Pancreas patch-seq provides a single-cell survey of function-transcriptome pairing in 1,369 islet cells from donors with and without diabetes • Expression of a specific subset of genes predicts b-cell electrophysiology in transcriptomefunction networks.• Compromised b-cell function in T2D correlates with altered ETV1 expression and inflammatory pathways• Functional heterogeneity in a-cells maps to ER stress and islet lineage markers• Application of patch-seq to cells from rare cryopreserved islets from donors with T1D SummaryPancreatic islet cells regulate glucose homeostasis through insulin and glucagon secretion; dysfunction of these cells leads to severe diseases like diabetes. Prior single-cell transcriptome studies have shown heterogeneous gene expression in major islet cell-types; however it remains challenging to reconcile this transcriptomic heterogeneity with observed islet cell functional variation. Here we achieved electrophysiological profiling and single-cell RNA sequencing in the same islet cell (pancreas patch-seq) thereby linking transcriptomic phenotypes to physiologic properties. We collected 1,369 cells from the pancreas of donors with or without diabetes and assessed function-gene expression networks. We identified a set of genes and pathways that drive functional heterogeneity in b-cells and used these to predict b-cell electrophysiology. We also report specific transcriptional programs that correlate with dysfunction in type 2 diabetes (T2D) and extend this approach to cryopreserved cells from donors with type 1 diabetes (T1D), generating a valuable resource for understanding islet cell heterogeneity in health and disease..

Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (23 full-term and 15 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.

Single-stranded DNA (ssDNA) plays a major role in several biological processes. It is therefore of fundamental interest to understand how the elastic response and the formation of secondary structures are modulated by the interplay between base pairing and electrostatic interactions. Here we measure force-extension curves (FECs) of ssDNA molecules in optical tweezers set up over two orders of magnitude of monovalent and divalent salt conditions, and obtain its elastic parameters by fitting the FECs to semiflexible models of polymers. For both monovalent and divalent salts, we find that the electrostatic contribution to the persistence length is proportional to the Debye screening length, varying as the inverse of the square root of cation concentration. The intrinsic persistence length is equal to 0.7 nm for both types of salts, and the effectivity of divalent cations in screening electrostatic interactions appears to be 100-fold as compared with monovalent salt, in line with what has been recently reported for single-stranded RNA. Finally, we propose an analysis of the FECs using a model that accounts for the effective thickness of the filament at low salt condition and a simple phenomenological description that quantifies the formation of non-specific secondary structure at low forces.

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

Temperature is a well-defined quantity for systems in equilibrium. For glassy systems, it has been extended to the non-equilibrium regime, showing up as an e ective quantity in a modified version of the fluctuation-dissipation theorem. However, experimental evidence supporting this definition remains scarce. Here, we present the first direct experimental demonstration of the e ective temperature by measuring correlations and responses in single molecules in non-equilibrium steady states generated under external random forces. We combine experiment, analytical theory and simulations for systems with di erent levels of complexity, ranging from a single bead in an optical trap to two-state and multiple-state DNA hairpins. From these data, we extract a unifying picture for the existence of an e ective temperature based on the relative order of various timescales characterizing intrinsic relaxation and external driving. Our study thus introduces driven small systems as a fertile ground to address fundamental concepts in statistical physics, condensed-matter physics and biophysics. F rom living cells to stars, natural processes occur under non-equilibrium conditions. Non-equilibrium systems have been classified, phenomenological patterns identified and theoretical frameworks developed, yet our current understanding of the fundamentals of the non-equilibrium problem still remains incomplete, undoubtedly far beyond what we know for equilibrium systems. Major advances in the field are the understanding of linear irreversible thermodynamics and, more recently, the development of fluctuation theorems 1-4 and macroscopic fluctuation theories 5,6 .Temperature, the measure of warmth or coldness of a system, is a genuine statistical concept. Related to erratic, agitated and unpredictable molecular motion, it is quantified through the average kinetic energy of molecules and ultimately grounded by the atomistic character of matter. Brownian motion, the shaken motion of the grains of pollen (diffusion) observed by Robert Brown in 1827, results from the bombardment of molecules experienced by the grains. The larger the average kinetic energy of such tiny colliding objects, the higher the temperature of the system. However, thermal forces are not the only way matter can be shaken, this can be also achieved by the slow release of accumulated stress in slowly relaxing systems such as structural glasses and granular media 7 , by injecting energy (for example, through external gradients) to steady-state systems 8 or through chemical reactions in self-propelled particles in active matter 9 . Can the erratic motion caused by such forces of non-thermal origin still be described in terms of the equilibriumbased concept of temperature? Spin-glass theories applied to systems exhibiting slow relaxation to equilibrium and ageing have persistently shown how the observed non-equilibrium behaviour can be described in terms of a non-equilibrium parameter, also called effective temperature. The effective temperature quantifies violations of the fluc...

We review the current knowledge on the use of single-molecule force spectroscopy techniques to extrapolate the elastic properties of nucleic acids. We emphasize the lesser-known elastic properties of single-stranded DNA. We discuss the importance of accurately determining the elastic response in pulling experiments, and we review the simplest models used to rationalize the experimental data as well as the experimental approaches used to pull single-stranded DNA. Applications used to investigate DNA conformational transitions and secondary structure formation are also highlighted. Finally, we provide an overview of the effects of salt and temperature and briefly discuss the effects of contour length and sequence dependence.

Maternal morbidity and mortality continue to rise, and pre-eclampsia is a major driver of this burden1. Yet the ability to assess underlying pathophysiology before clinical presentation to enable identification of pregnancies at risk remains elusive. Here we demonstrate the ability of plasma cell-free RNA (cfRNA) to reveal patterns of normal pregnancy progression and determine the risk of developing pre-eclampsia months before clinical presentation. Our results centre on comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data include 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks (s.d., 4.5 weeks) before delivery. We show that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal and fetal levels and can robustly predict pre-eclampsia, with a sensitivity of 75% and a positive predictive value of 32.3% (s.d., 3%), which is superior to the state-of-the-art method2. cfRNA signatures of normal pregnancy progression and pre-eclampsia are independent of clinical factors, such as maternal age, body mass index and race, which cumulatively account for less than 1% of model variance. Further, the cfRNA signature for pre-eclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of pre-eclampsia.

Our method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.