m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that N6-methyladenosine (m6A) RNA methylation by Mettl3 is required for developmental pausing in mouse blastocysts and embryonic stem (ES) cells. Mettl3 enforces transcriptional dormancy through two interconnected mechanisms: (1) it promotes global mRNA destabilization and (2) it suppresses global nascent transcription by destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a crucial anti-pausing factor. Knockdown of N-Myc rescues pausing in Mettl3-/- ES cells, and forced demethylation and stabilization of Mycn mRNA in paused wild-type ES cells largely recapitulates the transcriptional defects of Mettl3-/- ES cells. These findings uncover Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during developmental pausing, with implications for dormancy in adult stem cells and cancer.

Development of a NASA roadmap for planetary protection to prepare for the first human missions to Mars.

As part of planning for future space exploration, COSPAR (The Committee on Space Research) together with participating space agencies, organized and held interdisciplinary meetings to consider next steps in addressing knowledge gaps for planetary protection for future human missions to Mars. Beginning with the results of these meetings and earlier work by NASA, ESA, and COSPAR (e.g., Criswell et al., 2005; Hogan et al., 2006; Rummel et al., 2008) as a base the authors of this paper carried out a follow-on NASA planning activity to identify the necessary steps to be accomplished to close knowledge gaps. We identified significant overlap between the planetary protection needs and other sets of Mars preparation roadmaps (1) microbial monitoring requirements for crew health and medical systems, (2) studies of the microbiome of the built environment, (3) environmental control and life support systems (ECLSS), (4) waste management, and (5) planetary surface operations. In many cases, efforts to mature exploration class systems for Mars that are occurring in other domains can be leveraged with minor changes to address planetary protection gaps as well. In other cases, work planned for testing on the International Space Station (ISS) as an analog for crew Mars transit, or on the lunar surface as an analog for Mars surface operations can be used to close planetary protection technology and knowledge gaps. An overall strategic framework that combines these domains has the advantage of being more comprehensive, efficient, and timely for closing gaps. This approach has led to the development of a NASA roadmap for addressing planetary protection integrated with other related roadmaps. NASA's development and execution of the planetary protection is now viewed in an integrated way with related technology development and testing. Key features of the integrated capabilities roadmap include.

Persistence and fading of the cognitive and socio-emotional benefits of preschool education in a low-resource setting: Group differences and dose-dependent associations in longitudinal data from Vietnam.

Introduction: Four analytic approaches examined the effectiveness of preschool education in Vietnam, which provides a context in which national curricula and teaching standards for preschools and schools, high levels of preschool attendance, and fee subsidies for disadvantaged children, limit the heterogeneity in children's experiences that often obscure the outcomes associated with preschool attendance. Methods: The Young Lives Study provided longitudinal data on children's receptive vocabulary, mathematics, and life satisfaction at 5, 8, 12, and 15 years of age, and on their self-concept and relationships at 12 and 15 years. Results: The first analysis found that children who attended preschool (n = 1,562 at 5 years of age) had larger vocabularies at 5, 8, 12, and 15 years, greater mathematics knowledge at 5, 8, and 12 years, and higher life satisfaction at 5 and 12 years of age than the small number of children who did not attend preschool (n = 164 at 5 years of age). The second, found that the dose of preschool education (hours per week × 4 × months) received by children who attended preschool was positively associated with their receptive vocabulary and mathematics scores at 5, 8, 12, and 15 years of age, and with their life satisfaction at 5 and 15 years of age. Although the magnitude of the effect for vocabulary declined over time, it remained stable for mathematics. The third analysis found that a high dose of preschool education allowed disadvantaged rural children to achieve comparable or better scores than their urban peers for receptive vocabulary at 8, 12, and 15 years, mathematics at 12 years, and life satisfaction at all ages. The final analysis found that even a low dose of preschool education improved rural children's receptive vocabulary at 5, 8, and 15 years, and their numeracy/mathematics scores at 5, 8, and 12 years. Discussion: Together, the results suggest that preschool attendance had a small but meaningful positive association with Vietnamese children's cognitive skills and life satisfaction that persisted for at least 10 years. These findings provide insights into the scale, scope, and longevity of effects that can be achieved from scaled-up preschool programs under resource-constrained conditions.

m 6 A RNA methylation orchestrates transcriptional dormancy during developmental pausing.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that m 6 A RNA methylation by Mettl3 is required for developmental pausing in mice by maintaining dormancy of paused embryonic stem cells and blastocysts. Mettl3 enforces transcriptional dormancy via two interconnected mechanisms: i) it promotes global mRNA destabilization and ii) suppresses global nascent transcription by specifically destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a critical anti-pausing factor. Our findings reveal Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during pausing, with implications for dormancy in stem cells and cancer.

Regulation, functions and transmission of bivalent chromatin during mammalian development.

Cells differentiate and progress through development guided by a dynamic chromatin landscape that mediates gene expression programmes. During development, mammalian cells display a paradoxical chromatin state: histone modifications associated with gene activation (trimethylated histone H3 Lys4 (H3K4me3)) and with gene repression (trimethylated H3 Lys27 (H3K27me3)) co-occur at promoters of developmental genes. This bivalent chromatin modification state is thought to poise important regulatory genes for expression or repression during cell-lineage specification. In this Review, we discuss recent work that has expanded our understanding of the molecular basis of bivalent chromatin and its contributions to mammalian development. We describe the factors that establish bivalency, especially histone-lysine N-methyltransferase 2B (KMT2B) and Polycomb repressive complex 2 (PRC2), and consider evidence indicating that PRC1 shapes bivalency and may contribute to its transmission between generations. We posit that bivalency is a key feature of germline and embryonic stem cells, as well as other types of stem and progenitor cells. Finally, we discuss the relevance of bivalent chromtin to human development and cancer, and outline avenues of future research.

Urban-rural differences in the relationship between stunting, preschool attendance, home learning support, and school readiness: A study in Côte d'Ivoire.

Background: Stunted physical growth during early childhood is a marker of chronic undernutrition, and the adverse life circumstances that underlie it. These have the potential to disrupt normal brain development and the acquisition of foundational cognitive, language, social and motor skills. Stunting is prevalent in most low-and middle-income countries. Because the prevention of stunting requires large-scale structural and attitudinal changes, several psycho-educational interventions have been developed to mitigate the adverse association between early stunting and skill development. However, the resource-intensive nature of custom-designed interventions limit their sustainability and scalability in resource-limited settings. This study explored the possibility that available resources that promote positive development (existing preschool education programs, and no- or low-cost home-based learning activities and resources) may protect against any negative association between stunting and the acquisition of foundational skills required for academic learning and adaptation at school. Method: Data for 36-to 59-month-old children (n = 3,522; M = 46.7 months; 51.2% male; 74.1% rural) were drawn from the most recent Multiple Indicator Cluster Survey conducted in Côte d'Ivoire (MICS5, 2016). Stunting was assessed using the WHO Child Growth Standards. Preschool attendance and home learning activities and resources were assessed by maternal report. School readiness was assessed using the 8-item form of the Early Child Development Index (ECDI). Results: A high percentage of children met the criteria for stunting (28.5%; 19.7% moderate; 8.8% severe). There were marked urban-rural differences in the prevalence of stunting, rates of preschool attendance, home learning activities and resources, children's school readiness scores, and the relationships between stunting, the protective factors and school readiness scores. These urban-rural differences in ECDI scores could be fully explained by differences between these settings in stunting and the protective factors. However, only two protective factors (access to books and home-based activities that promote learning) made independent contributions to variance in ECDI scores. There was tentative evidence that stunted children whose homes provided highly diverse learning activities and multiple types of learning resources were more likely than those who did not to have a high level of school readiness. Conclusion: Capitalizing on the existing practices of families that show positive deviance in caregiving may provide a basis for culturally appropriate, low-cost interventions to improve school readiness among children in low- and middle-income countries, including children with stunted growth.

Cdc42 reactivation at growth sites is regulated by local cell-cycle-dependent loss of its GTPase-activating protein Rga4 in fission yeast.

In fission yeast, polarized cell growth stops during division and resumes after cytokinesis completes and cells separate. It is unclear how growth reactivation is timed to occur immediately after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth during septation, displaying a polar elongation simultaneous with septation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, during septation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. Although the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that, for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

Cdc42 promotes Bgs1 recruitment for septum synthesis and glucanase localization for cell separation during cytokinesis in fission yeast.

Cytokinesis in fission yeast involves actomyosin ring constriction concurrent to septum synthesis followed by septum digestion resulting in cell separation. A recent report indicates that endocytosis is required for septum synthesis and cell separation. The conserved GTPase Cdc42 is required for membrane trafficking and promotes endocytosis. Cdc42 is activated by Guanine nucleotide exchange factors (GEFs). Cdc42 GEFs have been shown to promote timely initiation of septum synthesis and proper septum morphology. Here we show that Cdc42 promotes the recruitment of the major primary septum synthesizing enzyme Bgs1 and consequent ring constriction. Cdc42 is also required for proper localization of the septum digesting glucanases at the division site. Thus, Cdc42 is required to promote multiple steps during cytokinesis.

Relationship between the Length of Sperm Tail Mitochondrial Sheath and Fertility Traits in Boars Used for Artificial Insemination.

The length of sperm tail midpiece, occupied by the mitochondrial sheath (MS), has been correlated with reproductive traits of mice, fish, and birds; however, it is not known whether such a correlation exists in higher order species such as domestic pigs. As the mitochondria provide for sperm motility and generate the fertility-affecting reactive oxygen species (ROS), we hypothesized that MS length correlates with boar semen parameters and artificial insemination (AI) fertility. Sperm samples collected from 57 boars and used for single sire AI were labeled with ProteoStat Aggresome probe (AGG; Enzo Life Sciences) for MS imaging by epifluorescence microscopy and image-based flow cytometry (IBFC). The mean boar MS length was 7.26 ± 0.2 µm, ranging from 6.94 ± 0.18 µm to 7.65 ± 0.31 µm. The absolute longest MS measured was 9.19 µm and the shortest was 5.83 µm. Boars in the high tertile of MS length had significantly higher conception rate (CR; p = 0.05) and sperm parameters. Boars within the high tertile of average number piglets born per litter had significantly shorter MS and more varied MS length than boars in the low tertile (p = 0.04). MS length data correlated with conventional sperm parameters including percent viable and intact acrosomes (p = 0.03), basal:induced oxidation ratio (measure of intracellular ROS levels; p = 0.02) and Comp DNA (chromatin integrity; p = 0.06) along with many flow cytometric AGG parameters in IBFC. Sperm head AGG intensity median absolute deviation had a negative correlation with total born (r = -0.423 p = 0.004). These data reveal a complex relationship between sperm MS length and aggresome abundance to sperm parameters and boar reproductive success in AI service.

Patient satisfaction after conversion from warfarin to direct oral anticoagulants for patients on extended duration of anticoagulation for venous thromboembolism - The SWAN Study.

BACKGROUND: Warfarin is an anticoagulant medication proven effective in the initial treatment and secondary prevention of venous thromboembolism. Anti-Xa direct oral anticoagulants are alternatives to warfarin; however there is limited data assessing satisfaction after switching from warfarin to an anti-Xa direct oral anticoagulant in patients for treatment of venous thromboembolism. OBJECTIVES: To assess medication satisfaction in patients requiring anticoagulation for venous thromboembolism after conversion from warfarin to an anti-Xa direct oral anticoagulant. METHODS: A retrospective cohort study with prospective assessment of satisfaction and review of adverse events following anti-Xa direct oral anticoagulant replacement of warfarin for treatment of venous thromboembolism. Out of 165 patients who had switched from warfarin to rivaroxaban or apixaban from an outpatient haematology practice, 126 patients consented for a survey of patient's relative satisfaction of anti-Xa direct oral anticoagulant therapy compared with previous warfarin therapy using the Anti-Clot Burden and Benefits Treatment Scale and SWAN Score. RESULTS: The mean Anti-Clot Burden and Benefits and SWAN Score was 93% (56/60) and 83% (24.8/30) respectively reflecting high satisfaction with anti-Xa direct oral anticoagulants. 120 patients stated preference for anti-Xa direct oral anticoagulants over warfarin. Leading perceptions driving this was the reduction in frequency of medical contact and fewer bleeding side effects. Thirteen patients (10.3%) experienced an adverse event after the anti-Xa direct oral anticoagulant switch (majority were non-major bleeding) but most remained on anti-Xa direct oral anticoagulant treatment after management options were implemented with continued high satisfaction scores. CONCLUSIONS: Patient satisfaction with anti-Xa direct oral anticoagulant therapy for the treatment and prevention of venous thromboembolism after switching from warfarin in routine clinical practice appeared high. Improved patient convenience including reduced frequency of medical contact and fewer unpredictable side effects were perceived as significant advantages of anti-Xa direct oral anticoagulants compared to warfarin.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Influence of combined vitamin D3 supplementation and resistance exercise training on musculoskeletal health in older men and women (EXVITD): protocol for a randomised controlled trial.

INTRODUCTION: Sarcopenia is a progressive loss in muscle mass, strength and function, the adverse consequences of which are severe, affecting quality of life and placing an increasing burden on social and healthcare systems. Vitamin D status is known to be associated with markers of sarcopenia, namely muscle mass, strength and function. Also, resistance exercise training (RET) is currently the only proven intervention to treat sarcopenia. However, very little data exist on the influence of combining the two interventions of vitamin D supplementation and resistance exercise training, although a recent systematic review provides tentative support for the current study's hypothesis that the combined intervention may further improve musculoskeletal function above exercise training alone. The aim of the present study is to determine whether vitamin D3 supplementation is any more effective in improving musculoskeletal function when combined with RET compared with exercise training alone in older adults. METHODS AND ANALYSIS: This double-blinded randomised placebo-controlled trial will recruit a target of 127 eligible men and women aged ≥65 years living independently or in sheltered housing within the Birmingham area to two groups: (1) 6 months RET and placebo or (2) 6 months RET and 800 IU/d vitamin D3. Measures of muscle power (Nottingham Power Rig), body composition (dual energy X-ray absorptiometry), muscle function (short physical performance battery, timed up and go), falls and fractures as events will be assessed. Assessments will take place at baseline and postintervention, with intermittent monitoring of bone turnover, calcium and vitamin D. The primary outcome will be lower limb extensor power output. Analyses of within-group changes and between-group differences in outcome measures are planned. ETHICS AND DISSEMINATION: The EXVITD study has ethical approval granted by the Black Country National Health Service Research Ethics Committee (14/WM/1220). Results of this trial will be submitted for publication in peer-reviewed journals and presented at conferences. The study is being conducted according to the principles of the Declaration of Helsinki.Trial registration numberNCT02467153; Post-results.

The effect of pH on antibody retention in multimodal cation exchange chromatographic systems.

The present paper builds upon previous work on mAb domain contributions to multimodal (MM) chromatography by examining how pH can impact mAb surface properties and retention in these systems. Linear salt gradient experiments were carried out between pH 5-7 for several mAbs with different pI and surface hydrophobicities in four different MM CEX resins at two ligand densities. mAb retention showed an inverse, non-linear correlation with pH. Changing pH affected the elution order, creating unique windows of selectivity in each of the MM CEX resins. One mAb showed a pH-dependent spectrum of domain contributions, demonstrating that pH can be used to tune the relative importance of the (Fab)2 and Fc domains for some mAbs in MM systems. Positive, negative, and hydrophobic patches were calculated between pH 5-7 for the mAbs. Visualizing these patches on the protein surface demonstrated that each mAb showed a unique distribution of surface charge and hydrophobicity that changed with pH. The sum of patch areas was tracked across this pH range to quantitatively understand how pH impacted these important surface properties. The quantitative analysis then was narrowed to consider only patches in the CDR loops, which were hypothesized to be an important interaction site for some mAbs in these systems. Interestingly, differences in the titration of CDR loop patches for each mAb were shown to be a result of Histidine titrations and patches in this region were qualitatively correlated with experimental trends including the observed elution order reversals. These results indicate that pH potentially can be employed as a lever for the strategic design of multimodal steps to create flow through, bind and elute, or weak partitioning operations with important implications for the design of integrated and/or continuous downstream purification processes. Furthermore, the ability to tune domain contributions in MM separations using pH creates intriguing possibilities for current downstream challenges such as the removal of product-related impurities, as well as the purification of bispecific mAbs.

A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis in fission yeast.

Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. Why the cell needs two GEFs is unclear, given that they are partially redundant and activate the same GTPase. Using the GEF localization pattern during cytokinesis as a paradigm, we report a novel interplay between Gef1 and Scd1 that spatially modulates Cdc42. We find that Gef1 promotes Scd1 localization to the division site during cytokinesis through recruitment of the scaffold protein Scd2, via a Cdc42 feedforward pathway. Similarly, during interphase Gef1 promotes Scd1 recruitment at the new end to enable the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation, and to maintain cell shape during interphase. Our findings reveal an elegant regulatory pattern in which Gef1 primes Cdc42 activation at new sites to initiate Scd1-dependent polarized growth, while Scd1 restricts Gef1 to sites of polarization. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes.This article has an associated First Person interview with the first author of the paper.

Investigating the impact of aromatic ring substitutions on selectivity for a multimodal anion exchange prototype library.

The increasing prevalence of low pI non-mAb therapeutics as well as current challenges in mAb-aggregate separations and low recoveries motivate further development in the multimodal anion exchange (MM AEX) space. In this work, linear salt gradient experiments at pH 7 were used to evaluate the retention of model proteins (with pI from 3.4 to 6.8) in 17 novel MM AEX prototype systems. The ligands were organized into three series. Series 1 extended previous work in multimodal ligand design and included a hydroxyl variant and linker length variants. Series 2 and 3 investigated the nature of hydrophobicity in MM AEX systems by adding hydrophobic (series 2) or fluorine (series 3) substituents to a solvent exposed phenyl ring. Compared to the commercial resin Capto Adhere, the series 1 and 3 ligands exhibited weaker binding, while some of the series 2 aliphatic prototypes showed dramatically increased retention and unique selectivities. Within series 1, the model proteins eluted earlier in the gradient as the charge-hydrophobic group distance on the ligand was increased from 4.9 Å to 8.5 Å. For the aliphatic variants in series 2, proteins that eluted early in the salt gradient were not affected by the increase in ligand hydrophobicity, while the later eluting proteins bound stronger as the length of the aliphatic substituent increased. The series 3 variants indicated that phenyl ring fluorination created subtle changes in protein elution in these MM AEX systems. Retention data from the three series was used to generate a partial least squares QSAR model based on both protein and ligand descriptors which accurately predicted protein retention with a training R2 of 0.81 and a test R2 of 0.76. The retention characteristics of some prototypes such as the earlier elution and unique selectivities compared to Capto Adhere suggest that they could potentially provide unique selectivities and increased recovery for the downstream processing of both mAb and non-mAb biotherapeutics.

Domain contributions to antibody retention in multimodal chromatography systems.

Although a platform process has been established for purification of antibodies, a deep, fundamental understanding of how these molecules interact with chromatography resins has yet to be developed. The increasing prevalence of antibody-related therapeutics and associated purification challenges further motivate research into these molecular level interactions. The objective of this work is to understand the nature (i.e. size and properties) of preferred protein-ligand binding regions for large, multi-domain molecules such as antibodies. In this work, three antibodies with pI 7.5-8.3 and varying hydrophobicity were enzymatically digested to create (Fab)2, Fab, and FC fragments. Linear salt gradient chromatography experiments from 0 to 1M NaCl were carried out with the full mAbs and the fragments in several multimodal chromatography systems at pH 6. The retention of the constituent fragments was then compared to that of the mAb to gain insight into the relative importance of these different domains and the contribution of each domain to the binding of the full mAb in these systems. While some mAbs were dominated by contribution from the FC constant region, others were primarily driven by the (Fab)2 interactions. The domain contributions for each mAb were connected to the unique distribution of surface charge and hydrophobicity using protein surface property maps. This work lays the foundation for identifying the key surface patches on large, multi-domain molecules that are important interaction sites in various multimodal systems. Finally, this work has important implications for the separation of product related variants as well as the design of complex therapeutics for biomanufacturability.

Pregnancy-Related Coronary Artery Dissection: Recognition of a Life Threatening Process.

Pregnancy-related spontaneous coronary artery dissection (P-SCAD) is a rare but life-threatening condition of the peripartum and postpartum mother. The gold standard of diagnosing P-SCAD is a left cardiac catheterization; however, this diagnostic tool may not be used early because myocardial infarction is not typically a top differential diagnosis for women and especially young pregnant women presenting with acute chest pain. Providers and registered nurses, particularly those in the prehospital setting, the emergency department, and labor and delivery units, should be aware of signs, symptoms, potential risk factors, and diagnostic results that could indicate P-SCAD and initiate early and appropriate treatment to improve maternal outcomes.