High-confidence calling of normal epithelial cells allows identification of a novel stem-like cell state in the colorectal cancer microenvironment.

Single-cell analyses can be confounded by assigning unrelated groups of cells to common developmental trajectories. For instance, cancer cells and admixed normal epithelial cells could adopt similar cell states thus complicating analyses of their developmental potential. Here, we develop and benchmark CCISM (for Cancer Cell Identification using Somatic Mutations) to exploit genomic single nucleotide variants for the disambiguation of cancer cells from genomically normal non-cancer cells in single-cell data. We find that our method and others based on gene expression or allelic imbalances identify overlapping sets of colorectal cancer versus normal colon epithelial cells, depending on molecular characteristics of individual cancers. Further, we define consensus cell identities of normal and cancer epithelial cells with higher transcriptome cluster homogeneity than those derived using existing tools. Using the consensus identities, we identify significant shifts of cell state distributions in genomically normal epithelial cells developing in the cancer microenvironment, with immature states increased at the expense of terminal differentiation throughout the colon, and a novel stem-like cell state arising in the left colon. Trajectory analyses show that the new cell state extends the pseudo-time range of normal colon stem-like cells in a cancer context. We identify cancer-associated fibroblasts as sources of WNT and BMP ligands potentially contributing to increased plasticity of stem cells in the cancer microenvironment. Our analyses advocate careful interpretation of cell heterogeneity and plasticity in the cancer context and the consideration of genomic information in addition to gene expression data when possible.

European Society of Anaesthesiology and Intensive Care consensus document on sustainability: 4 scopes to achieve a more sustainable practice.

Climate change is a defining issue for our generation. The carbon footprint of clinical practice accounts for 4.7% of European greenhouse gas emissions, with the European Union ranking as the third largest contributor to the global healthcare industry's carbon footprint, after the United States and China. Recognising the importance of urgent action, the European Society of Anaesthesiology and Intensive Care (ESAIC) adopted the Glasgow Declaration on Environmental Sustainability in June 2023. Building on this initiative, the ESAIC Sustainability Committee now presents a consensus document in perioperative sustainability. Acknowledging wider dimensions of sustainability, beyond the environmental one, the document recognizes healthcare professionals as cornerstones for sustainable care, and puts forward recommendations in four main areas: direct emissions, energy, supply chain and waste management, and psychological and self-care of healthcare professionals. Given the urgent need to cut global carbon emissions, and the scarcity of evidence-based literature on perioperative sustainability, our methodology is based on expert opinion recommendations. A total of 90 recommendations were drafted by 13 sustainability experts in anaesthesia in March 2023, then validated by 36 experts from 24 different countries in a two-step Delphi validation process in May and June 2023. To accommodate different possibilities for action in high- versus middle-income countries, an 80% agreement threshold was set to ease implementation of the recommendations Europe-wide. All recommendations surpassed the 80% agreement threshold in the first Delphi round, and 88 recommendations achieved an agreement >90% in the second round. Recommendations include the use of very low fresh gas flow, choice of anaesthetic drug, energy and water preserving measures, "5R" policies including choice of plastics and their disposal, and recommendations to keep a healthy work environment or on the importance of fatigue in clinical practice. Executive summaries of recommendations in areas 1, 2 and 3 are available as cognitive aids that can be made available for quick reference in the operating room.

Fatigue amongst anaesthesiology and intensive care trainees in Europe: A matter of concern.

BACKGROUND: There is a growing awareness of the effects of fatigue on trainee wellbeing and health. Trainees in anaesthesiology and intensive care work long hours, switching work schedules frequently with insufficient rest. This may have unwanted long-term effects on mental and physical health and emotional well being, resulting in burnout and affecting patient safety. OBJECTIVE: The study aimed to evaluate the prevalence, severity, causes and effects of work-related fatigue in trainees in anaesthesiology and intensive care. DESIGN: Online survey of trainees in anaesthesiology and intensive care. SETTINGS: A total of 31 countries within Europe were included in the survey. PARTICIPANTS: European anaesthesiology and intensive care trainees who responded to an invitation to take part by electronic mail or through social media. MAIN OUTCOME MEASURES: Responses from a 29-item online survey to assess the realities within European countries with regards to work-related fatigue. RESULTS: One thousand and two hundred trainees from 31 European countries answered the survey demonstrating that an alarming number of trainees were fatigued by their working patterns and night shifts. Trainees reported effects on personal well being, safe commuting and potential for clinical errors. Respondents described a lack of support from hospitals and management for recovery during and after night shifts. CONCLUSION: Fatigue among trainees in anaesthesiology and intensive care has a significant impact on their well being and potentially, on the incidence of clinical errors. Current measures from authorities and hospital management are not sufficient to prevent serious fatigue, and therefore a fatigue risk management system should be considered. Failure to address this issue might lead to a further decline in trainees' wellbeing, their capacity to work in the speciality in the future, and potentially increase patient care errors.

The single-cell transcriptional landscape of lung carcinoid tumors.

Lung carcinoid tumors, also referred to as pulmonary neuroendocrine tumors or lung carcinoids, are rare neoplasms of the lung with a more favorable prognosis than other subtypes of lung cancer. Still, some patients suffer from relapsed disease and metastatic spread. Several recent single-cell studies have provided detailed insights into the cellular heterogeneity of more common lung cancers, such as adeno- and squamous cell carcinoma. However, the characteristics of lung carcinoids on the single-cell level are yet completely unknown. To study the cellular composition and single-cell gene expression profiles in lung carcinoids, we applied single-cell RNA sequencing to three lung carcinoid tumor samples and normal lung tissue. The single-cell transcriptomes of carcinoid tumor cells reflected intertumoral heterogeneity associated with clinicopathological features, such as tumor necrosis and proliferation index. The immune microenvironment was specifically enriched in noninflammatory monocyte-derived myeloid cells. Tumor-associated endothelial cells were characterized by distinct gene expression profiles. A spectrum of vascular smooth muscle cells and pericytes predominated the stromal microenvironment. We found a small proportion of myofibroblasts exhibiting features reminiscent of cancer-associated fibroblasts. Stromal and immune cells exhibited potential paracrine interactions which may shape the microenvironment via NOTCH, VEGF, TGFß and JAK/STAT signaling. Moreover, single-cell gene signatures of pericytes and myofibroblasts demonstrated prognostic value in bulk gene expression data. Here, we provide first comprehensive insights into the cellular composition and single-cell gene expression profiles in lung carcinoids, demonstrating the noninflammatory and vessel-rich nature of their tumor microenvironment, and outlining relevant intercellular interactions which could serve as future therapeutic targets.

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue.

PURPOSE: Single-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. So far, single-cell studies have been constrained by the requirement for prospectively collected fresh or cryopreserved tissue. This limitation might be overcome by recent technical developments enabling single-cell analysis of FFPE tissue. METHODS: We benchmark single-cell profiles from patient-matched fresh, cryopreserved and archival FFPE cancer tissue. RESULTS: We find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathway, hallmark, and clinically useful signatures, although expression of single genes varied due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting. CONCLUSION: Our analysis highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and increases the applicability in translational research.

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos.

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology.

Palaeogenomic analysis of black rat (Rattus rattus) reveals multiple European introductions associated with human economic history.

The distribution of the black rat (Rattus rattus) has been heavily influenced by its association with humans. The dispersal history of this non-native commensal rodent across Europe, however, remains poorly understood, and different introductions may have occurred during the Roman and medieval periods. Here, in order to reconstruct the population history of European black rats, we first generate a de novo genome assembly of the black rat. We then sequence 67 ancient and three modern black rat mitogenomes, and 36 ancient and three modern nuclear genomes from archaeological sites spanning the 1st-17th centuries CE in Europe and North Africa. Analyses of our newly reported sequences, together with published mitochondrial DNA sequences, confirm that black rats were introduced into the Mediterranean and Europe from Southwest Asia. Genomic analyses of the ancient rats reveal a population turnover in temperate Europe between the 6th and 10th centuries CE, coincident with an archaeologically attested decline in the black rat population. The near disappearance and re-emergence of black rats in Europe may have been the result of the breakdown of the Roman Empire, the First Plague Pandemic, and/or post-Roman climatic cooling.

Drip-and-Ship for Thrombectomy Treatment in Patients With Acute Ischemic Stroke Leads to Inferior Clinical Outcomes in a Stroke Network Covering Vast Rural Areas Compared to Direct Admission to a Comprehensive Stroke Center.

Introduction: Organizing regional stroke care considering thrombolysis as well as mechanical thrombectomy (MTE) remains challenging in light of a wide range of regional population distribution. To compare outcomes of patients in a stroke network covering vast rural areas in southwestern Germany who underwent MTE via direct admission to a single comprehensive stroke center [CSC; mothership (MS)] with those of patients transferred from primary stroke centers [PSCs; drip-and-ship (DS)], we undertook this analysis of consecutive stroke patients with MTE. Materials and Methods: Patients who underwent MTE at the CSC between January 2013 and December 2016 were included in the analysis. The primary outcome measure was 90-day functional independence [modified Rankin score (mRS) 0-2]. Secondary outcome measures included time from stroke onset to recanalization/end of MTE, angiographic outcomes, and mortality rates. Results: Three hundred and thirty-two consecutive patients were included (MS 222 and DS 110). Median age was 74 in both arms of the study, and there was no significant difference in baseline National Institutes of Health Stroke Scale scores (median MS 15 vs. 16 DS). Intravenous (IV) thrombolysis (IVT) rates differed significantly (55% MS vs. 70% DS, p = 0.008). Time from stroke onset to recanalization/end of MTE was 112 min shorter in the MS group (median 230 vs. 342 min, p < 0.001). Successful recanalization [thrombolysis in cerebral infarction (TICI) 2b-3] was achieved in 72% of patients in the MS group and 73% in the DS group. There was a significant difference in 90-day functional independence (37% MS vs. 24% DS, p = 0.017), whereas no significant differences were observed for mortality rates at 90 days (MS 22% vs. DS 17%, p = 0.306). Discussion: Our data suggest that patients who had an acute ischemic stroke admitted directly to a CSC may have better 90-day outcomes than those transferred secondarily for thrombectomy from a PSC.

Diversity and Paleodemography of the Addax (Addax nasomaculatus), a Saharan Antelope on the Verge of Extinction.

Since the 19th century, the addax (Addax nasomaculatus) has lost approximately 99% of its former range. Along with its close relatives, the blue antelope (Hippotragus leucophaeus) and the scimitar-horned oryx (Oryx dammah), the addax may be the third large African mammal species to go extinct in the wild in recent times. Despite this, the evolutionary history of this critically endangered species remains virtually unknown. To gain insight into the population history of the addax, we used hybridization capture to generate ten complete mitochondrial genomes from historical samples and assembled a nuclear genome. We found that both mitochondrial and nuclear diversity are low compared to other African bovids. Analysis of mitochondrial genomes revealed a most recent common ancestor ~32 kya (95% CI 11-58 kya) and weak phylogeographic structure, indicating that the addax likely existed as a highly mobile, panmictic population across its Sahelo-Saharan range in the past. PSMC analysis revealed a continuous decline in effective population size since ~2 Ma, with short intermediate increases at ~500 and ~44 kya. Our results suggest that the addax went through a major bottleneck in the Late Pleistocene, remaining at low population size prior to the human disturbances of the last few centuries.

Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma.

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also the tumor microenvironment can guide precision medicine. A comprehensive and in-depth characterization of the tumor microenvironment is challenging since its cell populations are diverse and can be important even if scarce. To identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to ten human lung adenocarcinomas and ten normal control tissues. Our analyses revealed heterogeneous carcinoma cell transcriptomes reflecting histological grade and oncogenic pathway activities, and two distinct microenvironmental patterns. The immune-activated CP²E microenvironment was composed of cancer-associated myofibroblasts, proinflammatory monocyte-derived macrophages, plasmacytoid dendritic cells and exhausted CD8+ T cells, and was prognostically unfavorable. In contrast, the inert N³MC microenvironment was characterized by normal-like myofibroblasts, non-inflammatory monocyte-derived macrophages, NK cells, myeloid dendritic cells and conventional T cells, and was associated with a favorable prognosis. Microenvironmental marker genes and signatures identified in single-cell profiles had progonostic value in bulk tumor profiles. In summary, single-cell RNA profiling of lung adenocarcinoma provides additional prognostic information based on the microenvironment, and may help to predict therapy response and to reveal possible target cell populations for future therapeutic approaches.

Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer.

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single-cell transcriptome analysis of tumors and matched non-cancerous tissues of twelve colorectal cancer patients. We defined patient-overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen-activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient-derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen-activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR-BRAF-MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non-genetic cancer cell heterogeneity and re-routing of trajectories as a response to targeted therapy.

Author Correction: Spotted phenotypes in horses lost attractiveness in the Middle Ages.

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

Decline of genetic diversity in ancient domestic stallions in Europe.

Present-day domestic horses are immensely diverse in their maternally inherited mitochondrial DNA, yet they show very little variation on their paternally inherited Y chromosome. Although it has recently been shown that Y chromosomal diversity in domestic horses was higher at least until the Iron Age, when and why this diversity disappeared remain controversial questions. We genotyped 16 recently discovered Y chromosomal single-nucleotide polymorphisms in 96 ancient Eurasian stallions spanning the early domestication stages (Copper and Bronze Age) to the Middle Ages. Using this Y chromosomal time series, which covers nearly the entire history of horse domestication, we reveal how Y chromosomal diversity changed over time. Our results also show that the lack of multiple stallion lineages in the extant domestic population is caused by neither a founder effect nor random demographic effects but instead is the result of artificial selection-initially during the Iron Age by nomadic people from the Eurasian steppes and later during the Roman period. Moreover, the modern domestic haplotype probably derived from another, already advantageous, haplotype, most likely after the beginning of the domestication. In line with recent findings indicating that the Przewalski and domestic horse lineages remained connected by gene flow after they diverged about 45,000 years ago, we present evidence for Y chromosomal introgression of Przewalski horses into the gene pool of European domestic horses at least until medieval times.

Optimized DNA sampling of ancient bones using Computed Tomography scans.

The prevalence of contaminant microbial DNA in ancient bone samples represents the principal limiting factor for palaeogenomic studies, as it may comprise more than 99% of DNA molecules obtained. Efforts to exclude or reduce this contaminant fraction have been numerous but also variable in their success. Here, we present a simple but highly effective method to increase the relative proportion of endogenous molecules obtained from ancient bones. Using computed tomography (CT) scanning, we identify the densest region of a bone as optimal for sampling. This approach accurately identifies the densest internal regions of petrous bones, which are known to be a source of high-purity ancient DNA. For ancient long bones, CT scans reveal a high-density outermost layer, which has been routinely removed and discarded prior to DNA extraction. For almost all long bones investigated, we find that targeted sampling of this outermost layer provides an increase in endogenous DNA content over that obtained from softer, trabecular bone. This targeted sampling can produce as much as 50-fold increase in the proportion of endogenous DNA, providing a directly proportional reduction in sequencing costs for shotgun sequencing experiments. The observed increases in endogenous DNA proportion are not associated with any reduction in absolute endogenous molecule recovery. Although sampling the outermost layer can result in higher levels of human contamination, some bones were found to have more contamination associated with the internal bone structures. Our method is highly consistent, reproducible and applicable across a wide range of bone types, ages and species. We predict that this discovery will greatly extend the potential to study ancient populations and species in the genomics era.

The origin of ambling horses.

Horseback riding is the most fundamental use of domestic horses and has had a huge influence on the development of human societies for millennia. Over time, riding techniques and the style of riding improved. Therefore, horses with the ability to perform comfortable gaits (e.g. ambling or pacing), so-called 'gaited' horses, have been highly valued by humans, especially for long distance travel. Recently, the causative mutation for gaitedness in horses has been linked to a substitution causing a premature stop codon in the DMRT3 gene (DMRT3_Ser301STOP) [1]. In mice, Dmrt3 is expressed in spinal cord interneurons and plays an important role in the development of limb movement coordination [1]. Genotyping the position in 4396 modern horses from 141 breeds revealed that nowadays the mutated allele is distributed worldwide with an especially high frequency in gaited horses and breeds used for harness racing [2]. Here, we examine historic horse remains for the DMRT3 SNP, tracking the origin of gaitedness to Medieval England between 850 and 900 AD. The presence of the corresponding allele in Icelandic horses (9(th)-11(th) century) strongly suggests that ambling horses were brought from the British Isles to Iceland by Norse people. Considering the high frequency of the ambling allele in early Icelandic horses, we believe that Norse settlers selected for this comfortable mode of horse riding soon after arrival. The absence of the allele in samples from continental Europe (including Scandinavia) at this time implies that ambling horses may have spread from Iceland and maybe also the British Isles across the continent at a later date.

Spotted phenotypes in horses lost attractiveness in the Middle Ages.

Horses have been valued for their diversity of coat colour since prehistoric times; this is especially the case since their domestication in the Caspian steppe in ~3,500 BC. Although we can assume that human preferences were not constant, we have only anecdotal information about how domestic horses were influenced by humans. Our results from genotype analyses show a significant increase in spotted coats in early domestic horses (Copper Age to Iron Age). In contrast, medieval horses carried significantly fewer alleles for these phenotypes, whereas solid phenotypes (i.e., chestnut) became dominant. This shift may have been supported because of (i) pleiotropic disadvantages, (ii) a reduced need to separate domestic horses from their wild counterparts, (iii) a lower religious prestige, or (iv) novel developments in weaponry. These scenarios may have acted alone or in combination. However, the dominance of chestnut is a remarkable feature of the medieval horse population.