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In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 106 cells, and analyzed their characteristics using transcriptomics. A comparison between clonal cultures revealed restriction to either proximal or distal kidney sub-lineages with distinct cellular and molecular characteristics; rapidly amplifying de-differentiated clones and a stably proliferating cuboidal epithelial-appearing clones, respectively. Furthermore, each showed distinct molecular features including cell-cycle, epithelial-mesenchymal transition, oxidative phosphorylation, BMP signaling pathway and cell surface markers. In addition, analysis of clonal versus bulk cultures show early clones to be more quiescent, with elevated expression of renal developmental genes and overall reduction in renal identity markers, but with an overlapping expression of nephron segment identifiers and multiple identity. Thus, ex-vivo clonal growth mimics the in-vivo situation displaying lineage-restricted precursor characteristics of mature renal cells. These data suggest that for reconstruction of varying renal lineages with human adult kidney based organoid technology and kidney regeneration ex-vivo, use of multiple heterogeneous precursors is warranted.

ScenarioTools is an Eclipse-based tool suite for the scenario-based modeling and analysis of reactive systems. ScenarioTools especially targets the modeling and analysis of systems where the behavior of the components is sensitive to changes in the component structure that can occur at run-time. For example, in a system of communicating cars, the cars' relationships can change due to their movement and influence how cars must interact. The modeling in ScenarioTools is based on the Scenario Modeling Language (SML), an extended variant of Live Sequence Charts (LSCs). For modeling structural changes and conditions, graph transformation rules can be combined with SML. The specifications are executable and can be analyzed by simulation. ScenarioTools further supports a formal synthesis procedure that can find specification inconsistencies or prove the specification's realizability. In this article, we illustrate the features of ScenarioTools by an example and describe its architecture.

The authors present an initial wise development framework: a development environment that proactively and interactively assists the software engineer in modeling complex reactive systems. Their framework repeatedly analyzes models of the system under development at various levels of abstraction, and then reasons about these models in order to detect possible errors, to derive emergent properties of interest, and to assist in system testing and debugging. Upon request, the environment can instrument the system model in order to monitor or test the execution for certain behaviors, or even augment it in order to repair or avoid detected behavior that is undesired. The direction and prioritization of the analysis and related tasks is based on the relevance of the observed properties and the expected impact of actions to be taken, and is performed by specialized automated and human-assisted techniques that have been incorporated into the framework. The authors' development environment is an initial step in the direction of their recent Wise Computing vision, which calls for turning the computer (namely, the development environment) into an equal member of the development team: knowledgeable, independent, concerned and proactively involved in the development process. They have implemented their tool within the context of behavioral programming (BP) – a scenario-based modeling approach, in which components are aligned with how humans often describe desired system behavior. The authors' work thus further enhances the naturalness and incrementality of developing in BP.

Background Spontaneous calcium release evoking delayed afterdepolarization is believed to cause catecholaminergic polymorphic ventricular tachycardia (CPVT), a lethal human arrhythmia provoked by exercise or emotional stress. β-Adrenergic blockers are the drug of choice, but fail to achieve complete arrhythmia control in some patients. These individuals often require flecainide, device implantation, and/or sympathetic denervation. Objective To optimize the arrhythmia therapy by pharmacological inhibition of the sympathetic nervous system in the homozygous calsequestrin knockout (CASQ2 Δ/Δ ) mouse model of CPVT2. Methods A heart telemetry device was implanted for continuous electrocardiographic recording at rest and during provocation testing. Calcium transients and abnormal calcium release were studied in cardiomyocytes isolated from adult mice. Adrenergic receptor expression was determined by using Western blotting and confocal microscopy. Results Adult CASQ2 Δ/Δ mice suffer from complex ventricular arrhythmia at rest and ventricular tachycardia during treadmill exercise and after epinephrine injection. β-Adrenergic blockers, propranolol and metoprolol, attenuated arrhythmia at rest but not after stress. Reserpine had no efficacy in controlling arrhythmia. Agents with α-blocking activity, phentolamine or labetalol, abolished both exercise- and epinephrine-induced arrhythmia. In contrast, injection of α-adrenergic agonist phenylephrine reproducibly provoked ventricular tachycardia. Isolated cardiomyocytes from CASQ2 Δ/Δ mice had delayed calcium release waves upon exposure to sympathetic agonists, which were abolished by phentolamine. Hearts of calsequestrin-mutant mice expressed more α1-adrenergic receptor than did wild type control mice ( P Conclusion We identified a contribution of the α-adrenergic pathway to the pathogenesis of catecholamine-induced arrhythmia. α-Blockade emerges as an effective therapy in the murine model of CPVT2 and should be tried in humans resistant to β-blockers.

As part of expanding the implementation and use of the behavioral programming (BP) approach in a variety of languages and configurations, we tackle some of the challenges associated with applying the approach in a truly distributed, decentralized manner, where different modules run on separate machines. BP supports the development of reactive applications from modules that are aligned with the desired and undesired scenarios of system behaviors as described, say, in a requirements document, in an enhancement request, or a field problem report. A key advantage of this approach is that it facilitates incremental development where loosely coupled modules are added as requirements are introduced, and meaningful prototype execution can be carried out from early stages of development. In BP, each behavioral module (called a behavior thread) takes care of a separate facet of the requirements, thus control is conceptually decentralized. However, as the underlying principles of BP call for constant synchronization of, and "consultation" with, all behavior threads, efficient implementation in a physically distributed environment is a significant challenge on the road to broader acceptance of BP as a viable new way to develop systems. We begin by describing an implementation of BP in Erlang, where the coordination protocol is implemented via message passing. We demonstrate through examples how developing distributed systems in Erlang can benefit from BP advantages of incremental development and alignment of the code modules with the requirements. Next, we propose general BP design patterns (not limited to Erlang) for using BP in distributed applications, showing how to design applications using BP without forcing full synchronization among all threads at each step of the execution. This allows modules to run at different time scales and to wait for external input without stalling the entire system. Finally, we propose ways to alter the execution mechanism of BP so that execution can progress without necessarily waiting for the synchronization of all the threads. The enhanced execution algorithm has the potential of accelerating the distributed execution of behavioral programs. We present a design pattern for programming behaviorally in Erlang.We present a proof-of-concept for a BP execution infrastructure in Erlang.We present BP design patterns for interfacing with unsynchronized environments.We present BP design patterns for coordinating behaviors at different time scales.We present mechanisms for eager execution in BP.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a primary electrical myocardial disease characterized by exercise- and stress-related ventricular tachycardia manifested as syncope and sudden death. The disease has a heterogeneous genetic basis, with mutations in the cardiac Ryanodine Receptor channel (RyR2) gene accounting for an autosomal-dominant form (CPVT1) in approximately 50% and mutations in the cardiac calsequestrin gene (CASQ2) accounting for an autosomal-recessive form (CPVT2) in up to 2% of CPVT cases. Both RyR2 and calsequestrin are important participants in the cardiac cellular calcium homeostasis. We review the physiology of the cardiac calcium homeostasis, including the cardiac excitation contraction coupling and myocyte calcium cycling. The pathophysiology of cardiac arrhythmias related to myocyte calcium handling and the effects of different modulators are discussed. The putative derangements in myocyte calcium homeostasis responsible for CPVT, as well as the clinical manifestations and therapeutic options available, are described.