In situ analysis of osmolyte mechanisms of proteome thermal stabilization.

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

The changing career paths of PhDs and postdocs trained at EMBL.

Individuals with PhDs and postdoctoral experience in the life sciences can pursue a variety of career paths. Many PhD students and postdocs aspire to a permanent research position at a university or research institute, but competition for such positions has increased. Here, we report a time-resolved analysis of the career paths of 2284 researchers who completed a PhD or a postdoc at the European Molecular Biology Laboratory (EMBL) between 1997 and 2020. The most prevalent career outcome was Academia: Principal Investigator (636/2284=27.8% of alumni), followed by Academia: Other (16.8%), Science-related Non-research (15.3%), Industry Research (14.5%), Academia: Postdoc (10.7%) and Non-science-related (4%); we were unable to determine the career path of the remaining 10.9% of alumni. While positions in Academia (Principal Investigator, Postdoc and Other) remained the most common destination for more recent alumni, entry into Science-related Non-research, Industry Research and Non-science-related positions has increased over time, and entry into Academia: Principal Investigator positions has decreased. Our analysis also reveals information on a number of factors - including publication records - that correlate with the career paths followed by researchers.

Multicellular factor analysis of single-cell data for a tissue-centric understanding of disease.

Biomedical single-cell atlases describe disease at the cellular level. However, analysis of this data commonly focuses on cell-type-centric pairwise cross-condition comparisons, disregarding the multicellular nature of disease processes. Here, we propose multicellular factor analysis for the unsupervised analysis of samples from cross-condition single-cell atlases and the identification of multicellular programs associated with disease. Our strategy, which repurposes group factor analysis as implemented in multi-omics factor analysis, incorporates the variation of patient samples across cell-types or other tissue-centric features, such as cell compositions or spatial relationships, and enables the joint analysis of multiple patient cohorts, facilitating the integration of atlases. We applied our framework to a collection of acute and chronic human heart failure atlases and described multicellular processes of cardiac remodeling, independent to cellular compositions and their local organization, that were conserved in independent spatial and bulk transcriptomics datasets. In sum, our framework serves as an exploratory tool for unsupervised analysis of cross-condition single-cell atlases and allows for the integration of the measurements of patient cohorts across distinct data modalities.

Principles and challenges of modeling temporal and spatial omics data.

Studies with temporal or spatial resolution are crucial to understand the molecular dynamics and spatial dependencies underlying a biological process or system. With advances in high-throughput omic technologies, time- and space-resolved molecular measurements at scale are increasingly accessible, providing new opportunities to study the role of timing or structure in a wide range of biological questions. At the same time, analyses of the data being generated in the context of spatiotemporal studies entail new challenges that need to be considered, including the need to account for temporal and spatial dependencies and compare them across different scales, biological samples or conditions. In this Review, we provide an overview of common principles and challenges in the analysis of temporal and spatial omics data. We discuss statistical concepts to model temporal and spatial dependencies and highlight opportunities for adapting existing analysis methods to data with temporal and spatial dimensions.

FISHFactor: a probabilistic factor model for spatial transcriptomics data with subcellular resolution.

MOTIVATION: Factor analysis is a widely used tool for unsupervised dimensionality reduction of high-throughput datasets in molecular biology, with recently proposed extensions designed specifically for spatial transcriptomics data. However, these methods expect (count) matrices as data input and are therefore not directly applicable to single molecule resolution data, which are in the form of coordinate lists annotated with genes and provide insight into subcellular spatial expression patterns. To address this, we here propose FISHFactor, a probabilistic factor model that combines the benefits of spatial, non-negative factor analysis with a Poisson point process likelihood to explicitly model and account for the nature of single molecule resolution data. In addition, FISHFactor shares information across a potentially large number of cells in a common weight matrix, allowing consistent interpretation of factors across cells and yielding improved latent variable estimates. RESULTS: We compare FISHFactor to existing methods that rely on aggregating information through spatial binning and cannot combine information from multiple cells and show that our method leads to more accurate results on simulated data. We show that our method is scalable and can be readily applied to large datasets. Finally, we demonstrate on a real dataset that FISHFactor is able to identify major subcellular expression patterns and spatial gene clusters in a data-driven manner. AVAILABILITY AND IMPLEMENTATION: The model implementation, data simulation and experiment scripts are available under https://www.github.com/bioFAM/FISHFactor.

Spatial multiomics map of trophoblast development in early pregnancy.

The relationship between the human placenta-the extraembryonic organ made by the fetus, and the decidua-the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal-fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell-cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.

Identifying temporal and spatial patterns of variation from multimodal data using MEFISTO.

Factor analysis is a widely used method for dimensionality reduction in genome biology, with applications from personalized health to single-cell biology. Existing factor analysis models assume independence of the observed samples, an assumption that fails in spatio-temporal profiling studies. Here we present MEFISTO, a flexible and versatile toolbox for modeling high-dimensional data when spatial or temporal dependencies between the samples are known. MEFISTO maintains the established benefits of factor analysis for multimodal data, but enables the performance of spatio-temporally informed dimensionality reduction, interpolation, and separation of smooth from non-smooth patterns of variation. Moreover, MEFISTO can integrate multiple related datasets by simultaneously identifying and aligning the underlying patterns of variation in a data-driven manner. To illustrate MEFISTO, we apply the model to different datasets with spatial or temporal resolution, including an evolutionary atlas of organ development, a longitudinal microbiome study, a single-cell multi-omics atlas of mouse gastrulation and spatially resolved transcriptomics.

Adaptive penalization in high-dimensional regression and classification with external covariates using variational Bayes.

Penalization schemes like Lasso or ridge regression are routinely used to regress a response of interest on a high-dimensional set of potential predictors. Despite being decisive, the question of the relative strength of penalization is often glossed over and only implicitly determined by the scale of individual predictors. At the same time, additional information on the predictors is available in many applications but left unused. Here, we propose to make use of such external covariates to adapt the penalization in a data-driven manner. We present a method that differentially penalizes feature groups defined by the covariates and adapts the relative strength of penalization to the information content of each group. Using techniques from the Bayesian tool-set our procedure combines shrinkage with feature selection and provides a scalable optimization scheme. We demonstrate in simulations that the method accurately recovers the true effect sizes and sparsity patterns per feature group. Furthermore, it leads to an improved prediction performance in situations where the groups have strong differences in dynamic range. In applications to data from high-throughput biology, the method enables re-weighting the importance of feature groups from different assays. Overall, using available covariates extends the range of applications of penalized regression, improves model interpretability and can improve prediction performance.

Developmental Gene Expression Differences between Humans and Mammalian Models.

Identifying the molecular programs underlying human organ development and how they differ from model species is key for understanding human health and disease. Developmental gene expression profiles provide a window into the genes underlying organ development and a direct means to compare them across species. We use a transcriptomic resource covering the development of seven organs to characterize the temporal profiles of human genes associated with distinct disease classes and to determine, for each human gene, the similarity of its spatiotemporal expression with its orthologs in rhesus macaque, mouse, rat, and rabbit. We find clear associations between spatiotemporal profiles and the phenotypic manifestations of diseases. We also find that half of human genes differ from their mouse orthologs in their temporal trajectories in at least one of the organs. These include more than 200 genes associated with brain, heart, and liver disease for which mouse models should undergo extra scrutiny.

MOFA+: a statistical framework for comprehensive integration of multi-modal single-cell data.

Technological advances have enabled the profiling of multiple molecular layers at single-cell resolution, assaying cells from multiple samples or conditions. Consequently, there is a growing need for computational strategies to analyze data from complex experimental designs that include multiple data modalities and multiple groups of samples. We present Multi-Omics Factor Analysis v2 (MOFA+), a statistical framework for the comprehensive and scalable integration of single-cell multi-modal data. MOFA+ reconstructs a low-dimensional representation of the data using computationally efficient variational inference and supports flexible sparsity constraints, allowing to jointly model variation across multiple sample groups and data modalities.

Survey of ex vivo drug combination effects in chronic lymphocytic leukemia reveals synergistic drug effects and genetic dependencies.

Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by "omics" profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components (e.g., BTK, PI3K, SYK) had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target's position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations.

Mechanistic insights into transcription factor cooperativity and its impact on protein-phenotype interactions.

Recent high-throughput transcription factor (TF) binding assays revealed that TF cooperativity is a widespread phenomenon. However, a global mechanistic and functional understanding of TF cooperativity is still lacking. To address this, here we introduce a statistical learning framework that provides structural insight into TF cooperativity and its functional consequences based on next generation sequencing data. We identify DNA shape as driver for cooperativity, with a particularly strong effect for Forkhead-Ets pairs. Follow-up experiments reveal a local shape preference at the Ets-DNA-Forkhead interface and decreased cooperativity upon loss of the interaction. Additionally, we discover many functional associations for cooperatively bound TFs. Examination of the link between FOXO1:ETV6 and lymphomas reveals that their joint expression levels improve patient clinical outcome stratification. Altogether, our results demonstrate that inter-family cooperative TF binding is driven by position-specific DNA readout mechanisms, which provides an additional regulatory layer for downstream biological functions.

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.

Multi-Omics Factor Analysis-a framework for unsupervised integration of multi-omics data sets.

Multi-omics studies promise the improved characterization of biological processes across molecular layers. However, methods for the unsupervised integration of the resulting heterogeneous data sets are lacking. We present Multi-Omics Factor Analysis (MOFA), a computational method for discovering the principal sources of variation in multi-omics data sets. MOFA infers a set of (hidden) factors that capture biological and technical sources of variability. It disentangles axes of heterogeneity that are shared across multiple modalities and those specific to individual data modalities. The learnt factors enable a variety of downstream analyses, including identification of sample subgroups, data imputation and the detection of outlier samples. We applied MOFA to a cohort of 200 patient samples of chronic lymphocytic leukaemia, profiled for somatic mutations, RNA expression, DNA methylation and ex vivo drug responses. MOFA identified major dimensions of disease heterogeneity, including immunoglobulin heavy-chain variable region status, trisomy of chromosome 12 and previously underappreciated drivers, such as response to oxidative stress. In a second application, we used MOFA to analyse single-cell multi-omics data, identifying coordinated transcriptional and epigenetic changes along cell differentiation.

Drug-perturbation-based stratification of blood cancer.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.