Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.
D.04 Initiative to set up a new research area in Slovenia
COBISS.SI-ID: 31163353The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. Beta-Adrenergic receptors (beta-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of beta-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of beta-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of beta-AR, rapidly increased [cAMP]i (15 s). The FRET signal response, mediated via beta-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP ()35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon -AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, beta-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease. GLIA 2014.
D.04 Initiative to set up a new research area in Slovenia
COBISS.SI-ID: 31149273Super resolution microscopy is the cutting edge technology with the resolution that is at list twice better than the resolution of conventional far field microscopy. One of the key instuments of super resolution microscopy is STED technology based instrument, which is now located also in Slovenia. It represents the most advanced technology in the field of optical microscopy, comprised of most advanced optics, mechanics and electronics. It enables observation of living objects at resolution between 35 and 40 nm with the potential to visualize objects beyond these limits. The conference with a workshop of the demonstration of the super-resolution microscopy is dedicated to the introduction of this technological novelty.
F.04 Increase of the technological level
COBISS.SI-ID: 2814543Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate.Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at theEuropean Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groupsto approach the EMA and the CAT as a regulatory advisor during development.
F.05 Ability to launch new technological development cycle
COBISS.SI-ID: 26685401Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolicfree glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600mu. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with theone recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration inadipocytes by a factor of 3.6; however, we recorded no effect on delta ratio(DeltaR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p ( 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.
F.02 Acquisition of new scientific knowledge
COBISS.SI-ID: 28263129