Epigenetic regulators controlling osteogenic lineage commitment and bone formation.

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

LINC01638 Sustains Human Mesenchymal Stem Cell Self-Renewal and Competency for Osteogenic Cell Fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC cell growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2,000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

Sperm and northern bottlenose whale interactions with deep-water trawlers in the western North Atlantic.

Commercial fisheries have increased in all the world's oceans with diverse unintended impacts on marine ecosystems. As a result of resource overlap, interactions between cetaceans and fisheries are a common occurrence and, in many cases, can give rise to significant conservation issues. Research on the distribution and types of such interactions is important for efficient management. In this study, we describe the behaviors of two whale species: sperm whales (Physeter macrocephalus) and northern bottlenose whales (Hyperoodon ampullatus), interacting with benthic trawlers fishing off the eastern Grand Banks of the western North Atlantic in 2007. Whale interactions were only observed when vessels were targeting Greenland halibut (Reinhardtius hippoglossoides) in deep-water fishing areas and were most common during net hauling. Sperm whales and northern bottlenose whales appeared to engage in feeding behavior close to the surface during hauling, especially during the latter stages, suggesting they targeted fish escapees rather than discards. Using photo-identification methods, seven individual sperm whales were identified with multiple resights of six individuals being recorded over an almost two month period. The maximum distance between two resights was 234 km, suggesting individual sperm whales were repeatedly targeting and even following fishing vessels over multiple days and between fishing areas. By contrast, there were no photographic resights of individual northern bottlenose whales within this study, or with substantial photo-identification catalogues from other adjacent high density areas, suggesting that individuals of this species may be less likely to follow vessels or move between areas. This study documents the earliest confirmed records of northern bottlenose whales in this remote region. These interactions and high encounter rates may indicate that adjacent populations are recovering from the previous century of commercial whaling. Our study provides new insights and details on whale-fisheries interactions, which can inform future research and help managers understand the real and perceived impacts of depredation behaviour on fisheries and whales.

Protein arginine methyltransferases PRMT1, PRMT4/CARM1 and PRMT5 have distinct functions in control of osteoblast differentiation.

Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.

Oxygen Uptake Efficiency Slope and Prognosis in Heart Failure With Reduced Ejection Fraction.

The prognostic utility of the oxygen uptake efficiency slope (OUES) in heart failure with reduced ejection fraction is uncertain. In this post hoc analysis of the HF-ACTION (Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training) trial (n = 2,074), we tested for associations of OUES and peak oxygen uptake (VO2) with heart failure hospitalization or cardiovascular death in multivariable Cox regression models, adjusting for minute ventilation/carbon dioxide production (VE/VCO2) slope and other important confounders. Harrell's C-statistics assessed the discriminatory performance of OUES and peak VO2. Lower OUES was associated with increased risk of the outcome (quartile 1 vs 4: hazard ratio 2.1 [1.5 to 2.9, p <0.001]). Peak VO2 had greater discrimination than OUES in comparable models (e.g., C-statistic = 0.73 vs 0.70, p <0.001, respectively). In the subgroup with respiratory exchange ratio <1 (n = 358), peak VO2 was associated with the outcome (p <0.001) but OUES was not (p = 0.96). In conclusion, whereas OUES was associated with clinical outcomes independently of VE/VCO2 slope, its prognostic utility was inferior to that of peak VO2, even when measured at submaximal effort.

Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.

Possible biased virulence attenuation in the Senegal strain of Ehrlichia ruminantium by ntrX gene conversion from an inverted segmental duplication.

Ehrlichia ruminantium is a tick-borne intracellular pathogen of ruminants that causes heartwater, a disease present in Sub-saharan Africa, islands in the Indian Ocean and the Caribbean, inducing significant economic losses. At present, three avirulent strains of E. ruminantium (Gardel, Welgevonden and Senegal isolates) have been produced by a process of serial passaging in mammalian cells in vitro, but unfortunately their use as vaccines do not offer a large range of protection against other strains, possibly due to the genetic diversity present within the species. So far no genetic basis for virulence attenuation has been identified in any E. ruminantium strain that could offer targets to facilitate vaccine production. Virulence attenuated Senegal strains have been produced twice independently, and require many fewer passages to attenuate than the other strains. We compared the genomes of a virulent and attenuated Senegal strain and identified a likely attenuator gene, ntrX, a global transcription regulator and member of a two-component system that is linked to environmental sensing. This gene has an inverted partial duplicate close to the parental gene that shows evidence of gene conversion in different E. ruminantium strains. The pseudogenisation of the gene in the avirulent Senegal strain occurred by gene conversion from the duplicate to the parent, transferring a 4 bp deletion which is unique to the Senegal strain partial duplicate amongst the wild isolates. We confirmed that the ntrX gene is not expressed in the avirulent Senegal strain by RT-PCR. The inverted duplicate structure combined with the 4 bp deletion in the Senegal strain can explain both the attenuation and the faster speed of attenuation in the Senegal strain relative to other strains of E. ruminantium. Our results identify nrtX as a promising target for the generation of attenuated strains of E. ruminantium by random or directed mutagenesis that could be used for vaccine production.

Fleet's Geode: A Breakthrough Sensor for Real-Time Ambient Seismic Noise Tomography over DtS-IoT.

As most of the outcropping and shallow mineral deposits have been found, new technology is imperative to finding the hidden critical mineral deposits required to transition to renewable energy. One such new technique, called ambient seismic noise tomography, has shown promise in recent years as a low-cost, low environmental impact method that can image under cover and at depth. Wireless and compact nodal seismic technology has been instrumental to enable industry applications of ambient noise tomography, but these devices are designed for the active seismic reflection method and do not have the required sensitivity at low frequencies for ambient noise tomography, and real-time data transmission in remote locations requires significant infrastructure to be installed. In this paper, we show the development and testing of the Geode-a real-time seismic node purpose-built by Fleet Space Technologies for ambient seismic noise tomography on exploration scales. We discuss the key differences between current nodal technology and the Geode and show results of a field trial where the performance of the Geode is compared with a commercially popular nodal geophone. The use of a 2 Hz high sensitivity geophone and low noise digitiser results in an instrument noise floor that is more than 30 dB lower below 5 Hz than nodes that are commonly used in the industry. The increased sensitivity results in signal-to-noise ratios in the cross-correlation functions in the field trial that are more than double that of commercially available nodal geophone at low frequencies. When considering the full bandwidth of retrievable correlations in our study, using the Geode would reduce the required recording time from 75 h to 32 h to achieve an average signal-to-noise ratio in the cross-correlation functions of 10. We also discuss the integration of a real-time direct-to-satellite Internet of Things (DtS-IoT) modem in the Geode, which, together with edge processing of seismic data directly on the Geode, enables us to image the subsurface in real-time. During the field trial, the Geodes successfully transmitted more than 90% of the available preprocessed data packets. The Geode is compact enough so that several devices can be carried and installed by one field technician, whilst the array of stations do not require a base station to transmit data to the cloud for further processing. We believe this is the future of passive seismic surveys and will result in faster and more dynamic seismic imaging capabilities analogous to the medical imaging community, increasing the pace at which new mineral deposits are discovered.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Fidelity of Mechanisms Governing the Cell Cycle.

The cell cycle is governed by stringent epigenetic mechanisms that, in response to intrinsic and extrinsic regulatory cues, support fidelity of DNA replication and cell division. We will focus on (1) the complex and interdependent processes that are obligatory for control of proliferation and compromised in cancer, (2) epigenetic and topological domains that are associated with distinct phases of the cell cycle that may be altered in cancer initiation and progression, and (3) the requirement for mitotic bookmarking to maintain intranuclear localization of transcriptional regulatory machinery to reinforce cell identity throughout the cell cycle to prevent malignant transformation.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Cell and Tissue Structure, Function, and Phenotype.

Epigenetic gene regulatory mechanisms play a central role in the biological control of cell and tissue structure, function, and phenotype. Identification of epigenetic dysregulation in cancer provides mechanistic into tumor initiation and progression and may prove valuable for a variety of clinical applications. We present an overview of epigenetically driven mechanisms that are obligatory for physiological regulation and parameters of epigenetic control that are modified in tumor cells. The interrelationship between nuclear structure and function is not mutually exclusive but synergistic. We explore concepts influencing the maintenance of chromatin structures, including phase separation, recognition signals, factors that mediate enhancer-promoter looping, and insulation and how these are altered during the cell cycle and in cancer. Understanding how these processes are altered in cancer provides a potential for advancing capabilities for the diagnosis and identification of novel therapeutic targets.

A novel method for identifying coded tags recorded on aquatic acoustic monitoring systems.

Aquatic biotelemetry increasingly relies on using acoustic transmitters ('tags') that enable passive detection of tagged animals using fixed or mobile receivers. Both tracking methods are resource-limited, restricting the spatial area in which movements of highly mobile animals can be measured using proprietary detection systems. Transmissions from tags are recorded by underwater noise monitoring systems designed for other purposes, such as cetacean monitoring devices, which have been widely deployed in the marine environment; however, no tools currently exist to decode these detections, and thus valuable additional information on animal movements may be missed. Here, we describe simple hybrid methods, with potentially wide application, for obtaining information from otherwise unused data sources. The methods were developed using data from moored, acoustic cetacean detectors (C-PODs) and towed passive receiver arrays, often deployed to monitor the vocalisations of cetaceans, but any similarly formatted data source could be used. The method was applied to decode tag detections that were found to have come from two highly mobile fish species, bass (Dicentrarchus labrax) and Twaite shad (Alosa fallax), that had been tagged in other studies. Decoding results were validated using test tags; range testing data were used to demonstrate the relative efficiency of these receiver methods in detecting tags. This approach broadens the range of equipment from which acoustic tag detections can be decoded. Novel detections derived from the method could add significant value to past and present tracking studies at little additional cost, by providing new insights into the movement of mobile animals at sea.

Evidence from sperm whale clans of symbolic marking in non-human cultures.

Culture, a pillar of the remarkable ecological success of humans, is increasingly recognized as a powerful force structuring nonhuman animal populations. A key gap between these two types of culture is quantitative evidence of symbolic markers-seemingly arbitrary traits that function as reliable indicators of cultural group membership to conspecifics. Using acoustic data collected from 23 Pacific Ocean locations, we provide quantitative evidence that certain sperm whale acoustic signals exhibit spatial patterns consistent with a symbolic marker function. Culture segments sperm whale populations into behaviorally distinct clans, which are defined based on dialects of stereotyped click patterns (codas). We classified 23,429 codas into types using contaminated mixture models and hierarchically clustered coda repertoires into seven clans based on similarities in coda usage; then we evaluated whether coda usage varied with geographic distance within clans or with spatial overlap between clans. Similarities in within-clan usage of both "identity codas" (coda types diagnostic of clan identity) and "nonidentity codas" (coda types used by multiple clans) decrease as space between repertoire recording locations increases. However, between-clan similarity in identity, but not nonidentity, coda usage decreases as clan spatial overlap increases. This matches expectations if sympatry is related to a measurable pressure to diversify to make cultural divisions sharper, thereby providing evidence that identity codas function as symbolic markers of clan identity. Our study provides quantitative evidence of arbitrary traits, resembling human ethnic markers, conveying cultural identity outside of humans, and highlights remarkable similarities in the distributions of human ethnolinguistic groups and sperm whale clans.

Epigenetic and transcriptome responsiveness to ER modulation by tissue selective estrogen complexes in breast epithelial and breast cancer cells.

Selective estrogen receptor modulators (SERMs), including the SERM/SERD bazedoxifene (BZA), are used to treat postmenopausal osteoporosis and may reduce breast cancer (BCa) risk. One of the most persistent unresolved questions regarding menopausal hormone therapy is compromised control of proliferation and phenotype because of short- or long-term administration of mixed-function estrogen receptor (ER) ligands. To gain insight into epigenetic effectors of the transcriptomes of hormone and BZA-treated BCa cells, we evaluated a panel of histone modifications. The impact of short-term hormone treatment and BZA on gene expression and genome-wide epigenetic profiles was examined in ERαneg mammary epithelial cells (MCF10A) and ERα+ luminal breast cancer cells (MCF7). We tested individual components and combinations of 17ß-estradiol (E2), estrogen compounds (EC10) and BZA. RNA-seq for gene expression and ChIP-seq for active (H3K4me3, H3K4ac, H3K27ac) and repressive (H3K27me3) histone modifications were performed. Our results show that the combination of BZA with E2 or EC10 reduces estrogen-mediated patterns of histone modifications and gene expression in MCF-7ERα+ cells. In contrast, BZA has minimal effects on these parameters in MCF10A mammary epithelial cells. BZA-induced changes in histone modifications in MCF7 cells are characterized by altered H3K4ac patterns, with changes at distal enhancers of ERα-target genes and at promoters of non-ERα bound proliferation-related genes. Notably, the ERα target gene GREB1 is the most sensitive to BZA treatment. Our findings provide direct mechanistic-based evidence that BZA induces epigenetic changes in E2 and EC10 mediated control of ERα regulatory programs to target distinctive proliferation gene pathways that restrain the potential for breast cancer development.

LncMIR181A1HG is a novel chromatin-bound epigenetic suppressor of early stage osteogenic lineage commitment.

Bone formation requires osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) and lineage progression of committed osteoblast precursors. Osteogenic phenotype commitment is epigenetically controlled by genomic (chromatin) and non-genomic (non-coding RNA) mechanisms. Control of osteogenesis by long non-coding RNAs remains a largely unexplored molecular frontier. Here, we performed comprehensive transcriptome analysis at early stages of osteogenic cell fate determination in human MSCs, focusing on expression of lncRNAs. We identified a chromatin-bound lncRNA (MIR181A1HG) that is highly expressed in self-renewing MSCs. MIR181A1HG is down-regulated when MSCs become osteogenic lineage committed and is retained during adipogenic differentiation, suggesting lineage-related molecular functions. Consistent with a key role in human MSC proliferation and survival, we demonstrate that knockdown of MIR181A1HG in the absence of osteogenic stimuli impedes cell cycle progression. Loss of MIR181A1HG enhances differentiation into osteo-chondroprogenitors that produce multiple extracellular matrix proteins. RNA-seq analysis shows that loss of chromatin-bound MIR181A1HG alters expression and BMP2 responsiveness of skeletal gene networks (e.g., SOX5 and DLX5). We propose that MIR181A1HG is a novel epigenetic regulator of early stages of mesenchymal lineage commitment towards osteo-chondroprogenitors. This discovery permits consideration of MIR181A1HG and its associated regulatory pathways as targets for promoting new bone formation in skeletal disorders.

Mechanical and interventional support for heart failure with preserved ejection fraction: A review.

INTRODUCTION: Restrictive cardiomyopathy (RCM) and hypertrophic cardiomyopathy (HCM) are two disease processes that are known to progress to heart failure with preserved ejection fraction (HFpEF). Pharmacologic therapies for HFpEF have not improved patient outcomes or reduced mortality in this patient cohort; thus, there continues to be substantial interest in other treatment strategies, including surgical interventions and devices. In this article, we explore and report the current utility of percutaneous therapies and surgically implanted mechanical support in the treatment of patients with HFpEF. RESULTS: Treatment strategies include percutaneous interventions with interatrial shunts, left atrial assist devices (LAADs), and ventricular assist devices (VADs) in various configurations. Although VADs have been employed to treat patients with heart failure with reduced ejection fraction, their efficacy is limited in those with RCM and HCM. A left atrial-to-aortic VAD has been proposed to directly unload the left atrium, but data is limited. Alternatively, a LAAD could be placed in the mitral position and simultaneously unload the left atrium, while filling the left ventricle. CONCLUSION: A left atrial assist device in the mitral position is a promising solution to address the hemodynamic abnormalities in RCM and HCM; these pumps, however, are still under development.

Titanium with nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by regulating histone methylation.

The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants.

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ.

Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

Medical care and first aid: an interassociation consensus framework for organised non-elite sport during the COVID-19 pandemic.

The cessation of amateur and recreational sport has had significant implications globally, impacting economic, social and health facets of population well-being. As a result, there is pressure to resume sport at all levels. The ongoing prevalence of SARS-CoV-2 and subsequent 'second waves' require urgent best practice guidelines to be developed to return recreational (non-elite) sports as quickly as possible while prioritising the well-being of the participants and support staff.This guidance document describes the need for such advice and the process of collating available evidence. Expert opinion is integrated into this document to provide uniform and pragmatic recommendations, thereby optimising on-field and field-side safety for all involved persons, including coaches, first responders and participants.The nature of SARS-CoV-2 transmission means that the use of some procedures performed during emergency care and resuscitation could potentially be hazardous, necessitating the need for guidance on the use of personal protective equipment, the allocation of predetermined areas to manage potentially infective cases and the governance and audit of the process.