Search

Objectives: Understanding variations in knowledge and attitudes of psychiatrists to psilocybin therapy is important for the collective discourse about the potential impact on clinical practice and public health in Ireland., Methods: A 28-item questionnaire was designed based on previous studies and distributed to psychiatrists in Ireland via online mailing lists and at in-person academic events., Results: 151 psychiatrists completed the questionnaire (73.3% were under 40 years of age, 76.0% were trainees, and 49.0% were female). In the total sample, 81.5% agreed that psilocybin therapy shows promise in the treatment of psychiatric disorders and 86.8% supported funding research, 86.8% would be willing to refer a patient if it was licensed and indicated, and 78.1% would consider the treatment for themselves, if indicated. Conversely, 6.6% agreed that psilocybin therapy was unsafe even under medical supervision, and 21.9% thought it was potentially addictive. 15.9% of the total sample reported at least one concern including, lack of robust evidence, long-term effectiveness, superiority to current interventions, potential harmful effects, cost and accessibility, and impartiality. Less than half of respondents felt knowledgeable (40.0%) and 9.9% felt adequately prepared to participate in psilocybin therapy. Consultant psychiatrists trended towards less optimism for a potential role in bipolar depression and emotionally unstable personality disorder compared to trainee psychiatrists., Conclusion: Overall psychiatrists in Ireland held positive attitudes towards psilocybin therapy. However, there was a lack of knowledge evident. Addressing the knowledge gap and aligning with the best available evidence will be key if psychedelic therapy is to prevail in a clinical setting.

The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro., (© 2024. The Author(s).)

In the companion Perspective 'Past and future of human developmental biology' (Hopwood, 2024), historian Nick Hopwood proposes that the field of human developmental biology has gone through periods of attention and neglect. Development invited researchers from the field to respond to this idea. In this article, published to coincide with the 10th anniversary of Development's 'From Stem Cells to Human Development' meeting, researchers from eight countries comment on how they believe their local legal, political, regulatory, societal and technological frameworks are influencing the field's trajectory., Competing Interests: Competing interests A.T.C. is past-president and board member of the International Society for Stem Cell Research. J.H.H. was granted (through the Yeda–Weizmann Institute of Science) patents relevant to the findings and technologies discussed here (naive and naive-like pluripotency and mouse and human bona fide synthetic whole embryo models). J.H.H. is a co-founder and chief scientific advisor of Renewal Bio, which has licensed technologies mentioned above., (© 2024. Published by The Company of Biologists Ltd.)

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The next 50 years of developmental biology will illuminate exciting new discoveries but are also poised to provide solutions to important problems society faces. Ten scientists whose work intersects with developmental biology in various capacities tell us about their vision for the future., Competing Interests: Declaration of interests J.L. has filed patents related to printing vascularized human tissues and organs from stem cell-derived multicellular building blocks. She is a member of the scientific advisory board of Trestle Biotherapeutics, Inc. (a startup company). Trestle has also licensed IP on kidney bioprinting from Harvard, in which the author is listed as an inventor. M.S. has filed patents for reducing aneuploidy in eggs. J.H.H. was granted (through Yeda–Weizmann Institute of Science) patents relevant to the findings and technologies discussed herein (naive and naive-like pluripotency and mouse and human structure-complete embryo models [SEMs]). J.H.H. is a co-founder and chief scientific advisor of Renewal Bio, which has licensed technologies mentioned above. M.Z.G. is an inventor on patents of stem cell-derived models of human and mouse embryos. S.M. is a founder and supervisory and scientific board member of HeartBeat.bio, an IMBA spin-off, based on submitted cardioid (2020) and multi-chamber cardioid (2022) patent applications., (Copyright © 2024. Published by Elsevier Inc.)

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N 6 -methyladenosine (m 6 A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m 6 A requirement in MNs, we depleted the m 6 A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m 6 A effects, and their silencing phenocopies METTL3 depletion. Among the m 6 A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m 6 A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation., Competing Interests: Declaration of interests F.L. is a co-founder and president of a startup company working on antisense oligonucleotide therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation 1 . Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking 1,2 . Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs) 3 . Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions) 4 . Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development., (© 2023. The Author(s).)

N 6 -methyladenosine (m 6 A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m 6 A profiling in bulk sequencing, single-cell technologies for analyzing m 6 A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m 6 A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m 6 A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m 6 A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes., (© 2023. The Author(s).)

Drug-checking services (DCS) provide people who use drugs (PWUD) the opportunity to have their substances tested before consumption. Though some suggest they may have adverse consequences, DCS have been introduced as a harm reduction (HR) strategy. A systematized review of the literature regarding drug checking (DC) methods and testing locations, advantages and disadvantages, and legal frameworks with an emphasis on HR was conducted referencing PRISMA guidelines. The primary search of PsychInfo, PubMed, Medline, CINHAL, CORE, and Web of Science was conducted between the 4 th and 10 th of September 2020, and 51 literature pieces were included in the final article. Most of the literature focuses on the benefits of currently available DCS. The services identified varied significantly in terms of testing methods, location of operation, primary goal, and the surrounding legal framework. The results suggest using multiple DC methods to be most beneficial. Further, DCS and the personalized interventions they provide can positively influence behavior change, minimize harm, and reduce mortality. DCS are a viable public health intervention that requires cross-sector support beyond the legal frameworks and testing methods. Services will need to be tailored to meet the needs of their chosen setting, local drug market, and target audience.

Research on early postimplantation mammalian development has been limited by the small size and intrauterine confinement of the developing embryos. Owing to the inability to observe and manipulate living embryos at these stages in utero, the establishment of robust ex utero embryo-culture systems that capture prolonged periods of mouse development has been an important research goal. In the last few years, these methods have been significantly improved by the optimization and enhancement of in vitro culture systems sustaining embryo development during peri-implantation stages for several species, and more recently, proper growth of natural mouse embryos from pregastrulation to late organogenesis stages and of embryonic stem cell (ES)-derived synthetic embryo models until early organogenesis stages. Here, we discuss the most recent ex utero embryo-culture systems established to date for rodents, nonhuman primates, and humans. We emphasize their technical aspects and developmental timeframe and provide insights into the new opportunities that these methods will contribute to the study of natural and synthetic mammalian embryogenesis and the stem-cell field., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor β (TGF-β) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in vitro conversion into extra-embryonic trophoblast lineages., Competing Interests: Conflict of interests J.H.H. has submitted patent applications relevant to the findings reported here, related to generation of mammalian synthetic embryoid models, and is a chief scientific advisor of Renewal Bio Ltd., which has licensed technologies described here., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Membranous nephropathy is a rare autoimmune kidney disease whose increasing prevalence in industrialized countries pleads for the involvement of an environmental factor in the development of the disease. In addition, the predominance of men in membranous nephropathy, classically attributed to biologic or genetic differences between men and women, could also be due to different occupational exposures. To support this hypothesis, we sought to describe the toxic occupational exposures of patients with membranous nephropathy., Design, Setting, Participants, & Measurements: In this observational epidemiologic study, we compared the occupations and toxic occupational exposures of 100 patients with membranous nephropathy with those of the general population, consisting of two cohorts of 26,734,000 and 26,500 French workers. We then compared the characteristics of patients exposed to an occupational toxic substance with those of unexposed patients., Results: Patients with membranous nephropathy worked more frequently in the construction sector than the general population (33% versus 7%, P <0.001). This difference remained significant by age and sex. They were also more frequently exposed to toxic substances, such as asbestos (16% versus 5%, P <0.001), lead (9% versus 1%, P <0.001), or organic solvents (37% versus 15%, P <0.001), than the general population. The predominance of men in the subgroup of patients occupationally exposed to toxic substances was not observed in unexposed individuals (organic solvents: 80% men versus 41%, P <0.001; asbestos: 90% men versus 55%, P =0.004). In addition, patients with phospholipase A2 receptor 1 (PLA2R1) epitope spreading were more frequently exposed to asbestos and organic solvents than patients without epitope spreading (32% versus 7%, P =0.02 and 74% versus 43%, P =0.02, respectively), with a dose-dependent effect., Conclusions: Patients with membranous nephropathy were more frequently exposed to certain occupational toxic substances, such as asbestos and organic solvents, than the general population. This occupational exposure was more frequent in men and in patients with PLA2R1 epitope spreading., Clinical Trial Registry Name and Registration Number: Immunopathological Analysis in a French National Cohort of Membranous Nephropathy (IHMN), NCT04326218., Podcast: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022&#95;10&#95;25&#95;CJN02930322.mp3., (Copyright © 2022 by the American Society of Nephrology.)

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro 1-5 , but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells 6-11 . Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis., (© 2022. The Author(s).)

In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation., Competing Interests: Declaration of interests J.H.H. has submitted patent applications relevant to the findings reported herein and is a chief scientific advisor of Renewal Bio Inc., which has licensed technologies described herein., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell asked LGBTQ+ scientists around the world about how their identity shapes their experiences in STEM. Here we share six unique perspectives of researchers highlighting how their area of expertise, research focus, institutions, and geographical location have played a role in this regard. We thank them for sharing their voices and continued efforts toward making science more inclusive., Competing Interests: Declaration of interests J.H.H. is a founding member and chief scientific officer of Renewal Bio Ltd. A.S. has additional affiliations: (1) Director, Human Solidarity Foundation and (2) Consultant, Sangath India. E.O. is director of Squirrel Nation Ltd., a not-for-profit company creating artworks., (Copyright © 2022. Published by Elsevier Inc.)

Background: Festival drug-related deaths are a growing public health concern., Aim: To examine drug use and related harm-reduction practices and attitudes towards utilisation of drug safety testing services., Methods: Data collection took place over the 2019 festival season (June-October). The questionnaire was self-reported. Data was gathered via the online survey, which was promoted through online and social media platforms and outlets. Social media communication methods were used to reach the targeted population more effectively., Results: A total of 1193 Irish festival attendees over the age of 18 completed an anonymous online survey. Alcohol, MDMA powder/crystals, ecstasy pills and cocaine were the highest reported drugs used by Irish festival attendees. The vast majority of participants reported polysubstance use (86.8%/n = 1036). Forty percent of participants (39.98%/n = 477) reported having had sex following the use of a drug at a festival; of these, 66% (n = 316) said that the sex was unprotected. Most participants (84.0%/n = 1003) engaged in some form of harm reduction when taking drugs at festivals. Overwhelmingly, participants reported a willingness to engage with drug-checking services. The vast majority (96.3%; n = 1149) and would use drug checking services more than three-quarters (75.1%/n = 897) reported that they would use an 'amnesty bin' for drugs if it were part of an alert system to notify if dangerous drugs are in circulation. A chi-square test of Independence was conducted to examine whether age and utilisation of drug safety testing service a festival were independent. Moreover, when all cases are taken together, the difference between testing modalities (onsite, offsite and amnesty bin) shows a significant difference p < 001 between those who would use onsite and offsite drug testing facilities., Conclusion: The evidence from this survey indicates that those young people who use drugs at festivals would be prepared to utilise drug checking services and amnesty bins should help inform the public health response to this important area., (© 2021. The Author(s).)

Antibody-mediated immunity is initiated by B cell differentiation into multiple cell subsets, including plasmablast, memory, and germinal center (GC) cells. B cell differentiation trajectories are determined by transcription factors, yet very few mechanisms that specifically determine early B cell fates have been described. Here, we report a post-transcriptional mechanism that suppresses the plasmablast genetic program and promotes GC B cell fate commitment. Single-cell RNA-sequencing analysis reveals that antigen-specific B cell precursors at the pre-GC stage upregulate YTHDF2, which enhances the decay of methylated transcripts. Ythdf2-deficient B cells exhibit intact proliferation and activation, whereas differentiation into GC B cells is blocked. Mechanistically, B cells require YTHDF2 to attenuate the plasmablast genetic program during GC seeding, and transcripts of key plasmablast-regulating genes are methylated and bound by YTHDF2. Collectively, this study reveals how post-transcriptional suppression of gene expression directs appropriate B cell fate commitment during initiation of the adaptive immune response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Persons experiencing homelessness (PEH) are at heightened risk for infection, morbidity, and mortality from COVID-19. However, health consequences of the pandemic extend far beyond those directly caused by the virus. This scoping review aimed to explore the impacts of the COVID-19 pandemic on the health and well-being of PEH in North America and Europe. A systematic search of academic and grey literature was conducted in September 2021. To be included, studies had to include primary data related to the impact of the pandemic on health or well-being of PEH and be written in English. All potentially relevant references were independently screened by two reviewers, and minor conflicts were settled with input of a third reviewer. A total of 96 articles met criteria for inclusion. Data extraction was completed for all included studies, and findings synthesised and presented thematically. Numerous health impacts of the pandemic on PEH were identified, including SARS-CoV-2 infection, morbidity, mortality, and hospitalisation, fear of infection, access to housing, hygiene, PPE, food, as well as mental health, substance use, other health-related outcomes and treatment services. Gaps in the literature relating to persons using alcohol, access to mental health support, and violence were also identified. Implications for future research are discussed.

Undergraduate university students are at a critical stage of development in terms of their academic, social, psychological and behavioural health. Patterns established during these formative years can last a lifetime. eHealth tools have the potential to be engaging, convenient and accessible to a wide range of students by providing health information and enhancing the uptake of positive health behaviours. The 'Healthy Trinity Online Tool' (H-TOT) was developed in collaboration with students and a transdisciplinary team with decades of experience between them in terms of research, clinical responsibility and service delivery. Developmental steps undertaken included: a literature review to formulate the topic content choices; a survey of students to check the relevance and suitability of topics identified; and, the tacit experience of the development team. This co-design model led to the development of content encompassing academic life, healthy eating, physical activity, mood, financial matters, alcohol, tobacco, drugs and relaxation. Qualitative focus groups were subsequently conducted for in-depth exploration of the usage and functionality of H-TOT. The theoretical underpinnings include the locus of control and social cognitive theory. Evidence-based behavioural change techniques are embedded throughout. During early pre-piloting of H-TOT, the team identified and solved content functionality problems. The tone of the content was also revised to ensure it was non-judgemental. To make the H-TOT as interactive as possible, video scenarios were included and all content was audio-recorded to allow playback for students with visual or learning difficulties. Evaluation plans for the pilot year of H-TOT are outlined., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Regulatory T (T reg ) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how T reg cells are mechanistically induced in vitro (iT reg ) and stabilized via transcriptional regulation of T reg lineage-specifying factor Foxp3. We find that acetylation of histone tails at the Foxp3 promoter is required for inducing Foxp3 transcription. Upon induction, histone acetylation signals via bromodomain-containing proteins, particularly targets of inhibitor JQ1, and sustains Foxp3 transcription via a global or trans effect. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements, mainly enhancer CNS2, increases chromatin accessibility and protein binding, stabilizing Foxp3 transcription and obviating the need for the histone acetylation signal. These processes transform stochastic iT reg induction into a stable cell fate, with the former sensitive and the latter resistant to genetic and environmental perturbations. Thus, sequential histone acetylation and DNA demethylation in Foxp3 induction and maintenance reflect stepwise mechanical switches governing iT reg cell lineage specification., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: This study examines Irish undergraduate students' behaviours and motives regarding alcohol consumption. The study explores both levels and patterns of consumption., Method: A cross-sectional design using a convenience sample of (n = 213) students from a selection of different courses in Health Sciences at Trinity College Dublin was used to obtain this data. The study used a peer-led approach to design and data collection. Peer-led research is emerging as a robust methodology. Evidence supports it as an effective approach, particularly with sensitive questions, which may be shared with more ease between persons with common interests and experiences., Results: In terms of alcohol consumption levels and patterns, of those who drank almost three quarters (149/71%) met the threshold for binge drinking (i.e. six of more consecutive drinks in one session). Males (n = 36/73.4%) were more likely than females (n = 113/69.7%) to binge drink. Moreover, one in 5 males (n = 10/20.4%) said that they drank ten or more drinks in one session. Males were more likely to drink for conformity reasons. Despite this, a significant proportion (69.2%) of participants reported alcohol-related problems. The Drinking Motives Questionnaire-Revised (DMQR) results showed that overall students were more likely to drink for social and enhancement reasons rather than coping or conformity reasons, consistent with other studies. Nonetheless, males in the current study were more likely to drink for conformity reasons., Conclusion: Given the high rates of hazardous drinking, the development of an alcohol intervention may be justified, given the high response rates to peer-screening, a peer-led intervention for alcohol-related harms may yield positive results., (© 2021. Royal Academy of Medicine in Ireland.)

Background: Women diagnosed with substance use disorders (SUDs) have higher rates of major medical conditions compared to women without SUDs. Cervical cancer is the second leading cause of cancer death in women aged 20-39 years worldwide and women with SUDs have an increased risk of cervical cancer compared to women without SUD. The National Drug Treatment Centre (NDTC) cervical screening programme, derived from the national CervicalCheck programme, offers free cervical screening to patients attending for treatment of SUDs., Aims: This study aimed to audit adherence to the NDTC Cervical Screening guidelines before and after the implementation of an awareness-raising educational intervention., Methods: The electronic clinical records of women aged between 25 and 60 years attending the lead consultant's (M.S.) outpatient clinic were reviewed for documentary evidence indicating that information about the cervical screening programme had been discussed. This was completed before and one month after the implementation of an awareness-raising educational intervention., Results: All women (n = 46, mean age 36.3 (SD = 6.5) years) had an opioid use disorder; 85% had a benzodiazepine use disorder, and 24% had an alcohol use disorder. Of these, 80% had at least one chronic medical condition, 76% had a psychiatric disorder, and 59% were homeless. Adherence to the NDTC cervical screening guideline, as indicated by documentary evidence in clinical records, was 33% (14/43) at baseline, and rose to 88% (36/41) (p < 0.0001) one month after the intervention., Conclusions: This completed audit cycle shows that an awareness-raising educational intervention can significantly improve adherence to a cervical screening programme in women with SUDs., (© 2021. Royal Academy of Medicine in Ireland.)

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SμGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N 6 -methyladenosine (m 6 A) RNA modification drives recognition and 3' end processing of SμGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m 6 A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SμGLT. Direct suppression of m 6 A modification of SμGLT or of m 6 A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m 6 A modification, and m 6 A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation-related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Grenov et al.)

Objective: Despite positive effects of physical activity programs in the workplace, an appropriate measurement instrument to capture employees' decisional balance to exercise is lacking. This study sought to develop and validate a decisional balance scale for exercise adapted to the workplace., Methods: Four complementary studies were carried out, with a total sample of 2398 French volunteer employees, to develop a preliminary version and examine its dimensionality, temporal stability, and construct validity., Results: The Workplace Decisional Balance scale for Exercise (WDBex) consists of 21 items with two subfactors, respectively composed of 12 items (facilitators) and nine items (barriers); for each subfactor, three dimensions (ie, physical, psychological, and logistic) were distinguished and presented satisfactory psychometric properties., Conclusions: This instrument constitutes a reliable and valid instrument for studying the psychosocial determinants of physical activity engagement in the workplace and for developing health promotion strategies., Competing Interests: Conflicts of Interest: None declared., (Copyright © 2021 American College of Occupational and Environmental Medicine.)

Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency., Competing Interests: Declaration of interests Two patent applications based on the findings reported in this work have been filed by the relevant authors, and some of the findings are being commercialized., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Developmental and epileptic encephalopathies (DEE) are a group of disorders associated with intractable seizures, brain development, and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain-containing oxidoreductase (WWOX) are associated with a relatively mild autosomal recessive spinocerebellar ataxia-12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE). In this study, we generated an in vitro model for DEEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR-engineered human ES cells and from patient-derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt pathway and DNA damage response impairment. Furthermore, we provide a proof of concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies., (©2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Background and Aims: This work aims to assess the impact of the COVID-19 pandemic on hospital workers' psychological parameters and attitudes toward substance use, before and during the French COVID-19 lockdown. Methods: An online survey was proposed to the staff of the University Hospital of Nice and Sainte-Marie psychiatric hospital in France from May 18 to June 6, 2020 assessing changes in daily habits, psychological distress and changes in substance use, including smoking. Results: A total of 702 respondents (80.3% female) filled out the survey. Overall, most of the workers reported increased stress, irritability, sadness, decreased motivation, and a worse quality of sleep after the beginning of the COVID-19 lockdown. Additionally, hospital workers who were more likely to use tobacco during the COVID-19 lockdown reported increased sadness ( OR = 1.23, p < 0.001), loss of motivation ( OR = 0.86, p < 0.05), alcohol consumption ( OR = 3.12, p < 0.001), lower incomes ( OR = 1.69, p < 0.05), living alone ( OR = 1.77, p < 0.001) and doing less physical activity ( OR = 0.36, p < 0.001). Conclusion: During the first lockdown, significant psychological changes (sadness, distress, irritability) associated with changes in tobacco use and physical activity were reported. Such results should encourage hospital leaders to implement dedicated policies to better accompany hospital workers' psychological distress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mounir, Menvielle, Perlaza, Chênevert, Planchard, Fabre, Benoit, Benyamina, Rolland, Cherikh and David.)

Several studies suggest parental substance use, while not always the case, exposes children to a higher risk of physical, psychological, behavioral and emotional problems. Identifying interventions that best support the parent and child needs is imperative. The PuP programme aims to improve family functioning and child outcomes by supporting parents who are or have been drug or alcohol dependent. PuP combines psychological principles relating to parenting, child behavior and parental emotion regulation within a case-management model. The current study was the first evaluation of the PuP program in a residential setting globally. Twenty-three women took part in pre and post-program qualitative interviews. Guilt was a dominant theme across interviews. The principal expectation of participants was to improve their relationships and access to their children. The benefits of the PuP program to the women and their children, as reported by participants, were immediate and direct. As the women progressed through the program, they were visibly building belief in their abilities to parent.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro 1,2 , approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The N6-methyladenosine (m 6 A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m 6 A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types., (© 2020 Lasman et al.; Published by Cold Spring Harbor Laboratory Press.)

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

We sought to evaluate the outcomes of chronic phase (CP) chronic myeloid leukemia (CML) in an era where five tyrosine kinase inhibitors (TKIs) are commercially available for the treatment of CML. Records of patients diagnosed with CP CML, treated with TKIs and referred to our center were reviewed. Between January 2005 and April 2016, 206 patients were followed for a median of 48.8 (1.4-190.1) months. A total of 76 (37%) patients received one TKI, 73 (35%) received two TKIs and 57 (28%) were exposed to >3 TKIs (3 TKIs, n = 33; 4 TKIs, n = 17; 5 TKIs, n = 7). Nineteen (9.2%) patients progressed to advanced phases of CML (accelerated phase, n = 6; myeloid blastic phase, n = 4; lymphoid blastic phase, n = 9). One third ( n = 69) achieved complete molecular response (CMR) at first-line treatment. An additional 55 patients achieved CMR after second-line treatment. Twenty-five patients (12.1%) attempted TKI discontinuation and 14 (6.8%) stopped TKIs for a median of 6.3 months (range 1-53.4). The 10-year progression-free survival and overall survival (OS) rates were 81% and 87%, respectively. OS after 10-years, based on TKI exposure, was 100% (1 TKI), 82% (2 TKIs), 87% (3 TKIs), 75% (4 TKIs) and 55% (5 TKIs). The best OS was observed in patients tolerating and responding to first line TKI, but multiple TKIs led patients to gain treatment-free remission.

The rising field of RNA modifications is stimulating massive research nowadays. m 6 A, the most abundant mRNA modification is highly conserved during evolution. Through the last decade, the essential components of this dynamic mRNA modification machinery were found and classified into writer, eraser and reader proteins. m 6 A modification is now known to take part in diverse biological processes such as embryonic development, cell circadian rhythms and cancer stem cell proliferation. In addition, there is already firm evidence for the importance of m 6 A modification in stem cell differentiation and gametogenesis, both in males and females. This review attempts to summarize the important results of recent years studying the mechanism underlying stem cell differentiation and gametogenesis processes.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney 1,2 . However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages 3-7 . ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.

N6-methyladenosine (m 6 A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m 6 A stoichiometry and the lack of antibody-independent methodologies for interrogating m 6 A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m 6 A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m 6 A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m 6 A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m 6 A regulation in subcellular fractions, diverse cell types, and disease states., (Copyright © 2019 Elsevier Inc. All rights reserved.)