Biofabrication of spheroids fusion-based tumor models: computational simulation of glucose effects.

In vitrotumor models consisting of cell spheroids are increasingly used for mechanistic studies and pharmacological testing. However, unless vascularized, the availability of nutrients such as glucose to deeper layers of multicellular aggregates is limited. In addition, recent developments in cells-only biofabrication (e.g. 'scaffold-free bioprinting'), allow the creation of more complex spheroid-based structures, further exposing the cells to nutrient deprivation within these constructs. To explore the impact of glucose availability on such tumor-like structures, we used the CompuCell3D platform for modeling of tumor spheroids. By monitoring the types of cells, fusing pairs geometry and the distance between spheroids centers of mass, we made novel heuristic observations on how binary- and multi-spheroid fusions are impacted by glucose availability. At limiting glucose concentrations mimicking hypoglycemia we noted an abrupt collapse of the tumor spheroids, unexpectedly amplified by the contact with normal cell spheroids. At higher glucose concentrations, we found an increased intermixing of cancerous cells, strong anti-phase oscillations between proliferating and quiescent tumor cells and a structural instability of fusing tumor spheroids, leading to their re-fragmentation. In a model of tumor microenvironment composed of normal cell spheroids fusing around a tumoral one, the competition for glucose lead to either the tumor's disappearance, to a steady state, or to its expansion. Moreover, the invasion of this microenvironment by individual tumor cells was also strongly depended on the available glucose. In conclusion, we demonstrate the value of computational simulations for anticipating the properties of biofabricated tumor models, and in generating testable hypotheses regarding the relationship between cancer, nutrition and diabetes.

Equivalent Isopropanol Concentrations of Aromatic Amino Acids Interactions with Lipid Vesicles.

We show that the interaction of aromatic amino acids with lipid bilayers can be characterized by conventional 1D [Formula: see text]H NMR spectroscopy using reference spectra obtained in isopropanol-d8/D[Formula: see text]O solutions. We demonstrate the utility of this method with three different peptides containing tyrosine, tryptophan, or phenylalanine amino acids in the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid membranes. In each case, we determine an equivalent isopropanol concentration (EIC) for each hydrogen site of aromatic groups, in essence constructing a map of the chemical environment. These EIC maps provide information on relative affinities of aromatic side chains for either PC or PS bilayers and also inform on amino acid orientation preference when bound to membranes.

Effects of lipid interactions on model vesicle engulfment by alveolar macrophages.

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state (2)H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.

Functional effects of nanoparticle exposure on Calu-3 airway epithelial cells.

High concentrations of manufactured carbon nanoparticles (CNP) are known to cause oxidative stress, inflammatory responses and granuloma formation in respiratory epithelia. To examine the effects of lower, more physiologically relevant concentrations, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in transepithelial permeability and cellular function of airway epithelia after exposure to environmentally realistic concentrations of carbon nanoparticles. Three common carbon nanoparticles, fullerenes, single- and multi-wall carbon nanotubes (SWCNT, MWCNT) were used in these experiments. Electrophysiological measurements were performed to assay transepithelial electrical resistance (TEER) and epinephrine-stimulated chloride (Cl(-)) ion secretion of epithelial cell monolayers that had been exposed to nanoparticles for three different times (1 h, 24 h and 48 h) and over a 7 log unit range of concentrations. Fullerenes did not have any effect on the TEER or stimulated ion transport. However, the carbon nanotubes (CNT) significantly decreased TEER and inhibited epinephrine-stimulated Cl(-) secretion. The changes were time dependent and at more chronic exposures caused functional effects which were evident at concentrations substantially lower than have been previously examined. The functional changes manifested in response to physiologically relevant exposures would inhibit mucociliary clearance mechanisms and compromise the barrier function of airway epithelia.

Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases.

α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.

Investigation of a catalyst ink dispersion using both ultra-small-angle X-ray scattering and cryogenic TEM.

The dispersion of Nafion ionomer particles and Pt/C catalyst aggregates in liquid media was studied using both ultra-small-angle X-ray scattering (USAXS) and cryogenic TEM. A systematic approach was taken to study first the dispersion of each component (i.e., ionomer particles and Pt/C aggregates), then the combination of the components, and last the catalyst ink. Multiple-level curve fitting was used to extract the particle size, size distribution, and geometry of the Pt/C aggregates and the Nafion particles in liquid media from the scattering data. The results suggest that the particle size, size distribution, and geometry are not uniform throughout the systems but rather vary significantly. It was found that the interaction of each component (i.e., the Nafion ionomer particles and the Pt/C aggregates) occurs in the dispersion. Cryogenic TEM was used to observe the size and geometry of the particles in liquid directly and to validate the scattering results. The TEM results showed excellent agreement.

alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis.

alpha-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema.