A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.

Bone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)1,2. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined3-11. Recent advances in single-cell RNA sequencing have revealed transcriptional differences among HSCs, providing a possible explanation for their functional heterogeneity12-17. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Analysis of differential gene expression between clones with distinct behaviour revealed an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor TCF15 to be required and sufficient to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programmes associated with functional stem cell heterogeneity and identifies a mechanism for the maintenance of the self-renewing HSC state.

Revealing evolutionary constraints on proteins through sequence analysis.

Statistical analysis of alignments of large numbers of protein sequences has revealed "sectors" of collectively coevolving amino acids in several protein families. Here, we show that selection acting on any functional property of a protein, represented by an additive trait, can give rise to such a sector. As an illustration of a selected trait, we consider the elastic energy of an important conformational change within an elastic network model, and we show that selection acting on this energy leads to correlations among residues. For this concrete example and more generally, we demonstrate that the main signature of functional sectors lies in the small-eigenvalue modes of the covariance matrix of the selected sequences. However, secondary signatures of these functional sectors also exist in the extensively-studied large-eigenvalue modes. Our simple, general model leads us to propose a principled method to identify functional sectors, along with the magnitudes of mutational effects, from sequence data. We further demonstrate the robustness of these functional sectors to various forms of selection, and the robustness of our approach to the identification of multiple selected traits.

Entropy Production of Nanosystems with Time Scale Separation.

Energy flows in biomolecular motors and machines are vital to their function. Yet experimental observations are often limited to a small subset of variables that participate in energy transport and dissipation. Here we show, through a solvable Langevin model, that the seemingly hidden entropy production is measurable through the violation spectrum of the fluctuation-response relation of a slow observable. For general Markov systems with time scale separation, we prove that the violation spectrum exhibits a characteristic plateau in the intermediate frequency region. Despite its vanishing height, the plateau can account for energy dissipation over a broad time scale. Our findings suggest a general possibility to probe hidden entropy production in nanosystems without direct observation of fast variables.

CoSpar identifies early cell fate biases from single-cell transcriptomic and lineage information.

A goal of single-cell genome-wide profiling is to reconstruct dynamic transitions during cell differentiation, disease onset and drug response. Single-cell assays have recently been integrated with lineage tracing, a set of methods that identify cells of common ancestry to establish bona fide dynamic relationships between cell states. These integrated methods have revealed unappreciated cell dynamics, but their analysis faces recurrent challenges arising from noisy, dispersed lineage data. In this study, we developed coherent, sparse optimization (CoSpar) as a robust computational approach to infer cell dynamics from single-cell transcriptomics integrated with lineage tracing. Built on assumptions of coherence and sparsity of transition maps, CoSpar is robust to severe downsampling and dispersion of lineage data, which enables simpler experimental designs and requires less calibration. In datasets representing hematopoiesis, reprogramming and directed differentiation, CoSpar identifies early fate biases not previously detected, predicting transcription factors and receptors implicated in fate choice. Documentation and detailed examples for common experimental designs are available at https://cospar.readthedocs.io/ .

Emergence of collective oscillations in adaptive cells.

Collective oscillations of cells in a population appear under diverse biological contexts. Here, we establish a set of common principles by categorising the response of individual cells against a time-varying signal. A positive intracellular signal relay of sufficient gain from participating cells is required to sustain the oscillations, together with phase matching. The two conditions yield quantitative predictions for the onset cell density and frequency in terms of measured single-cell and signal response functions. Through mathematical constructions, we show that cells that adapt to a constant stimulus fulfil the phase requirement by developing a leading phase in an active frequency window that enables cell-to-signal energy flow. Analysis of dynamical quorum sensing in several cellular systems with increasing biological complexity reaffirms the pivotal role of adaptation in powering oscillations in an otherwise dissipative cell-to-cell communication channel. The physical conditions identified also apply to synthetic oscillatory systems.

Inferring energy dissipation from violation of the fluctuation-dissipation theorem.

The Harada-Sasa equality elegantly connects the energy dissipation rate of a moving object with its measurable violation of the Fluctuation-Dissipation Theorem (FDT). Although proven for Langevin processes, its validity remains unclear for discrete Markov systems whose forward and backward transition rates respond asymmetrically to external perturbation. A typical example is a motor protein called kinesin. Here we show generally that the FDT violation persists surprisingly in the high-frequency limit due to the asymmetry, resulting in a divergent FDT violation integral and thus a complete breakdown of the Harada-Sasa equality. A renormalized FDT violation integral still well predicts the dissipation rate when each discrete transition produces a small entropy in the environment. Our study also suggests a way to infer this perturbation asymmetry based on the measurable high-frequency-limit FDT violation.

Factors Associated with Blood Culture Contamination in the Emergency Department: Critical Illness, End-Stage Renal Disease, and Old Age.

BACKGROUND: Blood culture contamination in emergency departments (ED) that experience a high volume of patients has negative impacts on optimal patient care. It is therefore important to identify risk factors associated with blood culture contamination in EDs. METHODOLOGY/PRINCIPAL FINDINGS: A prospectively observational study in a university-affiliated hospital were conducted between August 2011 and December 2012. Positive monomicrobial and negative blood cultures drawn from adult patients in the ED were analyzed to evaluate the possible risk factors for contamination. A total of 1,148 positive monomicrobial cases, 391 contamination cases, and 13,689 cases of negative blood culture were identified. Compared to patients with negative blood cultures, patients in triage levels 1 and 2 (Incidence Rate Ratio, IRR = 2.24), patients with end-stage renal disease (ESRD) (IRR = 2.05), and older patients (IRR: 1.02 per year) were more likely to be associated with ED blood culture contamination. CONCLUSIONS/SIGNIFICANCE: Critical patients (triage levels 1 and 2), ESRD patients, and older patients were more commonly associated with blood culture contamination in the ED. Further studies to evaluate whether the characteristics of skin commensals contribute to blood culture contamination is warranted, especially in hospitals populated with high-risk patients.

Rapid Human Immunodeficiency Virus Screening in an Emergency Department in a Region with Low HIV Seroprevalence.

Human immunodeficiency virus (HIV) tests are commonly performed in emergency departments (EDs) in the United States (US), but the experience and effectiveness of conducting rapid HIV tests in EDs in regions with low HIV seroprevalence outside the US have seldom been reported. An observational cross-sectional opt-in rapid HIV test and counseling program was conducted at an ED in a teaching hospital in Taiwan, a country with low seroprevalence, to determine the acceptance of rapid HIV tests as well as risky behaviors and illness presentations of people who agreed to undergo the tests. Among 7,645 ED patients between 20 and 55 years of age, 2,138 (28%) agreed to undergo rapid HIV tests, and only 2 (0.09%) tested positive. Patients diagnosed with urinary tract infections, respiratory tract infections, infectious diarrhea, and pelvic inflammatory disease were more likely to be willing to undergo rapid HIV tests in the ED. Stratified analysis revealed that sexually active patients were more likely to consent to HIV testing. Therefore, non-targeted opt-in HIV testing and counseling in the ED was feasible but was not effective in a region with low HIV seroprevalence.