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

Manganese(II) Molecular Sources for Plasma-Assisted CVD of Mn Oxides and Fluorides: From Precursors to Growth Process

A viable route to manganese-based materials of high technological interest is plasma-assisted chemical vapor deposition (PA-CVD), offering various degrees of freedom for the growth of high-purity nanostructures from suitable precursors. In this regard, fluorinated \u3b2-diketonate diamine Mn(II) complexes of general formula Mn(dik)2\ub7TMEDA [TMEDA = N,N,N\u2032,N\u2032-tetramethylethylenediamine; Hdik = 1,1,1,5,5,5-hexafluoro-2,4-pentanedione (Hhfa), or 1,1,1-trifluoro-2,4-pentanedione (Htfa)] represent a valuable option in the quest of candidate molecular sources for PA-CVD environments. In this work, we investigate and highlight the chemico-physical properties of these compounds of importance for their use in PA-CVD processes, through the use of a comprehensive experimental\u2013theoretical investigation. Preliminary PA-CVD validation shows the possibility of varying the Mn oxidation state, as well as the system chemical composition from MnF2 to MnO2, by simple modulations of the reaction atmosphere, paving the way to a successful utilization of the target compounds in the growth of manganese-containing nanomaterials for different technological applications

Deciduoid mesothelioma of the thorax: A comprehensive review of the scientific literature.

AbstractObjectiveDeciduoid mesothelioma is a rare variant of malignant epithelioid mesothelioma. It often involves the peritoneum, but also thoracic cases have been reported. The aim of the present review is to describe the demographic, clinical, radiological, and pathological features of such a rare variant of thoracic mesothelioma, and the state of the art regarding the therapeutic approaches currently available.Data sourceEnglish‐language articles published from 1985 to June 2016, and related to thoracic deciduoid mesothelioma cases were retrieved using the Pubmed database.Study selectionThe search terms were "mesothelioma," "thoracic mesothelioma," "epithelial mesothelioma," "pleural mesothelioma," and "deciduoid mesothelioma."ResultsForty‐four cases included in 16 articles, published in the period under investigation, were analyzed in detail.ConclusionsThe mean age of the patients was 63 years, and the male to female ratio 1.7:1. Approximately 58% had exposure to asbestos, and 73% had a smoking history; familiarity was rarely reported. The most common anatomical site of origin was the right pleura, and the most frequent clinical manifestations were chest pain, dyspnea, cough, and weight loss. Thoracic X‐ray and computed tomography were the imaging techniques most employed for diagnosis and surgical planning. The pathological diagnosis was obtained by examination of surgical or biopsy specimens in most cases. The best treatment strategy of deciduoid mesothelioma is a matter of debate; nevertheless a multidisciplinary approach is currently the best option for the choice of the adequate therapeutic scheme

Response of alveolar type II pneumocytes to mechanical stimulation

The aim of this work was to study the responses of Alveolar type II (ATII) cells to mechanical stimulation. We focused in particular on stretch induced changes in the cytoplasmic Ca2+ concentration ([Ca2+]c) and on the underlying mechanisms. To overcome the methodological problems of live cell imaging at high temporal resolution during cell stretch, suitable techniques had to be developed. The first part of this work focused on design of new stretch devices. We used a computer controlled unidirectional stretch-compression device and a system with a micro-spatula for local stimulation of ATII cells. With the micro-spatula we achieved image acquisition interval of 30 - 35 ms and without any delay after the stretch. In the second part we focused on the fast [Ca2+]c increase as a response of ATII cells to mechanical stimulation with a special interest in the TRP channel family. Pharmacological evidence but especially the silencing of TRPV2 with siRNA techniques led to a significant reduction of the stretch-induced [Ca2+]c increase and identified TRPV2 as a mechanosensitive channel in ATII cells. Moreover, we found that mechanical gating of mechano sensitive channels in ATII cells is strictly dependent on the presence of stretch stimulus, pointing at a direct gating mechanism. TRPV2 is localized in close vicinity to FA as shown by co-immuno-labeling and immunoprecipitation experiments suggesting molecular interaction of TRPV2 with FA proteins. Indeed, we can show that integrins, FA proteins, and the actin cytoskeleton play a crucial role in transducing the mechanical signal and possibly activate the mechano-sensitive channels. For the first time our study provides molecular information on the mechanically induced Ca2+ response in ATII cells. This information may be used as a basis in the search for drugs preventing mechanically induced lung diseases

MODELING THE FIRST ACTIVATION STAGES OF THE Fe(hfa)2TMEDA CVD PRECURSOR ON A HEATED GROWTH SURFACE

Iron oxide-based functional nanostructures are important technological materials that can be fruitfully obtained with high purity and tailored phase composition/morphology by Chemical Vapor Deposition (CVD). In this context, previous works have experimentally demonstrated the suitability of the Fe(II) diketonate-diamine complex Fe(hfa)2TMEDA as CVD precursor. However, further progress in the field strongly depends on the understanding of mechanisms governing the molecule-to-materials conversion, a goal that can be achieved by advanced computational methods. In this work, structural optimization of the Fe(hfa)2TMEDA molecule on a model CVD growth surface was performed to provide an insight on the physisorption geometry at 0 K. The first stages of thermally activated surface processes were then investigated by first principles molecular dynamics simulation, which revealed interesting aspects of the precursor behaviour at temperatures conditions typically adopted in Fe2O3 deposition experiments. Whereas the physisorbed complex maintains its octahedral geometry, high temperature interactions with the surface lead to drastic perturbations of the molecular structure and significant weakening of the coordination bonds of the metal center with the diamine ligands. Our results provide key elements for the fundamental knowledge of the temperatureinduced behaviour of this precursor on a heated substrate, which may help the understanding of its CVD activation mechanisms and decomposition pathways

Modeling the first activation stages of the Fe(hfa)2TMEDA CVD precursor on a heated growth surface

Iron oxide-based functional nanostructures are important technological materials that can be fruitfully obtained with high purity and tailored phase composition/morphology by Chemical Vapor Deposition (CVD). In this context, previous works have experimentally demonstrated the suitability of the Fe(II) diketonate-diamine complex Fe(hfa)2TMEDA as CVD precursor. However, further progress in the field strongly depends on the understanding of mechanisms governing the molecule-to-materials conversion, a goal that can be achieved by advanced computational methods. In this work, structural optimization of the Fe(hfa)2TMEDA molecule on a model CVD growth surface was performed to provide an insight on the physisorption geometry at 0 K. The first stages of thermally activated surface processes were then investigated by first principles molecular dynamics simulation, which revealed interesting aspects of the precursor behaviour at temperatures conditions typically adopted in Fe2O3 deposition experiments. Whereas the physisorbed complex maintains its octahedral geometry, high temperature interactions with the surface lead to drastic perturbations of the molecular structure and significant weakening of the coordination bonds of the metal center with the diamine ligands. Our results provide key elements for the fundamental knowledge of the temperatureinduced behaviour of this precursor on a heated substrate, which may help the understanding of its CVD activation mechanisms and decomposition pathway

Mechanical strain of alveolar type II cells in culture: changes in the transcellular cytokeratin network and adaptations

Mechanical forces exert multiple effects in cells, ranging from altered protein expression patterns to cell damage and death. Despite undisputable biological importance, little is known about structural changes in cells subjected to strain ex vivo. Here, we undertake the first transmission electron microscopy investigation combined with fluorescence imaging on pulmonary alveolar type II cells that are subjected to equibiaxial strain. When cells are investigated immediately after stretch, we demonstrate that curved cytokeratin (CK) fibers are straightened out at 10% increase in cell surface area (CSA) and that this is accompanied by a widened extracellular gap of desmosomes–the insertion points of CK fibers. Surprisingly, a CSA increase by 20% led to higher fiber curvatures of CK fibers and a concurrent return of the desmosomal gap to normal values. Since 20% CSA increase also induced a significant phosphorylation of CK8-ser431, we suggest CK phosphorylation might lower the tensile force of the transcellular CK network, which could explain the morphological observations. Stretch durations of 5 min caused membrane injury in up to 24% of the cells stretched by 30%, but the CK network remained surprisingly intact even in dead cells. We conclude that CK and desmosomes constitute a strong transcellular scaffold that survives cell death and hypothesize that phosphorylation of CK fibers is a mechano-induced adaptive mechanism to maintain epithelial overall integrity

2-APB and capsazepine-induced Ca2+ influx stimulates clathrin-dependent endocytosis in alveolar epithelial cells

Calcium as a second messenger influences many cellular and physiological processes. In lung, alveolar type II (ATII) cells sense mechanical stress and respond by Ca2+ dependent release of surfactant, which is essential for respiratory function. Nevertheless, Ca2+ signaling mechanisms in these cells - in particular Ca2+ entry pathways are still poorly understood. Herein, we investigated pharmacological properties of non-voltage-gated Ca2+ channel modulators in ATII and NCI-H441 cells and demonstrate that 2-Aminoethoxydiphenyl-borinate (2-APB) and capsazepine (CPZ) activate Ca2+ entry with pharmacologically distinguishable components. Surprisingly, 2-APB and CPZ activated clathrin dependent endocytosis in ATII and NCI-H441 cells, which was dependent on Ca2+ entry. The internalized material accumulated in non-acidic granules distinct from surfactant containing lamellar bodies (LB). LB exocytosis was not observed under these conditions. Our study demonstrates that 2-APB/CPZ induces Ca2+ entry which unlike ATP- or stretch-induced Ca2+ entry in ATII cells does not activate exocytosis but an opposing endocytotic mechanism

Characterization and Pharmacological Inhibition of the Pore-Forming Clostridioides difficile CDTb Toxin

The clinically highly relevant Clostridioides (C.) difficile releases several AB-type toxins that cause diseases such as diarrhea and pseudomembranous colitis. In addition to the main virulence factors Rho/Ras-glycosylating toxins TcdA and TcdB, hypervirulent strains produce the binary AB-type toxin CDT. CDT consists of two separate proteins. The binding/translocation B-component CDTb facilitates uptake and translocation of the enzyme A-component CDTa to the cytosol of cells. Here, CDTa ADP-ribosylates G-actin, resulting in depolymerization of the actin cytoskeleton. We previously showed that CDTb exhibits cytotoxicity in the absence of CDTa, which is most likely due to pore formation in the cytoplasmic membrane. Here, we further investigated this cytotoxic effect and showed that CDTb impairs CaCo-2 cell viability and leads to redistribution of F-actin without affecting tubulin structures. CDTb was detected at the cytoplasmic membrane in addition to its endosomal localization if CDTb was applied alone. Chloroquine and several of its derivatives, which were previously identified as toxin pore blockers, inhibited intoxication of Vero, HCT116, and CaCo-2 cells by CDTb and CDTb pores in vitro. These results further strengthen pore formation by CDTb in the cytoplasmic membrane as the underlying cytotoxic mechanism and identify pharmacological pore blockers as potent inhibitors of cytotoxicity induced by CDTb and CDTa plus CDTb