BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

Acute depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate impairs specific steps in endocytosis of the G-protein-coupled receptor.

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] has been shown to be crucial for endocytosis of some cell surface receptors, such as EGF and transferrin receptors, but its role in G-protein-coupled receptor internalization has not been investigated. By using luciferase-labeled type 1 angiotensin II (AT1R), type 2C serotonin (5HT2CR) or ß(2) adrenergic (ß2AR) receptors and fluorescently tagged proteins (ß-arrestin-2, plasma-membrane-targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdIns(4,5)P(2) in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdIns(4,5)P(2). Here we show that ligand-induced interaction of AT1, 5HT2C and ß(2)A receptors with ß-arrestin-2 was unaffected by PtdIns(4,5)P(2) depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdIns(4,5)P(2). Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdIns(4,5)P(2) depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane, but did not enter the cells. Our data suggest that in the absence of PtdIns(4,5)P(2), these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

Percutaneous tracheostomy: Comparison of three different methods with respect to tracheal cartilage injury in cadavers-Randomized controlled study.

Background: Performing tracheostomy improves patient comfort and success rate of weaning from prolonged invasive mechanical ventilation. Data suggest that patients have more benefit of percutaneous technique than the surgical procedure, however, there is no consensus on the percutaneous method of choice regarding severe complications such as late tracheal stenosis. Aim of this study was comparing incidences of cartilage injury caused by different percutaneous dilatation techniques (PDT), including Single Dilator, Griggs' and modified (bidirectional) Griggs' method. Materials and methods: Randomized observational study was conducted on 150 cadavers underwent post-mortem percutaneous tracheostomy. Data of cadavers including age, gender and time elapsed from death until the intervention (more or less than 72 h) were collected and recorded. Primary and secondary outcomes were: rate of cartilage injury and cannula malposition respectively. Results: Statistical analysis revealed that method of intervention was significantly associated with occurrence of cartilage injury, as comparing either standard Griggs' with Single Dilator (p = 0.002; OR: 4.903; 95% CI: 1.834-13.105) or modified Griggs' with Single Dilator (p < 0.001; OR: 6.559; 95% CI: 2.472-17.404), however, no statistical difference was observed between standard and modified Griggs' techniques (p = 0.583; OR: 0.748; 95% CI: 0.347-1.610). We found no statistical difference in the occurrence of cartilage injury between the early- and late post-mortem group (p = 0.630). Neither gender (p = 0.913), nor age (p = 0.529) influenced the rate of cartilage fracture. There was no statistical difference between the applied PDT techniques regarding the cannula misplacement/malposition. Conclusion: In this cadaver study both standard and modified Griggs' forceps dilatational methods were safer than Single dilator in respect of cartilage injury.

Reliability and clinical correlations of semi-quantitative lung ultrasound on BLUE points in COVID-19 mechanically ventilated patients: The 'BLUE-LUSS'-A feasibility clinical study.

INTRODUCTION: Bedside lung ultrasound has gained a key role in each segment of the treatment chain during the COVID-19 pandemic. During the diagnostic assessment of the critically ill patients in ICUs, it is highly important to maximize the amount and quality of gathered information while minimizing unnecessary interventions (e.g. moving/rotating the patient). Another major factor is to reduce the risk of infection and the workload of the staff. OBJECTIVES: To serve these significant issues we constructed a feasibility study, in which we used a single-operator technique without moving the patient, only assessing the easily achievable lung regions at conventional BLUE points. We hypothesized that calculating this 'BLUE lung ultrasound score' (BLUE-LUSS) is a reasonable clinical tool. Furthermore, we used both longitudinal and transverse scans to measure their reliability and assessed the interobserver variability as well. METHODS: University Intensive Care Unit based, single-center, prospective, observational study was performed on 24 consecutive SARS-CoV2 RT-PCR positive, mechanically ventilated critically ill patients. Altogether 400 loops were recorded, rated and assessed off-line by 4 independent intensive care specialists (each 7+ years of LUS experience). RESULTS: Intraclass correlation values indicated good reliability for transversal and longitudinal qLUSS scores, while we detected excellent interrater agreement of both cLUSS calculation methods. All of our LUS scores correlated inversely and significantly to the P/F values. Best correlation was achieved in the case of longitudinal qLUSS (r = -0.55, p = 0.0119). CONCLUSION: Summarized score of BLUE-LUSS can be an important, easy-to-perform adjunct tool for assessing and quantifying lung pathology in critically ill ventilated patients at bedside, especially for the P/F ratio. The best agreement for the P/F ratio can be achieved with the longitudinal scans. Regarding these findings, assessing BLUE-points can be extended with the BLUE-LUSS for daily routine using both transverse and longitudinal views.

Development of Nonspecific BRET-Based Biosensors to Monitor Plasma Membrane Inositol Lipids in Living Cells.

There are several difficulties to face when investigating the role of phosphoinositides. Although they are present in most organelles, their concentration is very low, sometimes undetectable with the available methods; moreover, their level can quickly change upon several external stimuli. Here we introduce a newly improved lipid sensor tool-set based on the balanced expression of luciferase-fused phosphoinositide recognizing protein domains and a Venus protein targeted to the plasma membrane, allowing us to perform Bioluminescence Resonance Energy Transfer (BRET) measurements that reflect phosphoinositide changes in a population of transiently transfected cells. This method is highly sensitive, specific, and capable of semiquantitative characterization of plasma membrane phosphoinositide changes with high temporal resolution.

Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors.

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications.