Human Embryogenesis: A Comparative Perspective.

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.

Enhancer evolution across 20 mammalian species.

The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.

A single-cell transcriptome atlas of marsupial embryogenesis and X inactivation.

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted X chromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that-in organisms with early X chromosome inactivation-imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR, an RSX antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation.

Publisher Correction: A single-cell transcriptome atlas of marsupial embryogenesis and X inactivation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TAF4b transcription networks regulating early oocyte differentiation.

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.

Gene expression across mammalian organ development.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo.

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for âˆ¼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3-CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells - independent of rituximab - was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

Myths and Methodologies: Standardisation in human physiology research-should we control the controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

Extinction risk of the world's freshwater mammals.

The continued loss of freshwater habitats poses a significant threat to global biodiversity. We reviewed the extinction risk of 166 freshwater aquatic and semiaquatic mammals-a group rarely documented as a collective. We used the International Union for the Conservation of Nature Red List of Threatened Species categories as of December 2021 to determine extinction risk. Extinction risk was then compared among taxonomic groups, geographic areas, and biological traits. Thirty percent of all freshwater mammals were listed as threatened. Decreasing population trends were common (44.0%), including a greater rate of decline (3.6% in 20 years) than for mammals or freshwater species as a whole. Aquatic freshwater mammals were at a greater risk of extinction than semiaquatic freshwater mammals (95% CI -7.20 to -1.11). Twenty-nine species were data deficient or not evaluated. Large species (95% CI 0.01 to 0.03) with large dispersal distances (95% CI 0.03 to 0.15) had a higher risk of extinction than small species with small dispersal distances. The number of threatening processes associated with a species compounded their risk of extinction (95% CI 0.28 to 0.77). Hunting, land clearing for logging and agriculture, pollution, residential development, and habitat modification or destruction from dams and water management posed the greatest threats to these species. The basic life-history traits of many species were poorly known, highlighting the need for more research. Conservation of freshwater mammals requires a host of management actions centered around increased protection of riparian areas and more conscientious water management to aid the recovery of threatened species.

Riesgo de extinción de los mamíferos de agua dulce Resumen La pérdida continua de hábitats de agua dulce representa una amenaza importante para la biodiversidad mundial. Analizamos el riesgo de extinción de 166 especies de mamíferos acuáticos y semiacuáticos de agua dulce-un grupo que se documenta pocas veces como colectivo. Usamos las categorías de la Lista Roja de Especies Amenazadas de la Unión Internacional para la Conservación de la Naturaleza de diciembre 2021 para determinar el riesgo de extinción. Después comparamos este riesgo entre grupos taxonómicos, áreas geográficas y caracteres biológicos. El 30% de los mamíferos de agua dulce están categorizados como amenazados. La declinación de las tendencias poblacionales fue común (44.0%), incluyendo una mayor tasa de declinación (3.6% en 20 años) que para los mamíferos o las especies de agua dulce como conjunto. Los mamíferos acuáticos de agua dulce se encuentran en mayor riesgo de extinción que los mamíferos semiacuáticos (95% IC -7.20 a -1.11). Veintinueve especies no contaban con suficientes datos o no estaban evaluadas. Las especies grandes (95% IC 0.01 a 0.03) con distancias de dispersión amplias (95% IC 0.03 a 0.15) tuvieron un mayor riesgo de extinción que las especies pequeñas con menores distancias de dispersión. El número de procesos amenazantes asociados a alguna especie agravó su riesgo de extinción (95% CI 0.28 a 0.77). Las principales amenazas para estas especies fueron la cacería, el desmonte de tierras para tala y agricultura, la contaminación, los desarrollos residenciales y la destrucción o modificación del hábitat causados por presas o manejo hidrológico. Se sabe poco sobre los caracteres básicos de la historia de vida de muchas especies, lo que destaca la necesidad de más investigación al respecto. La conservación de mamíferos de agua dulce requiere una serie de acciones gestoras centradas en el incremento de la protección de las áreas ribereñas y una gestión hidrológica más consciente para ayudar a la recuperación de las especies amenazadas.

Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Myths and Methodologies: Standardisation in Human Physiology Research-Should We Control the Controllables?

The premise of research in human physiology is to explore a multifaceted system whilst identifying one or a few outcomes of interest. Therefore, the control of potentially confounding variables requires careful thought regarding the extent of control and complexity of standardisation. One common factor to control prior to testing is diet, as food and fluid provision may deviate from participants' habitual diets, yet a self-report and replication method can be flawed by under-reporting. Researchers may also need to consider standardisation of physical activity, whether it be through familiarisation trials, wash-out periods, or guidance on levels of physical activity to be achieved before trials. In terms of pharmacological agents, the ethical implications of standardisation require researchers to carefully consider how medications, caffeine consumption and oral contraceptive prescriptions may affect the study. For research in females, it should be considered whether standardisation between- or within-participants in regards to menstrual cycle phase is most relevant. The timing of measurements relative to various other daily events is relevant to all physiological research and so it can be important to standardise when measurements are made. This review summarises the areas of standardisation which we hope will be considered useful to anyone involved in human physiology research, including when and how one can apply standardisation to various contexts.

The establishment and value of peer group clinical supervision: A qualitative study of stakeholders' perspectives.

AIMS: Explore perspectives of steering group members and external clinical supervision facilitators of developing and establishing peer group clinical supervision. BACKGROUND: The climate of healthcare is complex which can lead to staff burnout and challenges to practice. Clinical supervision is suggested as an approach to managing and leadership of such complexities. DESIGN: Qualitative descriptive. METHODS: Focus group interviews with 19 members of the peer group clinical supervision steering groups and individual interviews with five external clinical supervision facilitators from the Western region of Ireland were conducted. Data analysis followed Elo and Kyngäs' content analysis method, involving preparation, organising and reporting, to extract meaning and identify patterns from the qualitative data collected. RESULTS: Developing peer group clinical supervision practice requires, clarity of purpose and function that address the pros and cons of clinical supervision. Organisational leadership is required to support and release staff for peer group clinical supervision and peer group clinical supervisors need to be credible and have a level of expertise in practice. When prepared and supported, the aspects of confidence, leadership, personal development and resilience develop. CONCLUSION: Peer group clinical supervisors need training and ongoing continual professional development for their role, scope of practice and responsibilities. Sustainability rests on staff awareness and familiarity with the purpose and format of peer group clinical supervision and the regularity of sessions. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: Peer group clinical supervision is a means of supporting improvement of patient care delivery while in parallel supporting personal and professional development of staff, building resilience in the workplace. IMPACT: This study explored the implementation of peer group clinical supervision for staff across nursing and midwifery disciplines. It found that implementing peer group clinical supervision had a positive impact on staff well-being and morality and on patient care delivery. These findings influence healthcare service providers in implementing peer group clinical supervision in a sustainable way enabling nurses to continue working in complex healthcare environments delivering safe person-centred care. REPORTING METHOD: The qualitative reporting guidelines Standards for Reporting Qualitative Research (SRQR) were followed. PATIENT OR PUBLIC CONTRIBUTION: Patient/public involvement was addressed in this study by staff, managers, planners, directors, leaders and educationalists being involved at all stages of the study (concept, design, analysis and reporting).

Meiotic Silencing in Mammals.

Meiosis is essential for reproduction in sexually reproducing organisms. A key stage in meiosis is the synapsis of maternal and paternal homologous chromosomes, accompanied by exchange of genetic material to generate crossovers. A decade ago, studies found that when chromosomes fail to synapse, the many hundreds of genes housed within them are transcriptionally inactivated. This process, meiotic silencing, is conserved in all mammals studied to date, but its purpose is not yet defined. Here, I review the molecular genetics of meiotic silencing and consider the many potential functions that it could serve in the mammalian germ line. In addition, I discuss how meiotic silencing influences sex differences in meiotic infertility and the profound impact that meiotic silencing has had on the evolution of mammalian sex chromosomes.

Modulating the RAGE-Induced Inflammatory Response: Peptoids as RAGE Antagonists.

While the primary pathology of Alzheimer's disease (AD) is defined by brain deposition of amyloid-ß (Aß) plaques and tau neurofibrillary tangles, chronic inflammation has emerged as an important factor in AD etiology. Upregulated cell surface expression of the receptor for advanced glycation end-products (RAGE), a key receptor of innate immune response, is reported in AD. In parallel, RAGE ligands, including Aß aggregates, HMGB1, and S100B, are elevated in AD brain. Activation of RAGE by these ligands triggers release of inflammatory cytokines and upregulates cell surface RAGE. Despite such observation, there are currently no therapeutics that target RAGE for treatment of AD-associated neuroinflammation. Peptoids, a novel class of potential AD therapeutics, display low toxicity, facile blood-brain barrier permeability, and resistance to proteolytic degradation. In the current study, peptoids were designed to mimic Aß, a ligand that binds the V-domain of RAGE, and curtail RAGE inflammatory activation. We reveal the nanomolar binding capability of peptoids JPT1 and JPT1a to RAGE and demonstrate their ability to attenuate lipopolysaccharide-induced pro-inflammatory cytokine production as well as upregulation of RAGE cell surface expression. These results support RAGE antagonist peptoid-based mimics as a prospective therapeutic strategy to counter neuroinflammation in AD and other neurodegenerative diseases.

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors.

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.

Erratum: Genome editing reveals a role for OCT4 in human embryogenesis.

This corrects the article DOI: 10.1038/nature24033.

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice.

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

Advances and challenges in genetic technologies to produce single-sex litters.

There is currently a requirement for single-sex litters for many applications, including agriculture, pest control, and reducing animal culling in line with the 3Rs principles: Reduction, Replacement, and Refinement. The advent of CRISPR/Cas9 genome editing presents a new opportunity with which to potentially generate all-female or all-male litters. We review some of the historical nongenetic strategies employed to generate single-sex litters and investigate how genetic and genome editing techniques are currently being used to produce all-male or all-female progeny. Lastly, we speculate on future technologies for generating single-sex litters and the possible associated challenges.

Photochemistry of transition metal carbonyls.

The purpose of this Tutorial Review is to outline the fundamental photochemistry of metal carbonyls, and to show how the advances in technology have increased our understanding of the detailed mechanisms, particularly how relatively simple experiments can provide deep understanding of complex problems. We recall some important early experiments that demonstrate the key principles underlying current research, concentrating on the binary carbonyls and selected substituted metal carbonyls. At each stage, we illustrate with examples from recent applications. This review first considers the detection of photochemical intermediates in three environments: glasses and matrices; gas phase; solution. It is followed by an examination of the theory underpinning these observations. In the final two sections, we briefly address applications to the characterization and behaviour of complexes with very labile ligands such as N2, H2 and alkanes, concentrating on key mechanistic points, and also describe some principles and examples of photocatalysis.