Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells.

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.

Do 'difficult to identify' pulmonary vein connections explain continuing high recurrence rates after atrial fibrillation ablation?

Despite the emerging technical evolution of the last two decades, the primary success rate of single-procedure pulmonary vein isolation (PVI), the cornerstone for any atrial fibrillation ablation procedure, is highly variable ranging from 53% to 92%. The recent development of ultra-high-density electroanatomic mapping systems, capable of acquiring and annotating multiple electrograms, with high spatiotemporal precision, which are processed by automated algorithms to generate activation and substrate maps to support and guide ablation procedures, has opened a new stage in cardiac electrophysiology. In this article, we review the existing evidence on the utility of high-density mapping on catheter-based PVI, the possibility to detect pulmonary vein potentials that remain undetected when using a standard approach and its potential relevance to the clinical outcome, and how this new technology is providing novel pathophysiological insights on complete PVI and atrial fibrillation ablation outcomes.

Identification of pulmonary vein reconnection gaps with high-density mapping in redo atrial fibrillation ablation procedures.

Aims: Maps obtained by means of electroanatomic high-density mapping (HDM) systems have shown their use in the identification of conduction gaps in experimental atrial linear lesion models. The objective of this study was to assess the use of HDM in the recognition of reconnection gaps in pulmonary veins (PV) in redo atrial fibrillation (AF) ablation procedures. Methods and results: One hundred and eight patients were included in a non-randomized study that assessed the recognition of reconnection gaps in PV by means of HDM compared to a control group that received conventional non-fluoroscopic guidance with a circular multipolar catheter (CMC). Among the HDM group, adequate recognition of reconnection gaps was obtained in 60.99% of the reconnected PVs (86 of 141), a figure significantly higher than that achieved with analysis of CMC recorded signals (39.66%, 48 of 121; P = 0.001). The number of applications and total radiofrequency time were also significantly lower in the HDM group (12.46 ± 6.1 vs. 15.63 ± 7.7 and 7.61 ± 3 vs. 9.29 ± 5; P = 0.02, and P = 0.03, respectively). At the 6-month follow-up, no statistically significant differences were found in recurrence of AF or any other atrial tachycardia between the HDM group (8 patients, 14.8%) and the control group in (16 patients, 29.6%; P = 0.104). Conclusion: An analysis of the high-density activation maps allows greater precision in the identification of reconnection gaps in PV, which results in lower radiofrequency time for the new isolation.

Atrial Fibrillation Ablation Guided by a Novel Nonfluoroscopic Navigation System.

INTRODUCTION AND OBJECTIVES: Rhythmia is a new nonfluoroscopic navigation system that is able to create high-density electroanatomic maps. The aim of this study was to describe the acute outcomes of atrial fibrillation (AF) ablation guided by this system, to analyze the volume provided by its electroanatomic map, and to describe its ability to locate pulmonary vein (PV) reconnection gaps in redo procedures. METHODS: This observational study included 62 patients who underwent AF ablation with Rhythmia compared with a retrospective cohort who underwent AF ablation with a conventional nonfluoroscopic navigation system (Ensite Velocity). RESULTS: The number of surface electrograms per map was significantly higher in Rhythmia procedures (12 125 ± 2826 vs 133 ± 21 with Velocity; P < .001), with no significant differences in the total procedure time. The Orion catheter was placed for mapping in 99.5% of PV (95.61% in the control group with a conventional circular mapping catheter; P = .04). There were no significant differences in the percentage of PV isolation between the 2 groups. In redo procedures, an ablation gap could be identified on the activation map in 67% of the reconnected PV (40% in the control group; P = .042). The measured left atrial volume was lower than that calculated by computed tomography (109.3 v 15.2 and 129.9 ± 13.2 mL, respectively; P < .001). There were no significant differences in the number of complications. CONCLUSIONS: The Rhythmia system is effective for AF ablation procedures, with procedure times and safety profiles similar to conventional nonfluoroscopic navigation systems. In redo procedures, it appears to be more effective in identifying reconnected PV conduction gaps.

Cooperativity to increase Turing pattern space for synthetic biology.

It is hard to bridge the gap between mathematical formulations and biological implementations of Turing patterns, yet this is necessary for both understanding and engineering these networks with synthetic biology approaches. Here, we model a reaction-diffusion system with two morphogens in a monostable regime, inspired by components that we recently described in a synthetic biology study in mammalian cells.1 The model employs a single promoter to express both the activator and inhibitor genes and produces Turing patterns over large regions of parameter space, using biologically interpretable Hill function reactions. We applied a stability analysis and identified rules for choosing biologically tunable parameter relationships to increase the likelihood of successful patterning. We show how to control Turing pattern sizes and time evolution by manipulating the values for production and degradation relationships. More importantly, our analysis predicts that steep dose-response functions arising from cooperativity are mandatory for Turing patterns. Greater steepness increases parameter space and even reduces the requirement for differential diffusion between activator and inhibitor. These results demonstrate some of the limitations of linear scenarios for reaction-diffusion systems and will help to guide projects to engineer synthetic Turing patterns.

Sender-receiver systems and applying information theory for quantitative synthetic biology.

Sender-receiver (S-R) systems abound in biology, with communication systems sending information in various forms. Information theory provides a quantitative basis for analysing these processes and is being applied to study natural genetic, enzymatic and neural networks. Recent advances in synthetic biology are providing us with a wealth of artificial S-R systems, giving us quantitative control over networks with a finite number of well-characterised components. Combining the two approaches can help to predict how to maximise signalling robustness, and will allow us to make increasingly complex biological computers. Ultimately, pushing the boundaries of synthetic biology will require moving beyond engineering the flow of information and towards building more sophisticated circuits that interpret biological meaning.

Genetically encoded sender-receiver system in 3D mammalian cell culture.

Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin-Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell-cell communication networks in 3D cell culture.