Slide-tags enables single-nucleus barcoding for multimodal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

Evolutionary fingerprints of EMT in pancreatic cancers.

Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.

Slide-tags: scalable, single-nucleus barcoding for multi-modal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed. Missing from these measurements, however, is the ability to routinely and easily spatially localise these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are 'tagged' with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 micron spatial resolution, and delivered whole-transcriptome data that was indistinguishable in quality from ordinary snRNA-seq. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil, and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualised receptor-ligand interactions driving B-cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to virtually any single-cell measurement technology. As proof of principle, we performed multiomic measurements of open chromatin, RNA, and T-cell receptor sequences in the same cells from metastatic melanoma. We identified spatially distinct tumour subpopulations to be differentially infiltrated by an expanded T-cell clone and undergoing cell state transition driven by spatially clustered accessible transcription factor motifs. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

Regulators of male and female sexual development are critical for the transmission of a malaria parasite.

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

The Application of Single-Cell RNA Sequencing in Vaccinology.

Single-cell RNA sequencing allows highly detailed profiling of cellular immune responses from limited-volume samples, advancing prospects of a new era of systems immunology. The power of single-cell RNA sequencing offers various opportunities to decipher the immune response to infectious diseases and vaccines. Here, we describe the potential uses of single-cell RNA sequencing methods in prophylactic vaccine development, concentrating on infectious diseases including COVID-19. Using examples from several diseases, we review how single-cell RNA sequencing has been used to evaluate the immunological response to different vaccine platforms and regimens. By highlighting published and unpublished single-cell RNA sequencing studies relevant to vaccinology, we discuss some general considerations how the field could be enriched with the widespread adoption of this technology.

The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle.

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites.

Single-Cell (Multi)omics Technologies.

Single-cell multiomics technologies typically measure multiple types of molecule from the same individual cell, enabling more profound biological insight than can be inferred by analyzing each molecular layer from separate cells. These single-cell multiomics technologies can reveal cellular heterogeneity at multiple molecular layers within a population of cells and reveal how this variation is coupled or uncoupled between the captured omic layers. The data sets generated by these techniques have the potential to enable a deeper understanding of the key biological processes and mechanisms driving cellular heterogeneity and how they are linked with normal development and aging as well as disease etiology. This review details both established and novel single-cell mono- and multiomics technologies and considers their limitations, applications, and likely future developments.

Ligando a estratégia preventiva de alto-risco ao mercado da indústria biomédica: implicações para a saúde pública / Linking high-risk preventive strategy to biomedical-industry market: implications for public health

Abstract This paper takes Geoffrey Rose's concepts on preventive strategy as the basis for theoretical framework to critically analyse the current approach to disease prevention. Rose's "continuum of risk and severity" has widened the scope for preventive actions and underpins two approaches: high-risk strategy (HRS) and population strategy (PS). Both of them produce paradoxes: HRS, despite having a good harm-benefit ratio, offers little impact on public health; PS has greater impact on public health, but offers minimal benefit at individual level. We argue that HRS is being misapplied by reducing cut-off points for preventive interventions to impact morbimortality attributed to specific diseases. This tends to medicalize prevention, producing more disease related phenomena through screening techniques, and inducing individual affective reactions, which require action in the present to secure better future health. This context has paved the way for speculative preventive medicine, which perceives health as a commodity but ignores its implications for public health services.

Resumo Este artigo aborda os conceitos de Geoffrey Rose sobre estratégia preventiva como base para análise teórico-crítica da abordagem atual para a prevenção de doenças. A proposta de Rose de um "continuum de risco e severidade" ampliou as possibilidades das ações preventivas ao classificar duas abordagens: a estratégia de alto risco (EAR) e a estratégia populacional (EP). Ambas produzem paradoxos: a EAR, apesar de apresentar boa relação dano-benefício, oferece pouco impacto para a saúde pública; a EP tem maior impacto sobre a saúde pública, mas oferece benefícios mínimos em nível individual. Argumentamos que a EAR tem sido mal utilizada ao reduzir pontos de corte para intervenções preventivas para impactar a morbimortalidade atribuída a doenças específicas. Isso tende a medicalizar a prevenção, produzindo mais fenômenos relacionados à doença por meio de técnicas de rastreamento, induzindo reações afetivas individuais que exigem ação no presente para garantir melhor saúde no futuro. Tal contexto abre caminho para a prática de uma medicina preventiva especulativa, que percebe a saúde como mercadoria, mas ignora suas implicações para os serviços de saúde pública.

The Quality and Outcomes Framework: Body commodification in UK general practice.

The UK's Quality and Outcomes Framework (QOF) is the largest pay-for-performance scheme in the world. This ethnographic study explored how QOF's monetary logic influences the approach to healthcare in UK general practice. From August 2013 to April 2014, we researched two UK general practice surgeries and one general practice training programme. These environments provided the opportunity for studying various spaces such as QOF meetings, consultation rooms, QOF recoding sessions, and the collection of computer-screen images depicting how patients' biomarkers are evaluated and costed through software systems. QOF as a biomedical technology has led to the commodification of patients and their bodies. This complex phenomenon breaks down into three main themes: commodification of patients, QOF as currency, and valuing commodities. Despite the ostensible aim of QOF being to improve healthcare in general practice, it is accompanied by a body commodification process. The interface between patients and care providers has been commodified, with QOF's pricing mechanism and fragmentation of care provision performing an important role in animating the UK economy.

What's 'difficult'? A multi-stage qualitative analysis of secondary care specialists' experiences with medically unexplained symptoms.

BACKGROUND: The term 'difficult' is pervasively used in relation to medically unexplained symptoms (MUS) and patients with MUS. This article scrutinises the use of the term by analysing interview data from a study of secondary care specialists' experiences with and attitudes towards patients suffering from MUS. DESIGN: Qualitative design employing semi-structured open-ended interviews systematically analysed in three stages: first, data were analysed according to the principles of content analysis. The analysis subsequently focused on the use of the term 'difficult'. Iterations of the term were extracted by summative analysis and thematic coding revealed its different meanings. Finally, alternative expressions were explored. SETTING: Three NHS trust secondary care hospitals in North-East England. PARTICIPANTS: 17 senior clinicians from seven medical and two surgical specialities. RESULTS: Unsolicited use of the term 'difficult' was common. 'Difficult' was rarely used as a patient characteristic or to describe the therapeutic relationship. Participants used 'difficult' to describe their experience of diagnosing, explaining, communicating and managing these conditions and their own emotional reactions. Health care system deficits and the conceptual basis for MUS were other facets of 'difficult'. Participants also reported experiences that were rewarding and positive. CONCLUSIONS: This study shows that blanket statements such as 'difficult patients' mask the complexity of doctors' experiences in the context of MUS. Our nuanced analysis of the use of 'difficult' challenges preconceived attitudes. This can help counter the unreflexive perpetuation of negative evaluations that stigmatize patients with MUS, encourage greater acknowledgement of doctors' emotions, and lead to more appropriate conceptualizations and management of MUS.

Using Ethical Reasoning to Amplify the Reach and Resonance of Professional Codes of Conduct in Training Big Data Scientists.

The use of Big Data--however the term is defined--involves a wide array of issues and stakeholders, thereby increasing numbers of complex decisions around issues including data acquisition, use, and sharing. Big Data is becoming a significant component of practice in an ever-increasing range of disciplines; however, since it is not a coherent "discipline" itself, specific codes of conduct for Big Data users and researchers do not exist. While many institutions have created, or will create, training opportunities (e.g., degree programs, workshops) to prepare people to work in and around Big Data, insufficient time, space, and thought have been dedicated to training these people to engage with the ethical, legal, and social issues in this new domain. Since Big Data practitioners come from, and work in, diverse contexts, neither a relevant professional code of conduct nor specific formal ethics training are likely to be readily available. This normative paper describes an approach to conceptualizing ethical reasoning and integrating it into training for Big Data use and research. Our approach is based on a published framework that emphasizes ethical reasoning rather than topical knowledge. We describe the formation of professional community norms from two key disciplines that contribute to the emergent field of Big Data: computer science and statistics. Historical analogies from these professions suggest strategies for introducing trainees and orienting practitioners both to ethical reasoning and to a code of professional conduct itself. We include two semester course syllabi to strengthen our thesis that codes of conduct (including and beyond those we describe) can be harnessed to support the development of ethical reasoning in, and a sense of professional identity among, Big Data practitioners.

The payment for performance model and its influence on British general practitioners' principles and practice.

This article explores some effects of the British payment for performance model on general practitioners' principles and practice, which may contribute to issues related to financial incentive modalities and quality of primary healthcare services in low and middle-income countries. Aiming to investigate what general practitioners have to say about the effect of the British payment for performance on their professional ethos we carried out semi-structured interviews with 13 general practitioner educators and leaders working in academic medicine across the UK. The results show a shift towards a more biomedical practice model and fragmented care with nurse practitioners and other health care staff focused more on specific disease conditions. There has also been an increased medicalisation of the patient experience both through labelling and the tendency to prescribe medications rather than non-pharmacological interventions. Thus, the British payment for performance has gradually strengthened a scientific-bureaucratic model of medical practice which has had profound effects on the way family medicine is practiced in the UK.

The payment for performance model and its influence on British general practitioners' principles and practice / O modelo de pagamento por desempenho e sua influencia nos principios e pratica dos medicos generalistas britanicos / El modelo de pago por desempeno y su influencia en los principios y practica de los medicos generales britanicos

This article explores some effects of the British payment for performance model on general practitioners’ principles and practice, which may contribute to issues related to financial incentive modalities and quality of primary healthcare services in low and middle-income countries. Aiming to investigate what general practitioners have to say about the effect of the British payment for performance on their professional ethos we carried out semi-structured interviews with 13 general practitioner educators and leaders working in academic medicine across the UK. The results show a shift towards a more biomedical practice model and fragmented care with nurse practitioners and other health care staff focused more on specific disease conditions. There has also been an increased medicalisation of the patient experience both through labelling and the tendency to prescribe medications rather than non-pharmacological interventions. Thus, the British payment for performance has gradually strengthened a scientific-bureaucratic model of medical practice which has had profound effects on the way family medicine is practiced in the UK.

Este artigo explora alguns efeitos do modelo de pagamento por desempenho nos princípios e prática dos médicos generalistas britânicos, podendo contribuir para o debate sobre a relação entre modalidades de incentivos financeiros e qualidade dos serviços na atenção primária à saúde em países de moderada e baixa renda. Objetivando investigar o que os médicos generalistas têm a dizer dos efeitos do pagamento por desempenho britânico sobre seu ethos profissional, conduzimos entrevistas semiestruturadas com 13 médicos generalistas, educadores e líderes no meio acadêmico da medicina no Reino Unido. Os resultados apontam um modelo de prática mais biomédica e fragmentação do cuidado, com enfermeiras e outros profissionais mais focados em doenças específicas. Houve também um aumento da medicalização da vivência dos pacientes, pela rotulação e tendência a prescrever mais medicação e menor uso de intervenções não farmacológicas. Assim, o pagamento por desempenho britânico tem gradualmente fortalecido um modelo científico-burocrático de prática médica que teve efeitos profundos sobre a forma como a medicina de família vem sendo praticada no Reino Unido.

Este artículo explora algunos efectos del modelo británico de pago por desempeño en los principios y práctica de médicos generales que pueden contribuir a cuestiones relacionadas con modalidades de incentivos financieros y calidad de servicios de atención primaria en países de bajos y medios ingresos. La investigación tuvo por objetivo lo que los médicos tienden a decir sobre el efecto del pago por desempeño británico en su ethos profesional; se realizaron entrevistas semi-estructuradas con 13 médicos generales, educadores y líderes en medicina académica del Reino Unido. Los resultados muestran cambios hacia un modelo de práctica más biomédica y atención fragmentada con enfermeras y otros profesionales enfocados en enfermedades específicas. También produjo un aumento en medicalización de la experiencia del paciente a través de rotulaciones y tendencia a prescribir medicamentos en lugar de intervenciones no farmacológicas. Así, el pago por desempeño británico ha reforzado gradualmente un modelo científico-burocrático de práctica que ha tenido profundos efectos en la forma en la que la medicina familiar está siendo practicada en el Reino Unido.

Challenging the biopsychosocial model in a chronic constipation clinic.

In this article we discuss the results of an ethnographic study of professionals' and patients' experiences within a specialist constipation clinic in England. Chronic constipation tends to be poorly understood and inadequately treated. Eleven patients were followed through their illness trajectory during a 5-month fieldwork period, involving 21 home interviews, clinic-based interviews, participant observation, and a focus group. Professionals were likewise observed and interviewed. The clinic could be broadly described as biopsychosocial in its approach. However, professionals expressed uncertainty about how best to provide biopsychosocial care and suggested that some patients were not "open" to psychosocial therapies or to discussing psychosocial aspects of their disease. Patients' concerns were with being taken seriously, receiving treatment, and narrating intersections of life events, emotional well-being, and the bowels. We situate these findings within the discourse of "functional" disorders and discuss why implementing a biopsychosocial approach is problematic in this case.

Distributions of enzyme residues yielding mutants with improved substrate specificities from two different directed evolution strategies.

A previous study of random mutations, mostly introduced by error-prone PCR (EPPCR) or DNA shuffling (DS), demonstrated that those closer to the enzyme active site were more effective than distant ones at improving enzyme activity, substrate specificity or enantioselectivity. Since then, many studies have taken advantage of this observation by targeting site-directed saturation mutagenesis (SDSM) to residues closer to or within enzyme active sites. Here, we have analysed a set of SDSM studies, in parallel to a similar set from EPPCR/DS, to determine whether the greater range of amino-acid types accessible by SDSM affects the distances at which the most effective sites occur. We have also analysed the relative effectiveness for obtaining beneficial mutants of residues with different degrees of natural sequence variation, as determined by their sequence entropy which is related to sequence conservation. These analyses attempt to answer the question-how well focused have targeted mutagenesis strategies been? We also compared two different sets of active-site atoms from which to measure distances and found that the inclusion of catalytic, substrate and cofactor atoms refined the analysis compared to using a single key catalytic atom. Using this definition, we found that EPPCR/DS is not effective for altering substrate specificity at sites that are within 5 A of the active-site atoms. In contrast, SDSM is most effective when targeted to residues at <5-6 A from the catalytic, substrate or cofactor atom, and also for residues with intermediate sequence entropies. Furthermore, SDSM is capable of altering substrate specificity at highly and completely conserved residues in the active site. The results suggest ways in which directed evolution by SDSM could be improved for greater efficiency in terms of reducing the library sizes required to obtain beneficial mutations that alter substrate specificity.