body CHG methylation was also recently shown to occur in both A. thaliana endosperm and embryos when wild-type plants were pollinated by diploid hypomethylated pollen. Diploid pollen creates triploid seeds with tetraploid endosperm that usually abort, but seed abortion is suppressed when the pollen is hypomethylated owing to mutations in met. Many of the genes that gain CHG methylation and have reduced expression in triploid rescued seeds are PEG. However, this phenotype appears to be distinct from what we observed; the CHG methylation gain is much more modest than what we have described in wild-type A. lyrata endosperm, and only one conserved PEG was affected. Our data further suggest that gene body CHG hypermethylation is not a state restricted to mutant tissues, but can occur in a developmentally regulated manner that could be important for maintaining gene expression programmes. This is the first study to compare imprinting between two closely related plant species that differ in breeding strategy. A. lyrata and A. thaliana homologous imprinted genes are epigenetically modified in a distinct manner despite the close relatedness of the species (Fig. 4). Allele-specific maintenance of gene repression by the PRC2 complex is an important component of the imprinting mechanism in A. thaliana and other species. The PRC2 complex silences the hypomethylated maternal allele of PEGs, and the methylated paternal allele is expressed. Several studies have suggested that H3K9me and H3K27me are repressive marks that can substitute for one another in mutant contexts. We suggest that this substitution can also occur in wild-type tissues, and favour the hypothesis that in A. lyrata endosperm the maternal allele of at least a subset of PEGs is repressed by CHG methylation/H3K9me. Overall, our results point to high conservation of imprinting accompanied by a distinct epigenetic signature, at least for PEGs. If the mechanism of imprinting is different but the genes that are imprinted are the same, this argues that imprinting is not simply a byproduct of endosperm methylation dynamics, but that imprinted expression of specific genes is under selection. Thus, the means by which monoallelic expression can be achieved are plastic, but the genes subject to this regulation are conserved., National Science Foundation (U.S.) (MCB 1121952), National Science Foundation (U.S.) (MCB 1453459)

We, as a group of international multi-disciplinary neurointerventional societies involved in the endovascular management of acute ischemic stroke, have put forth these training guidelines. We believe that a neuroscience background, dedicated neurointerventional training, and stringent peer review and quality assurance processes are critical to ensuring the best possible patient outcomes. Well-trained neurointerventionalists are a critical component of an organized and efficient team needed to deliver clinically effective mechanical thrombectomy for acute ischemic stroke patients.

Ischemic stroke is a leading cause of death and disability worldwide. Much of the long-term disability occurs in patients with Emergent Large Vessel Occlusion (ELVO). In fact, in these patients, occlusion of a major intracerebral artery results in a large area of brain injury often resulting in

Summary Host cell factor-1 (HCF-1) is a conserved regulator of the longevity and stress response functions of DAF-16/FOXO. SKN-1 transcription factor is an evolutionarily conserved xenobiotic stress regulator and a pro-longevity factor. Here, we demonstrate that SKN-1 contributes to the enhanced oxidative stress resistance incurred by hcf-1 mutation in C. elegans. HCF-1 prevents the nuclear accumulation of SKN-1 and represses the transcriptional activation of SKN-1 specifically at target genes involved in cellular detoxification pathways. Our findings reveal a novel and context-specific regulatory relationship between two highly conserved longevity and stress response factors HCF-1 and SKN-1.