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Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca2+ signals of low amplitude. Subsequently, also global Ca2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic. Upon stimulation of receptors, NAADP is formed as calcium-mobilizing second messenger in many cells. Here, the authors develop MASTER-NAADP, a membrane-permeant precursor of NAADP, and characterize its biological activity. [ABSTRACT FROM AUTHOR]

Ca2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatiotemporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community. [ABSTRACT FROM AUTHOR]

Local, sub-second Ca 2+ signals, termed Ca 2+ microdomains, are highly dynamic and short-lived Ca 2+ signals, which result in a global [Ca 2+ ]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca 2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca 2+ ]i. To capture these fast and dynamically occurring Ca 2+ signals, we developed a high-resolution imaging technique using a combination of two Ca 2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca 2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca 2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

Ca 2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca 2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca 2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca 2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca 2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatio-temporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca 2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community. Code available at: 10.5281/zenodo.10459243., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GD declared a past co-authorship with one of the authors B-PD, AG to the handling editor., (Copyright © 2024 Woelk, Kovacevic, Husseini, Förster, Gerlach, Möckl, Altfeld, Guse, Diercks and Werner.)

Objective: Adjusting pacemaker pulse amplitude influences the longevity of the pacemaker. Our aim was to establish the initial longevity gain., Methods: Forty randomly selected patients with implanted pacemakers were analyzed. Mean age was 65.58+/-13.7 years. All pacemakers were working on factory settings of pulse amplitude 3.5 V and pulse width of 0.4 ms for average of 3 years before the adjustment. Initial mean longevity was projected to 68.61+/-18.86 months, mean battery voltage 2.78 V, and mean battery current 14.21+/-2.61 microA., Results: Pulse amplitude threshold test was performed and average value of 0.632+/-0.22 V was obtained. Pulse amplitude was programmed to 2.5 V and pulse width was left unchanged. New readings of battery data were obtained. Battery voltage did not show immediate changes, and battery current decreased to 11.53+/-1.98 microA. New average longevity was projected to 81.03+/-19.82 months, which presents a 12.42 months of initial longevity gain with statistical significance at 95% confidence interval (p=0.003). Positive correlation was found between the new pulse amplitude and new values of battery current (p<0.01)., Conclusion: Pulse amplitude decrease of only 1 V provides significant initial longevity gain of more than a year. If found correlations would have any impact on further longevity gains over longer period of time is yet to be established.

Atrio-ventricular node reentry (AVNRT) is typically induced with an anterograde block over the fast pathway (FP) and conduction over the slow pathway (SP), with subsequent retrograde conduction over the FP. Rarely, a premature atrial complex (PAC) conducts simultaneously over the FP and SP to induce AVNRT. Previous publications have reported that conduction over the fast and slow pathway of the atrioventricular node can occur successively one after the other, thus leading to dual ventricular depolarization from what initially was a single atrial impulse. We report a case of an 18-year-old male patient referred for repeated bursts of ectopic activity. Evaluation of the patient's electrocardiographic recordings suggested the presence of dual ventricular activations for each atrial beat. The electrophysiological study revealed that the patient had simultaneous conduction over the fast and slow pathways of the atrioventricular node giving rise to a non-reentrant tachycardia, along with an absence of retrograde (ventriculoatrial) conduction, and a significant atrio-His bundle jump (A-H jump) through the slow pathway from the fast pathway during programmed electrical stimulation from the right atrium. Ablation of the slow pathway at the base of the Koch triangle yielded a cessation of the dual ventricular response, absence of the nonreentrant tachycardia and no A-H jump.