DNA methylation mutants in Physcomitrella patens elucidate individual roles of CG and non-CG methylation in genome regulation.

Cytosine (DNA) methylation in plants regulates the expression of genes and transposons. While methylation in plant genomes occurs at CG, CHG, and CHH sequence contexts, the comparative roles of the individual methylation contexts remain elusive. Here, we present Physcomitrella patens as the second plant system, besides Arabidopsis thaliana, with viable mutants with an essentially complete loss of methylation in the CG and non-CG contexts. In contrast to A. thaliana, P. patens has more robust CHH methylation, similar CG and CHG methylation levels, and minimal cross-talk between CG and non-CG methylation, making it possible to study context-specific effects independently. Our data found CHH methylation to act in redundancy with symmetric methylation in silencing transposons and to regulate the expression of CG/CHG-depleted transposons. Specific elimination of CG methylation did not dysregulate transposons or genes. In contrast, exclusive removal of non-CG methylation massively up-regulated transposons and genes. In addition, comparing two exclusively but equally CG- or CHG-methylated genomes, we show that CHG methylation acts as a greater transcriptional regulator than CG methylation. These results disentangle the transcriptional roles of CG and non-CG, as well as symmetric and asymmetric methylation in a plant genome, and point to the crucial role of non-CG methylation in genome regulation.

The Work of ASBH's Clinical Ethics Consultation Affairs Committee: Development Processes Behind Our Educational Materials.

The authors of this article are previous or current members of the Clinical Ethics Consultation Affairs (CECA) Committee, a standing committee of the American Society for Bioethics and Humanities (ASBH). The committee is composed of seasoned healthcare ethics consultants (HCECs), and it is charged with developing and disseminating education materials for HCECs and ethics committees. The purpose of this article is to describe the educational research and development processes behind our teaching materials, which culminated in a case studies book called A Case-Based Study Guide for Addressing Patient-Centered Ethical Issues in Health Care (hereafter, the Study Guide). In this article, we also enumerate how the Study Guide could be used in teaching and learning, and we identify areas that are ripe for future work.

The Polycomb group protein CLF emerges as a specific tri-methylase of H3K27 regulating gene expression and development in Physcomitrella patens.

Packaging of eukaryotic DNA largely depends on histone modifications that affect the accessibility of DNA to transcriptional regulators, thus controlling gene expression. The Polycomb group (PcG) chromatin remodeling complex deposits a methyl group on lysine 27 of histone 3 leading to repressed gene expression. Plants encode homologs of the Enhancer of zeste (E(z)), a component of the PcG complex from Drosophila, one of which is a SET domain protein designated CURLY LEAF (CLF). Although this SET domain protein exhibits a strong correlation with the presence of the H3K27me3 mark in plants, the methyl-transferase activity and specificity of its SET domain have not been directly tested in-vivo. Using the evolutionary early-diverged land plant model species Physcomitrella patens we show that abolishment of a single copy gene PpCLF, as well as an additional member of the PcG complex, FERTILIZATION-INDEPENDENT ENDOSPERM (PpFIE), results in a specific loss of tri-methylation of H3K27. Using site-directed mutagenesis of key residues, we revealed that H3K27 tri-methylation is mediated by the SET domain of the CLF protein. Moreover, the abolishment of H3K27me3 led to enhanced expression of transcription factor genes. This in turn led to the development of fertilization-independent sporophyte-like structures, as observed in PpCLF and PpFIE null mutants. Overall, our results demonstrate the role of PpCLF as a SET protein in tri-methylation of H3K27 in-vivo and the importance of this modification in regulating the expression of transcription factor genes involved in developmental programs of P. patens.

The Evolution of Spina Bifida Treatment Through a Biomedical Ethics Lens.

Spina bifida is a neurodevelopmental disorder that results in a broad range of disability. Over the last few decades, there have been significant advances in diagnosis and treatment of this condition, which have raised concerns regarding how clinicians prognosticate the extent of disability, determine quality of life, and use that information to make treatment recommendations. From the selective treatment of neonates in the 1970s, to the advent of maternal-fetal surgery today, the issues that have been raised surrounding spina bifida intervention invoke principles of medical bioethics such as beneficence and nonmaleficence, while also highlighting how quality of life judgments may drive care decisions. Such changes in treatment norms are also illustrative of how disability is viewed both within the medical community and by society at large. An examination of the changes in spina bifida treatment provides a model through which to understand how ethically complex decisions regarding care for children with disabilities has evolved, and the challenges faced when medical information is combined with value-based judgments to guide medical decision making.

FIE, a nuclear PRC2 protein, forms cytoplasmic complexes in Arabidopsis thaliana.

Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program.The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE-green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation.

DNA METHYLTRANSFERASE 1 is involved in (m)CG and (m)CCG DNA methylation and is essential for sporophyte development in Physcomitrella patens.

DNA methylation has a crucial role in plant development regulating gene expression and silencing of transposable elements. Maintenance DNA methylation in plants occurs at symmetrical (m)CG and (m)CHG contexts ((m) = methylated) and is maintained by DNA METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE (CMT) DNA methyltransferase protein families, respectively. While angiosperm genomes encode for several members of MET1 and CMT families, the moss Physcomitrella patens, serving as a model for early divergent land plants, carries a single member of each family. To determine the function of P. patens PpMET we generated ΔPpmet deletion mutant which lost (m)CG and unexpectedly (m)CCG methylation at loci tested. In order to evaluate the extent of (m)CCG methylation by MET1, we reexamined the Arabidopsis thaliana Atmet1 mutant methylome and found a similar pattern of methylation loss, suggesting that maintenance of DNA methylation by MET1 is conserved through land plant evolution. While ΔPpmet displayed no phenotypic alterations during its gametophytic phase, it failed to develop sporophytes, indicating that PpMET plays a role in gametogenesis or early sporophyte development. Expression array analysis revealed that the deletion of PpMET resulted in upregulation of two genes and multiple repetitive sequences. In parallel, expression analysis of the previously reported ΔPpcmt mutant showed that lack of PpCMT triggers overexpression of genes. This overexpression combined with loss of (m)CHG and its pleiotropic phenotype, implies that PpCMT has an essential evolutionary conserved role in the epigenetic control of gene expression. Collectively, our results suggest functional conservation of MET1 and CMT families during land plant evolution. A model describing the relationship between MET1 and CMT in CCG methylation is presented.

A single CMT methyltransferase homolog is involved in CHG DNA methylation and development of Physcomitrella patens.

C-5 DNA methylation is an essential mechanism controlling gene expression and developmental programs in a variety of organisms. Though the role of DNA methylation has been intensively studied in mammals and Arabidopsis, little is known about the evolution of this mechanism. The chromomethylase (CMT) methyltransferase family is unique to plants and was found to be involved in DNA methylation in Arabidopsis, maize and tobacco. The moss Physcomitrella patens, a model for early terrestrial plants, harbors a single homolog of the CMT protein family designated as PpCMT. Our phylogenetic analysis suggested that the CMT family is unique to embryophytes and its earliest known member PpCMT belongs to the CMT3 subfamily. Thus, P. patens may serve as a model to study the ancient functions of the CMT3 family. We have generated a ΔPpcmt deletion mutant which demonstrated that PpCMT is essential for P. patens protonema and gametophore development and is involved in CHG methylation as demonstrated at four distinct genomic loci. PpCMT protein accumulation pattern correlated with proliferating cells and was sub-localized to the nucleus as predicted from its function. Taken together, our results suggested that CHG DNA methylation mediated by CMT has been employed early in land plant evolution to control developmental programs during both the vegetative and reproductive haploid phases along the plant life cycle.

Knockout of DDM1 in Physcomitrium patens disrupts DNA methylation with a minute effect on transposon regulation and development.

The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a non-flowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.

Factors associated with attrition from a randomized controlled trial of meaning-centered group psychotherapy for patients with advanced cancer.

OBJECTIVE: The generalizability of palliative care intervention research is often limited by high rates of study attrition. This study examined factors associated with attrition from a randomized controlled trial comparing meaning-centered group psychotherapy (MCGP), an intervention designed to help advanced cancer patients sustain or enhance their sense of meaning to the supportive group psychotherapy (SGP), a standardized support group. METHODS: Patients with advanced solid tumor cancers (n = 153) were randomized to eight sessions of either the MCGP or SGP. They completed assessments of psychosocial, spiritual, and physical well-being pretreatment, midtreatment, and 2 months post-treatment. Attrition was assessed in terms of the percent of participants who failed to complete these assessments, and demographic, psychiatric, medical, and study-related correlates of attrition were examined for the participants in each of these categories. RESULTS: The rates of attrition at these time points were 28.1%, 17.7%, and 11.1%, respectively; 43.1% of the participants (66 of 153) completed the entire study. The most common reason for dropout was patients feeling too ill. Attrition rates did not vary significantly between study arms. The participants who dropped out pretreatment reported less financial concerns than post-treatment dropouts, and the participants who dropped out of the study midtreatment had poorer physical health than treatment completers. There were no other significant associations between attrition and any demographic, medical, psychiatric, or study-related variables. CONCLUSIONS: These findings highlight the challenge of maintaining advanced cancer patients in longitudinal research and suggest the need to consider alternative approaches (e.g., telemedicine) for patients who might benefit from group interventions but are too ill to travel.

The effect of values affirmation on psychological stress.

There is growing evidence for the efficacy of acceptance-based behavioral therapies, which aim to increase acceptance of internal experiences and values-consistent action. Further, experimental studies have demonstrated that acceptance decreases distress and increases willingness to engage in challenging tasks (e.g. Levitt, Brown, Orsillo, & Barlow, 2004). However, research demonstrating the positive effects of values articulation on psychological functioning is needed. The goal of the present study was to evaluate the efficacy of a brief intervention in reducing anxiety related to a stressful speech task. Contrary to predictions, engagement in values writing did not reduce anticipatory or posttask anxiety relative to engagement in a neutral writing task. However, self-esteem significantly predicted anxious response to the task. Experiential avoidance and valued living were also associated with anxious response to the task, although the contribution of these predictors was not statistically significant.

A case of luteinizing thecoma with sclerosing peritonitis: revisiting a link with anti-epileptic drugs.

Luteinizing thecoma with sclerosing peritonitis (LTSP) is a rare ovarian tumor of unclear etiology and pathogenesis. The diagnostic entity was proposed in 1994, but a number of earlier reports described possible cases, and some suggested an association with anti-epileptic drugs (AEDs). In presenting a new case we review the literature of previous cases to evaluate the possibility of such a link. When cases in reproductively immature patients are considered, evidence for an association between LTSP and AEDs is strongly suggested despite the rarity of the condition.

Multimedia article: endoscopically guided thoracoscopic esophagectomy for stricture in a child.

PURPOSE: Caustic ingestion is a common cause for acquired esophageal strictures in children. Thoracoscopic esophagectomy can be very useful in this setting, particularly for short segments of disease [1-4]. Thus far, the use of endoscopy to guide resection margins has not been described. METHODS: A 6-year-old boy developed a tight, short esophageal stricture from a lye ingestion injury at the age of 4 years. He had a gastrostomy tube placed at the time for supplemental feedings and subsequently failed attempts at antegrade and retrograde esophageal dilatation. This video demonstrates an endoscopically guided (endoscopes simultaneously passed retrograde via the gastrostomy and antegrade through the oropharynx) thoracoscopic esophagectomy and primary anastomosis management of the stricture. METHODS: In the left lateral decubitus position, four 5-mm ports were placed in the right chest in the fifth intercostal space, anterior-axillary line; sixth intercostal space, midaxillary line; fourth intercostal space, midaxillary line; and seventh intercostal space, posterior axillary line. A 5-mm 30 degrees Storz telescope and 4.9-mm and 9.6-mm Olympus endoscopes were used. The area of esophageal stricture was identified using the endoscopes and thoracoscopically dissected circumferentially. The vagus nerves were identified and circumferentially freed from the strictured esophageal segment. Primary resection and anastomosis was performed using intracorporeal sutures and then tested for leak using the endoscope via the gastrostomy site. RESULTS: Esophagram on postoperative day (POD) 7 revealed no leak and a widely patent anastomosis. He was discharged home on a soft diet on POD 8 and continues to feed orally over 1 year following his operation. CONCLUSION: As demonstrated by our video, endoscopy is a useful adjunct in the performance of a thoracoscopic esophagectomy for short esophageal stricture. It is particularly helpful because it provides direct visualization of the compromised lumen and allows for a more precise resection.

Polycomb group complexes self-regulate imprinting of the Polycomb group gene MEDEA in Arabidopsis.

Fertilization in flowering plants initiates the development of the embryo and endosperm, which nurtures the embryo. A few genes subjected to imprinting are expressed in endosperm from their maternal allele, while their paternal allele remains silenced. Imprinting of the FWA gene involves DNA methylation. Mechanisms controlling imprinting of the Polycomb group (Pc-G) gene MEDEA (MEA) are not yet fully understood. Here we report that MEA imprinting is regulated by histone methylation. This epigenetic chromatin modification is mediated by several Pc-G activities during the entire plant life cycle. We show that Pc-G complexes maintain MEA transcription silenced throughout vegetative life and male gametogenesis. In endosperm, the maternal allele of MEA encodes an essential component of a Pc-G complex, which maintains silencing of the paternal MEA allele. Hence, we conclude that a feedback loop controls MEA imprinting. This feedback loop ensures a complete maternal control of MEA expression from both parental alleles and might have provided a template for evolution of imprinting in plants.

RdDM-independent de novo and heterochromatin DNA methylation by plant CMT and DNMT3 orthologs.

To properly regulate the genome, cytosine methylation is established by animal DNA methyltransferase 3 s (DNMT3s). While altered DNMT3 homologs, Domains rearranged methyltransferases (DRMs), have been shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, the role of true-plant DNMT3 orthologs remains elusive. Here, we profile de novo (RPS transgene) and genomic methylation in the basal plant, Physcomitrella patens, mutated in each of its PpDNMTs. We show that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomically, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Overall, our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM; suggest that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2.

Publisher Correction: RdDM-independent de novo and heterochromatin DNA methylation by plant CMT and DNMT3 orthologs.

The original version of this Article contained an error in Fig. 5, in which the evolutionary origin of DRM2 was incorrectly placed prior to the divergence between gymnosperms and angiosperms . The correct evolutionary origin of DRM2 should be in angiosperms. In addition, in the "Percent methylation change" section of the Methods, Equation 1 was incorrect. This has been corrected in both the PDF and HTML versions of the Article.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Coincident myelomeningocele and gastroschisis: report of 2 cases.

Myelomeningocele and gastroschisis, on their own, are both relatively common entities encountered in pediatric surgical care. Coexistence of these pathologies, however, is exceedingly rare. The authors report on 2 patients who presented with myelomeningocele and gastroschisis at birth. They obtained blood for whole-exome analysis for one of the patients and identified 3 mutations that could be related to the underlying anomalies: homozygous mutations in FAM171B and ABCA1 and a hemizygous (X-linked) mutation in COL4A5. Of these, FAM171B and ABCA1 both have function that may be related to the underlying disease.

Informed Consent in Decision-Making in Pediatric Practice.

Informed consent should be seen as an essential part of health care practice; parental permission and childhood assent is an active process that engages patients, both adults and children, in their health care. Pediatric practice is unique in that developmental maturation allows, over time, for increasing inclusion of the child's and adolescent's opinion in medical decision-making in clinical practice and research. This technical report, which accompanies the policy statement "Informed Consent in Decision-Making in Pediatric Practice" was written to provide a broader background on the nature of informed consent, surrogate decision-making in pediatric practice, information on child and adolescent decision-making, and special issues in adolescent informed consent, assent, and refusal. It is anticipated that this information will help provide support for the recommendations included in the policy statement.

A single homeobox gene triggers phase transition, embryogenesis and asexual reproduction.

Plants characteristically alternate between haploid gametophytic and diploid sporophytic stages. Meiosis and fertilization respectively initiate these two different ontogenies(1). Genes triggering ectopic embryo development on vegetative sporophytic tissues are well described(2,3); however, a genetic control of embryo development from gametophytic tissues remains elusive. Here, in the moss Physcomitrella patens we show that ectopic overexpression of the homeobox gene BELL1 induces embryo formation and subsequently reproductive diploid sporophytes from specific gametophytic cells without fertilization. In line with this, BELL1 loss-of-function mutants have a wild-type phenotype, except that their egg cells are bigger and unable to form embryos. Our results identify BELL1 as a master regulator for the gametophyte-to-sporophyte transition in P. patens and provide mechanistic insights into the evolution of embryos that can generate multicellular diploid sporophytes. This developmental innovation facilitated the colonization of land by plants about 500 million years ago(4) and thus shaped our current ecosystems.