Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

Joint probabilistic modeling of paired transcriptome and proteome measurements in single cells

The paired measurement of RNA and surface protein abundance in single cells with CITE-seq is a promising approach to connect transcriptional variation with cell phenotypes and functions. However, each data modality exhibits unique technical biases, making it challenging to conduct a joint analysis and combine these two views into a unified representation of cell state. Here we present Total Variational Inference (totalVI), a framework for the joint probabilistic analysis of paired RNA and protein data from single cells. totalVI probabilistically represents the data as a composite of biological and technical factors such as limited sensitivity of the RNA data, background in the protein data, and batch effects. To evaluate totalVI, we performed CITE-seq on immune cells from murine spleen and lymph nodes with biological replicates and with different antibody panels measuring over 100 surface proteins. With this dataset, we demonstrate that totalVI provides a cohesive solution for common analysis tasks like the integration of datasets with matched or unmatched protein panels, dimensionality reduction, clustering, evaluation of correlations between molecules, and differential expression testing. totalVI enables scalable, end-to-end analysis of paired RNA and protein data from single cells and is available as open-source software.

Detection and quantification of left-to-right shunting using transpulmonary ultrasound dilution (TPUD): a validation study in neonatal lambs

Objectives:We investigated the accuracy of left-to-right shunt detection using transpulmonary ultrasound dilution (TPUD) and compared the agreement between pulmonary over systemic blood flow (Qp/Qs) ratio measured by TPUD [Qp/Qs(tpud)] and ultrasonic flow probes [Qp/Qs(ufp)].Methods:Seven newborn lambs under general anesthesia were connected to the TPUD monitor (COstatus™) after insertion of arterial and central venous catheters. A Gore-TexResults:Seventy-two paired measurement sessions were analyzed. Shunts were detected by TPUD with a positive predictive value of 86%, a negative predictive value of 100%, a sensitivity of 100% and a specificity of 83%. The Bland-Altman analysis comparing Qp/Qs(tpud) and Qp/Qs(ufp) showed an overall mean bias (SD) of 0.1 (0.3), limits of agreement (LOA) of ±0.6 and a percentage error of 34.8%.Conclusions:The qualitative diagnostic accuracy of TPUD for shunt detection is high. Modification of the algorithm seems required as shunt quantification by TPUD is accurate, but not yet very precise.

Analysis of bidirectional pattern synchrony of concentration-secretion pairs: implementation in the human testicular and adrenal axes

The hypothalamo-pituitary-testicular and hypothalamo-pituitary-adrenal axes are prototypical coupled neuroendocrine systems. In the present study, we contrasted in vivo linkages within and between these two axes using methods without linearity assumptions. We examined 11 young (21–31 yr) and 8 older (62–74 yr) men who underwent frequent (every 2.5 min) blood sampling overnight for paired measurement of LH and testosterone and 35 adults (17 women and 18 men; 26–77 yr old) who underwent adrenocorticotropic hormone (ACTH) and cortisol measurements every 10 min for 24 h. To mirror physiological interactions, hormone secretion was first deconvolved from serial concentrations with a waveform-independent biexponential elimination model. Feedforward synchrony, feedback synchrony, and the difference in feedforward-feedback synchrony were quantified by the cross-approximate entropy (X-ApEn) statistic. These were applied in a forward (LH concentration template, examining pattern recurrence in testosterone secretion), reverse (testosterone concentration template, examining pattern recurrence in LH secretion), and differential (forward minus reverse) manner, respectively. Analogous concentration-secretion X-ApEn estimates were calculated from ACTH-cortisol pairs. X-ApEn, a scale- and model-independent measure of pattern reproducibility, disclosed 1) greater testosterone-LH feedback coordination than LH-testosterone feedforward synchrony in healthy men and significant and symmetric erosion of both feedforward and feedback linkages with aging; 2) more synchronous ACTH concentration-dependent feedforward than feedback drive of cortisol secretion, independent of gender and age; and 3) enhanced detection of bidirectional physiological regulation by in vivo pairwise concentration-secretion compared with concentration-concentration analyses. The linking of relevant biological input to output signals and vice versa should be useful in the dissection of the reciprocal control of neuroendocrine systems or even in the analysis of other nonendocrine networks.

Evaluation of oestrus observation and conception rates in suckling beef cows using whole milk progesterone concentration

A 2-sample regime was used to measure whole milk progesterone concentration on the day of oestrus and insemination (Day 0) and 6 days later (Day 6) in a sample of 50 primiparous and 100 multiparous suckling beef cows. Exposure to teaser bulls and observation by cattlemen identified the occurrence of oestrus. Three sets of criteria used to define ovulatory oestrus were compared : a) milk progesterone concentration less than 6 nmol / l on Day 0 ; b) milk progesterone less than 6 nmol / l on Day 0 and rising to greater than 6 nmol / l on Day 6; c) milk progesterone less than 6 nmol / l on Day 0 and rising to greater than 6 nmol / l on Day 6, or cow diagnosed pregnant to 1st insemination. Using only a single milk sample on Day 0 (criterion a) would have resulted in the positive predictive value of heat detection being estimated at 98.7%. Using a paired measurement (criterion b) resulted in a significantly lower estimate of 84.7%. The inclusion of cows that conceived despite not showing a marked rise in milk progesterone concentration (criterion c) resulted in a more accurate estimate of 89.3%. Use of a 2-sample regime also allowed calculation of conception rates while eliminating the effect of heat detection errors. In the cows sampled, of those in ovulatory oestrus that were inseminated, 73.1% conceived to the 1st insemination. These results demonstrate that artificial insemination within a limited breeding season can be successful if nutrition is optimal and management is intensive. The use of a 2-sample milk progesterone test may be a valuable tool in investigating heat detection and conception problems in beef herds in which artificial insemination is used.

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption.

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.