Bioengineering bacterial outer membrane vesicles as delivery system for RNA therapeutics targeted to lung epithelial cytosols

Intact epithelia lining the airways and alveoli in the lung are essential to maintain lung function. Structural or functional damage of epithelial cells leads in severe diseases, including COPD/emphysema, ibrosis or ALI/ARDS. This central role of epithelia in pulmonary diseases identifies these cells as primary candidates for targeted therapy. With the exception of surface-expressed molecules, however, targeting intracellular components is severely restricted due to poor delivery. We aim to overcome this obstacle using topically administered, bioengineered, biocompatible bacterial outer membrane vesicles (OMVs) as recombinant drug delivery systems for novel biopharmaceuticals. Engineering recombinant surface expression of eukaryotic receptor ligands in ClearColi®, a commercial E.coli BL21 (DE3) strain deficient in lipopolysaccharide production, we have used red fluorescent protein reporters to track OMV loading, transgene expression, and eukaryotic cell trafficking. We demonstrate statistically significant differences in the levels of over 700 proteins between differentially engineered and purified OMV preps with additional differences in transcriptome and lipidome consistency. We also characterised visual and particle size differences observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Here we report early bioadhesion and culture of re-differentiated lung epithelia. This project aims to bridge the biotechnological gap in the intracellular biopharmaceutics drug delivery challenge for respiratory epithelia through highly controlled, and scalable bio-nanotechnology process. If successful, our work will unlock intracellular imaging and therapeutics research for respiratory diseases with a significant epithelial component, paving the way for other targeting ligands and potentially non-respiratory indications. cellular uptake results in A549 culture as well as air-liquid interface

Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Background Ca2+ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca2+ concentration ([Ca2+]c) in the prefusion phase, the occurrence and significance of Ca2+ signals in the postfusion phase have not been described before. Methodology/Principal Findings We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca2+- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca2+]c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t1/2 of decay = 3.2 s) rise of localized [Ca2+]c originating at the site of lamellar body fusion. [Ca2+]c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca2+ from, or addition of Ni2+ to the extracellular solution, strongly inhibited these [Ca2+]c transients, whereas Ca2+ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca2+]c. Both effects were reduced by the non-specific Ca2+ channel blocker SKF96365. Conclusions/Significance Fusion-activated Ca2+ entry (FACE) is a new mechanism that leads to [Ca2+]c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca2+ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions

A Mobile Assessment Tool for Collecting Data in Large-Scale Educational Studies

Abstract Conducting scientific studies is an often frustrating and tedious task. To minimize the usual problems, such as lack of concentration or willingness to participate, and instead promote interest in the study, a smart mobile device assessment tool was developed. The tablet-based assessment tool offers a wide range of visual tasks that can be employed when conducting studies utilizing the European Framework of Visual Literacy (ENViL). Furthermore, the assessment tool is highly configurable in the field using a centralized server and spreadsheet-based configuration files, thereby ensuring that no programming language is required to adapt the tasks on the mobile devices participating in the study. Finally, the presented framework and architecture are completely cross-platform and cross-device and can be re-used and extended for any number of similar studies and tasks

Exploring the Usability of the German COVID-19 Contact Tracing App in a Combined Eye Tracking and Retrospective Think Aloud Study

In the course of the corona virus (COVID-19) pandemic, many digital solutions for mobile devices (e.g., apps) were presented in order to provide additional resources supporting the control of the pandemic. Contact tracing apps (i.e., identify persons who may have been in contact with a COVID-19 infected) constitute one of the most popular as well as promising solutions. However, as a prerequisite for an effective application, such apps highly depend on being used by large numbers of the population. Consequently, it is important that these apps offer a high usability for everyone. We therefore conducted an exploratory study to learn more about the usability of the German COVID-19 contact tracing app Corona-Warn-App (CWA). More specifically, N = 15 participants assessed the CWA, relying on a combined eye tracking and retrospective think aloud approach. The results indicate, on the one hand, that the CWA leaves a promising impression for pandemic control, as essential functions are easily recognized. However, on the other hand, issues were revealed (e.g., privacy policy) that could be addressed in future updates more properly

NICOL: A Neuro-inspired Collaborative Semi-humanoid Robot that Bridges Social Interaction and Reliable Manipulation

Robotic platforms that can efficiently collaborate with humans in physical tasks constitute a major goal in robotics. However, many existing robotic platforms are either designed for social interaction or industrial object manipulation tasks. The design of collaborative robots seldom emphasizes both their social interaction and physical collaboration abilities. To bridge this gap, we present the novel semi-humanoid NICOL, the Neuro-Inspired COLlaborator. NICOL is a large, newly designed, scaled-up version of its well-evaluated predecessor, the Neuro-Inspired COmpanion (NICO). NICOL adopts NICO's head and facial expression display and extends its manipulation abilities in terms of precision, object size, and workspace size. Our contribution in this paper is twofold -- firstly, we introduce the design concept for NICOL, and secondly, we provide an evaluation of NICOL's manipulation abilities by presenting a novel extension for an end-to-end hybrid neuro-genetic visuomotor learning approach adapted to NICOL's more complex kinematics. We show that the approach outperforms the state-of-the-art Inverse Kinematics (IK) solvers KDL, TRACK-IK and BIO-IK. Overall, this article presents for the first time the humanoid robot NICOL, and contributes to the integration of social robotics and neural visuomotor learning for humanoid robots

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein alpha -subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough

Surfactant secretion in LRRK2 knock-out rats : changes in lamellar body morphology and rate of exocytosis

Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca2+ release upon ATP treatment and significant triggering of LB exocytosis. These findings are in line with the strong Ca2+-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca2+ signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP