Genetically encoded phosphatidylserine biosensor for in vitro, ex vivo and in vivo labelling.

BACKGROUND: The dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure. METHODS: For this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression. RESULTS: The biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice. CONCLUSIONS: We have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

Internet Usage Habits and Experienced Levels of Psychopathology: A Pilot Study on Association with Spontaneous Eye Blinking Rate.

Increasing availability of the internet has resulted in the increased prevalence of problematic online behaviors. Reliable and affordable neurobiological and psychological biomarkers that distinguish problematic internet use (PIU) from functional online activities are of utmost importance. Previous studies have shown a relationship between spontaneous eye blinking rate (sEBR) and changes in dopamine regulation in neurological and psychiatric disorders, including substance use disorders. In this study, we utilized sEBR to examine the potential link between individual differences in dopaminergic neurotransmission and PIU. In sum, 62 subjects participated in this study (median age 25, IQR 6 years, 34 females). The Problematic Internet Use Questionnaire (PIUQ-9), Beck Depression Inventory (BDI-II), Beck Anxiety Inventory (BAI), Clark-Beck Obsessive-Compulsive Inventory (CBOCI) and Barratt Impulsiveness Scale (BIS-11) were used for psychological assessment. The sEBRs were assessed with an electrooculogram recorded from above and below the left eye and from the right and left outer canthi. The group with PIU (PIUQ-9 > 20) expressed higher levels of impulsivity and compulsive behavior symptoms than the control group. In the group with PIU, impulsivity levels were inversely related to sEBR, and a trend of negative association of sEBR with compulsive behavior was observed. Future research should enroll subjects with high levels of PIU and strongly expressed psychopathology levels to further address the utility of sEBR as a potential biomarker.