1. Integrating live cell calcium imaging and tissue damage assessment in a novel model of acute pancreatitisPolona Kovačič, Maša Skelin, Eva Paradiž, Viktória Venglovecz, Loránd Kiss, Gabriella Mihalekné Fűr, Andraž Stožer, Jurij Dolenšek, 2025, objavljeni povzetek znanstvenega prispevka na konferenci Ključne besede: acute pancreatitis, calcium imaging, LiveDead assay, pancreatic tissue slices, histological analysis Objavljeno v DKUM: 31.03.2025; Ogledov: 0; Prenosov: 13
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2. Functional coatings with ethyl cellulose-calcium carbonate alkaline nanoparticles for deacidification and mechanical reinforcement of paper artifactsMatej Bračič, Jasna Malešič, Mihael Brunčko, Doris Bračič, Alenka Ojstršek, Tea Kapun, Sašo Gyergyek, Karin Stana-Kleinschek, Tamilselvan Mohan, 2025, izvirni znanstveni članek Opis: Paper artifacts susceptible to acid hydrolysis and mechanical stress require effective conservation methods to ensure their longevity. In this study, a novel approach for the deacidification of acidic paper using calcium carbonate (CaCO3) [1,2]-ethylcellulose nanoparticles (CaCO3-EC NPs) dispersed in a non-aqueous ethyl acetate solution is presented. The dispersions were carefully prepared and applied to model acidic paper samples using a dipcoating method and then analyzed for their effectiveness. Transmission electron microscopy showed the formation of agglomerates containing quadrangular alkaline nanoparticles with diameters of 40 to 100 nm and a total agglomerate size of 250 nm. Hydrodynamic analyzes indicate the presence of a swollen ethyl cellulose coating on these agglomerates, which facilitates their dispersion. The results show the effectiveness of the CaCO3-EC NPs system in neutralizing acidic components (change of paper pH from 4.3 to 7) due to the homogeneous distribution within the paper substrates, effectively arresting the degradation processes. Acid-base titration showed a linear correlation between the concentration of alkaline nanoparticles and the alkaline reserve, emphasizing the role of ethylcellulose in facilitating particle transport within the paper matrix. In addition, ethylcellulose was found to improve the mechanical properties of the treated paper, as demonstrated by the standard mechanical tests. Importantly, the optical properties remained unchanged after treatment, as no adverse changes in color were observed. These results underline the effectiveness of the developed deacidification dispersions for the treatment of acidic paper and potentially other cellulose-based cultural heritage documents prone to acidic degradation. This approach offers promising implications for preserving and restoring valuable historical materials. Ključne besede: ethylcellulose, calcium carbonate, functional coating, deacidification, strengthening, cultural heritage Objavljeno v DKUM: 20.03.2025; Ogledov: 0; Prenosov: 1
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3. Cataract progression associated with modifications in calcium signaling in human lens epithelia as studied by mechanical stimulationMarko Gosak, Dajana Gojić, Elena Spasovska, Marko Hawlina, Sofija Andjelić, 2021, izvirni znanstveni članek Ključne besede: human eye lens, lens epithelial cells, calcium signaling, mechanical stimulation, mechanical stimulation, calcium waves, paracrine signaling Objavljeno v DKUM: 14.02.2025; Ogledov: 0; Prenosov: 2
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4. Ultrafast multicellular calcium imaging of calcium spikes in mouse beta cells in tissue slicesJurij Dolenšek, Viljem Pohorec, Maša Skelin, Marko Gosak, Andraž Stožer, 2025, izvirni znanstveni članek Opis: Background: The crucial steps in beta cell stimulus-secretion coupling upon stimulation with glucose are oscillatory changes in metabolism, membrane potential, intracellular calcium concentration, and exocytosis. The changes in membrane potential consist of bursts of spikes, with silent phases between them being dominated by membrane repolarization and absence of spikes. Assessing intra- and intercellular coupling at the multicellular level is possible with ever-increasing detail, but our current ability to simultaneously resolve spikes from many beta cells remains limited to double-impalement electrophysiological recordings. Methods: Since multicellular calcium imaging of spikes would enable a better understanding of coupling between changes in membrane potential and calcium concentration in beta cell collectives, we set out to design an appropriate methodological approach. Results: Combining the acute tissue slice method with ultrafast calcium imaging, we were able to resolve and quantify individual spikes within bursts at a temporal resolution of >150 Hz over prolonged periods, as well as describe their glucose-dependent properties. In addition, by simultaneous patch-clamp recordings we were able to show that calcium spikes closely follow membrane potential changes. Both bursts and spikes coordinate across islets in the form of intercellular waves, with bursts typically displaying global and spikes more local patterns. Conclusions: This method and the associated findings provide additional insight into the complex signaling within beta cell networks. Once extended to tissue from diabetic animals and human donors, this approach could help us better understand the mechanistic basis of diabetes and find new molecular targets. Ključne besede: beta cell, calcium imaging, calcium oscillations, calcium spikes, physiology Objavljeno v DKUM: 24.01.2025; Ogledov: 0; Prenosov: 7
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5. From isles of Königsberg to islets of Langerhans: examining the function of the endocrine pancreas through network scienceAndraž Stožer, Marko Šterk, Eva Paradiž, Rene Markovič, Maša Skelin, Cara E. Ellis, Lidija Križančić Bombek, Jurij Dolenšek, Patrick E. MacDonald, Marko Gosak, 2022, pregledni znanstveni članek Opis: Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function. Electrical coupling between the insulin-secreting beta cells through gap junctions composed of connexin36 is particularly important, as it provides the required, most important, basis for coordinated responses of the beta cell population. The increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. In the last decade, several novel techniques have been proposed to assess cooperation between cells in islets, including the prosperous combination of multicellular imaging and network science. In the present contribution, we review recent advances related to the application of complex network approaches to uncover the functional connectivity patterns among cells within the islets. We first provide an accessible introduction to the basic principles of network theory, enumerating the measures characterizing the intercellular interactions and quantifying the functional integration and segregation of a multicellular system. Then we describe methodological approaches to construct functional beta cell networks, point out possible pitfalls, and specify the functional implications of beta cell network examinations. We continue by highlighting the recent findings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. Finally, we emphasize that the combination of these imaging techniques and network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus. Ključne besede: pancreatic islets, beta cells, calcium imaging, intercellular communication, functional networks, multilayer networks Objavljeno v DKUM: 20.12.2024; Ogledov: 0; Prenosov: 88
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6. Both electrical and metabolic coupling shape the collective multimodal activity and functional connectivity patterns in beta cell collectives : a computational model perspectiveMarko Šterk, Uroš Barać, Andraž Stožer, Marko Gosak, 2023, izvirni znanstveni članek Opis: Pancreatic beta cells are coupled excitable oscillators that synchronize their activity via different communication pathways. Their oscillatory activity manifests itself on multiple timescales and consists of bursting electrical activity, subsequent oscillations in the intracellular Ca 2 + , as well as oscillations in metabolism and exocytosis. The coordination of the intricate activity on the multicellular level plays a key role in the regulation of physiological pulsatile insulin secretion and is incompletely understood. In this paper, we investigate theoretically the principles that give rise to the synchronized activity of beta cell populations by building up a phenomenological multicellular model that incorporates the basic features of beta cell dynamics. Specifically, the model is composed of coupled slow and fast oscillatory units that reflect metabolic processes and electrical activity, respectively. Using a realistic description of the intercellular interactions, we study how the combination of electrical and metabolic coupling generates collective rhythmicity and shapes functional beta cell networks. It turns out that while electrical coupling solely can synchronize the responses, the addition of metabolic interactions further enhances coordination, the spatial range of interactions increases the number of connections in the functional beta cell networks, and ensures a better consistency with experimental findings. Moreover, our computational results provide additional insights into the relationship between beta cell heterogeneity, their activity profiles, and functional connectivity, supplementing thereby recent experimental results on endocrine networks. Ključne besede: pancreatic, beta cells, oscilators, calcium signaling, cells signaling Objavljeno v DKUM: 10.12.2024; Ogledov: 0; Prenosov: 5
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7. Network representation of multicellular activity in pancreatic islets : Technical considerations for functional connectivity analysisMarko Šterk, Yaowen Zhang, Viljem Pohorec, Eva Paradiž, Jurij Dolenšek, Richard K. P. Benninger, Andraž Stožer, Vira Kravets, Marko Gosak, 2024, izvirni znanstveni članek Opis: Within the islets of Langerhans, beta cells orchestrate synchronized insulin secretion, a pivotal aspect of metabolic homeostasis. Despite the inherent heterogeneity and multimodal activity of individual cells, intercellular coupling acts as a homogenizing force, enabling coordinated responses through the propagation of intercellular waves. Disruptions in this coordination are implicated in irregular insulin secretion, a hallmark of diabetes. Recently, innovative approaches, such as integrating multicellular calcium imaging with network analysis, have emerged for a quantitative assessment of the cellular activity in islets. However, different groups use distinct experimental preparations, microscopic techniques, apply different methods to process the measured signals and use various methods to derive functional connectivity patterns. This makes comparisons between findings and their integration into a bigger picture difficult and has led to disputes in functional connectivity interpretations. To address these issues, we present here a systematic analysis of how different approaches influence the network representation of islet activity. Our findings show that the choice of methods used to construct networks is not crucial, although care is needed when combining data from different islets. Conversely, the conclusions drawn from network analysis can be heavily affected by the pre-processing of the time series, the type of the oscillatory component in the signals, and by the experimental preparation. Our tutorial-like investigation aims to resolve interpretational issues, reconcile conflicting views, advance functional implications, and encourage researchers to adopt connectivity analysis. As we conclude, we outline challenges for future research, emphasizing the broader applicability of our conclusions to other tissues exhibiting complex multicellular dynamics. Ključne besede: islets of Langerhans, beta cells, calcium signaling, intercellular communication, functional networks, myosin model Objavljeno v DKUM: 09.12.2024; Ogledov: 0; Prenosov: 4
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8. Glucose-dependent activation, activity, and deactivation of beta cell networks in acute mouse pancreas tissue slicesAndraž Stožer, Maša Skelin, Marko Gosak, Lidija Križančić Bombek, Viljem Pohorec, Marjan Rupnik, Jurij Dolenšek, 2021, izvirni znanstveni članek Opis: Many details of glucose-stimulated intracellular calcium changes in [beta] cells during activation, activity, and deactivation, as well as their concentration-dependence, remain to be analyzed. Classical physiological experiments indicated that in islets, functional differences between individual cells are largely attenuated, but recent findings suggest considerable intercellular heterogeneity, with some cells possibly coordinating the collective responses. To address the above with an emphasis on heterogeneity and describing the relations between classical physiological and functional network properties, we performed functional multicellular calcium imaging in mouse pancreas tissue slices over a wide range of glucose concentrations. During activation, delays to activation of cells and any-cell-to-first-responder delays are shortened, and the sizes of simultaneously responding clusters increased with increasing glucose concentrations. Exactly the opposite characterized deactivation. The frequency of fast calcium oscillations during activity increased with increasing glucose up to 12 mM glucose concentration, beyond which oscillation duration became longer, resulting in a homogenous increase in active time. In terms of functional connectivity, islets progressed from a very segregated network to a single large functional unit with increasing glucose concentration. A comparison between classical physiological and network parameters revealed that the first-responders during activation had longer active times during plateau and the most active cells during the plateau tended to deactivate later. Cells with the most functional connections tended to activate sooner, have longer active times, and deactivate later. Our findings provide a common ground for recent differing views on [beta] cell heterogeneity and an important baseline for future studies of stimulus-secretion and intercellular coupling.
NEW & NOTEWORTHY: We assessed concentration-dependence in coupled [beta] cells, degree of functional heterogeneity, and uncovered possible specialized subpopulations during the different phases of the response to glucose at the level of many individual cells. To this aim, we combined acute mouse pancreas tissue slices with functional multicellular calcium imaging over a wide range from threshold (7 mM) and physiological (8 and 9 mM) to supraphysiological (12 and 16 mM) glucose concentrations, classical physiological, and advanced network analyses. Ključne besede: beta cells, calcium imaging, glucose-dependence, network analysis Objavljeno v DKUM: 15.10.2024; Ogledov: 0; Prenosov: 19
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9. Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ cyclingSenka Ljubojevic-Holzer, Simon Kraler, Nataša Djalinac, Mahmoud Abdellatif, Julia Voglhuber, Julia Schipke, Marlene Schmidt, Katharina-Maria Kling, Greta Therese Franke, Viktoria Herbst, Simon Sedej, 2022, izvirni znanstveni članek Opis: Aims: Autophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure.
Methods and results: RT-qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5-/-). Before manifesting cardiac dysfunction, Atg5-/- mice showed compromised cardiac reserve in response to β-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high-frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load, and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5-/- cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5-/- cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload.
Conclusion: Loss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve. Ključne besede: autophagy, beta-adrenergic signalling, calcium, cardiomyocytes, mitochondria Objavljeno v DKUM: 26.09.2024; Ogledov: 0; Prenosov: 2
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10. Glucose-stimulated calcium dynamics in beta cells from male C57BL/6J, C57BL/6N, and NMRI mice : a comparison of activation, activity, and deactivation properties in tissue slicesViljem Pohorec, Lidija Križančić Bombek, Maša Skelin, Jurij Dolenšek, Andraž Stožer, 2022, izvirni znanstveni članek Opis: Although mice are a very instrumental model in islet beta cell research, possible phenotypic differences between strains and substrains are largely neglected in the scientific community. In this study, we show important phenotypic differences in beta cell responses to glucose between C57BL/6J, C57BL/6N, and NMRI mice, i.e., the three most commonly used strains. High-resolution multicellular confocal imaging of beta cells in acute pancreas tissue slices was used to measure and quantitatively compare the calcium dynamics in response to a wide range of glucose concentrations. Strain- and substrain-specific features were found in all three phases of beta cell responses to glucose: a shift in the dose-response curve characterizing the delay to activation and deactivation in response to stimulus onset and termination, respectively, and distinct concentration-encoding principles during the plateau phase in terms of frequency, duration, and active time changes with increasing glucose concentrations. Our results underline the significance of carefully choosing and reporting the strain to enable comparison and increase reproducibility, emphasize the importance of analyzing a number of different beta cell physiological parameters characterizing the response to glucose, and provide a valuable standard for future studies on beta cell calcium dynamics in health and disease in tissue slices. Ključne besede: beta cell, mouse models, calcium imaging, glucose-dependence, tissue slice Objavljeno v DKUM: 15.07.2024; Ogledov: 148; Prenosov: 22
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