Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability

[[abstract]]Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>

Cinnamic Anilides as New Mitochondrial Permeability Transition Pore Inhibitors Endowed with Ischemia-Reperfusion Injury Protective Effect in Vivo

In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (<i>E</i>)-3-(4-fluoro-3-hydroxy-phenyl)-<i>N</i>-naphthalen-1-yl-acrylamide <b>22</b>, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction