Changes in intra- and extracellular potassium ion (K⁺) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K⁺ changes is severely limited by the lack of practicable detection methods. We developed K⁺-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K⁺ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K⁺ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K⁺ signals. Hitherto unknown Ca²⁺-triggered, organelle-specific K⁺ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K⁺ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K⁺ assays and open new avenues for live-cell K⁺ imaging.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor – referred to as pH-Lemon – optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.

Summary: Background: Probiotics may correct intestinal dysbiosis and proinflammatory conditions in patients with liver cirrhosis.Aim: To test the effects of a multispecies probiotic on innate immune function, bacterial translocation and gut permeability.Methods: In a randomised, double blind, placebo‐controlled study, stable cirrhotic out‐patients either received a daily dose of a probiotic powder containing eight different bacterial strains (Ecologic Barrier, Winclove, Amsterdam, The Netherlands) (n = 44) or a placebo (n = 36) for 6 months and were followed up for another 6 months.Results: We found a significant but subclinical increase in neutrophil resting burst (2.6–3.2%, P = 0.0134) and neopterin levels (7.7–8.4 nmol/L, P = 0.001) with probiotics but not with placebo. Probiotic supplementation did not have a significant influence on neutrophil phagocytosis, endotoxin load, gut permeability or inflammatory markers. Ten severe infections occurred in total; one during intervention in the placebo group, and five and four after the intervention has ended in the probiotic and placebo group, respectively. Liver function showed some improvement with probiotics but not with placebo.Conclusions: Probiotic supplementation significantly increased serum neopterin levels and the production of reactive oxygen species by neutrophils. These findings might explain the beneficial effects of probiotics on immune function. Furthermore, probiotic supplementation may be a well‐tolerated method to maintain or even improve liver function in stable cirrhosis. However, its influence on gut barrier function and bacterial translocation in cirrhotic patients is minimal.

Senescence is characterized by a gradual decline in cellular functions, including changes in energy homeostasis and decreased proliferation activity. As cellular power plants, contributors to signal transduction, sources of reactive oxygen species (ROS) and executors of programmed cell death, mitochondria are in a unique position to affect aging-associated processes of cellular decline. Notably, metabolic activation of mitochondria is tightly linked to Ca2+ due to the Ca2+ -dependency of several enzymes in the Krebs cycle, however, overload of mitochondria with Ca2+ triggers cell death pathways. Consequently, a machinery of proteins tightly controls mitochondrial Ca2+ homeostasis as well as the exchange of Ca2+ between the different cellular compartments, including Ca2+ flux between mitochondria and the endoplasmic reticulum (ER). In this study, we investigated age-related changes in mitochondrial Ca2+ homeostasis, mitochondrial-ER linkage and the activity of the main ROS production site, the mitochondrial respiration chain, in an in vitro aging model based on porcine aortic endothelial cells (PAECs), using high-resolution live cell imaging, proteomics and various molecular biological methods. We describe that in aged endothelial cells, increased ER-mitochondrial Ca2+ crosstalk occurs due to enhanced ER-mitochondrial tethering. The close functional inter-organelle linkage increases mitochondrial Ca2+ uptake and thereby the activity of the mitochondrial respiration, but also makes senescent cells more vulnerable to mitochondrial Ca2+-overload-induced cell death. Moreover, we identified the senolytic properties of the polyphenol resveratrol, triggering cell death via mitochondrial Ca2+ overload exclusively in senescent cells. By unveiling aging-related changes in the inter-organelle tethering and Ca2+ communications we have advanced the understanding of endothelial aging and highlighted a potential basis to develop drugs specifically targeting senescent cells.