Calsequestrin Type 2 (CASQ2) is a high-capacity, low-affinity, Ca²⁺-binding protein expressed in the sarcoplasmic reticulum (SR) of the cardiac myocyte. Mutations in CASQ2 have been linked to the arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT2) that occurs with acute emotional stress or exercise can result in sudden cardiac death (SCD). CASQ2^G112+5X is a 16 bp (339–354) deletion CASQ2 mutation that prevents the protein expression due to premature stop codon. Understanding the subcellular mechanisms of CPVT2 is experimentally challenging because the occurrence of arrhythmia is rare. To obtain an insight into the characteristics of this rare disease, simulation studies using a local control stochastic computational model of the Guinea pig ventricular myocyte investigated how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of β-adrenergic stimulation or in the slowing of heart rate afterward once β-adrenergic stimulation ceases. Adjustment of the computational model parameters based upon recent experiments explore the functional changes caused by the CASQ2 mutation. In the simulation studies under rapid pacing (6 Hz), electromechanically concordant cellular alternans appeared under β-adrenergic stimulation in the CPVT mutant but not in the wild-type nor in the non-β-stimulated mutant. Similarly, the simulations of accelerating pacing from slow to rapid and back to the slow pacing did not display alternans but did generate early afterdepolarizations (EADs) during the period of second slow pacing subsequent acceleration of rapid pacing.

Introduction:Decision aids have been found to improve patients’ knowledge of treatments and decrease decisional regrets. Despite these benefits, there is not widespread use of decision aids...

This Research to Practice Full Paper presents our experience in teaching an introductory programming course in Python by using a visual programming development environment based on flow-charts and active learning with an interactive eBook. The field of computer science education is always being challenged with the high attrition rates despite the ever growing industry demand for computing expertise. The lower rate of student retention is often associated with considerable dropout and failure rates in introductory programming courses during the freshmen year. The main challenge is getting students to write meaningful programs in a short time by focusing more on computational thinking principles and less on language details at that point in time. Nowadays, most students prefer to be engaged and discover course content through exploration, interaction, and collaboration that is relevant, useful, and fun compared to traditional blackboard-based lecturing styles. For evaluation of learning outcomes based on the quantifiable criteria with robust statistical analysis, eleven sections of the course over three semesters were considered. The initial evaluation of summative assessment and analysis of a survey result enable us to conclude that the proposed instructional approach increases student engagement, facilitate learning and contribute to the progress of students in this course.

Mutations in the ryanodine receptor (RyR2) gene have been linked to arrhythmia and possibly sudden cardiac death (SCD) during acute emotional stress, physical activities, or catecholamine perfusion. The most prevalent disorder is catecholaminergic polymorphic ventricular tachycardia (CPVT1). Four primary mechanisms have been proposed to describe CPVT1 with a RyR2 mutation: (a) gain-of-function, (b) destabilization of binding proteins, (c) store-overload-induced Ca²⁺ release (SOICR), and (d) loss of function. The goal of this study was to use computational models to understand these four mechanisms and how they might contribute to arrhythmia. To this end, we have developed a local control stochastic model of a ventricular cardiac myocyte and used it to investigate how the Ca²⁺ dynamics in the mutant RyR2 are responsible for the development of an arrhythmogenic episode under the condition of β-adrenergic (β-AR) stimulation or pauses afterward. Into the model, we have incorporated 20,000 distinct cardiac dyads consisting of stochastically gated L-type Ca²⁺ channels (LCCs) and ryanodine receptors (RyR2s) and the intervening dyadic cleft to analyze the alterations in Ca²⁺ dynamics. Recent experimental findings were incorporated into the model parameters to test these proposed mechanisms and their role in triggering arrhythmias. The model could not find any connection between SOICR and the destabilization of binding proteins as the arrhythmic mechanisms in the mutant myocyte. On the other hand, the model was able to observe loss-of-function and gain-of-function mutations resulting in EADs (Early Afterdepolarizations) and variations in action potential amplitudes and durations as the precursors to generate arrhythmia, respectively. These computational studies demonstrate how GOF and LOF mutations can lead to arrhythmia and cast doubt on the feasibility of SOICR as a mechanism of arrhythmia.

Although antibody derivatives, such as Fabs and scFvs, have revolutionized the cellular imaging, quantification and tracking of proteins, analogous tools and strategies are unavailable for cellular RNA visualization. Here, we developed four synthetic anti-RNA scFv (sarabody) probes and their green fluorescent protein (GFP) fusions and demonstrated their potential to visualize RNA in live mammalian cells. We expressed these sarabodies and sarabody–GFP modules, purified them as soluble proteins, characterized their binding interactions with their corresponding epitopes and finally employed two of the four modules, sara1-GFP and sara1c-GFP, to visualize a target messenger RNA in live U2OS cells. Our current RNA imaging strategy is analogous to the existing MCP-MS2 system for RNA visualization, but additionally, our approach provides robust flexibility for developing target RNA-specific imaging modules, as epitope-specific probes can be selected from a library generated by diversifying the sarabody complementarity determining regions. While we continue to optimize these probes, develop new probes for various target RNAs and incorporate other fluorescence proteins like mCherry and HaloTag, our groundwork results demonstrated that these first-of-a-kind immunofluorescent probes will have tremendous potential for tracking mature RNAs and may aid in visualizing and quantifying many cellular processes as well as examining the spatiotemporal dynamics of various RNAs.

Calcium sparks are the elementary Ca²⁺ release events in excitation-contraction coupling that underlie the Ca²⁺ transient. The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca²⁺ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca²⁺]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca²⁺]_myo transients increase in pacing rates consistent with the experiments. The [Ca²⁺]_myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca²⁺]_sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca²⁺] transients. However, the local control model also suggests that the reduction of the L-type Ca²⁺ current, with an increase in pacing frequency due to Ca²⁺-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca²⁺ transient in the FFR correlated with the highest numbers of SR Ca²⁺ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca²⁺ sparks.