Stefano Guido

Three-dimensional shape of a drop under simple shear flow

The three-dimensional deformation of an isolated drop in an immiscible liquid phase undergoing simple shear flow was investigated by using a parallel-plate apparatus. The drop was observed by video-enhanced contrast optical microscopy, either along the vorticity direction or along the velocity gradient direction of the shear flow. An experimental methodology based on image analysis was especially developed to study in a quantitative way the three-dimensional shape of the deformed drop, both under steady-state flow and during transients. Up to moderate deformations, the steady-state drop shape was well described within experimental error by an ellipsoid having three different axes. The deviation of drop shape from an ellipsoid at higher deformations was also characterized in a quantitative way. Good agreement was found between the experimental results of this work and numerical simulations reported in the literature.

Drop breakup and fragment size distribution in shear flow

We report a study on the deformation and breakup of drops in an impulsively started shear flow under Stokes flow conditions using boundary-integral simulations and video-microscopy experiments. Two independent techniques are used for determining the physical parameters of the system from the combined use of numerical simulations and experiments. Accurate breakup criteria (critical capillary numbers) are presented for a range of viscosity ratios. The time required for breakup events has a broad minimum corresponding to moderate shear rates. The size distribution of droplets produced by breakup events is shown to scale with the critical size drop for breakup in shear. A simplified model, based on this finding, is developed for the size distribution in a sheared emulsion. According to the model, the drop size distribution in a given emulsion depends only on the average initial drop size and the shear rate.

Lysosomal accumulation of gliadin p31–43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa

Background An unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31–43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines. Aim We addressed whether the epithelial uptake of p31–43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response. Methods The time course of intracellular delivery to lysosomes of p31–43, pα-2 or pα-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)γ ubiquitination and p42/44–mitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis. Results After 24 h of challenge p31–43, but not pα-2 or pα-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARγ. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARγ levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation. Conclusion p31–43 accumulation in lysosomes leads to epithelial activation via the ROS–TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARγ downregulation.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Deformation of a Newtonian drop in a viscoelastic matrix under steady shear flow: Experimental validation of slow flow theory

Abstract The small deformation of a single drop in a Boger fluid under steady-state slow shear flow is here investigated by video-enhanced microscopy and image analysis. Data are compared to predictions of a recent perturbation analysis, dealing with the drop-in-a-flow-field problem for viscoelastic fluid components [J. Non-Newt. Fluid Mech. 107 (2002) 111]. The main experimental result, in agreement with theory, is that drop orientation towards the flow direction is significantly enhanced as compared to the Newtonian case. In fact, based on this result, an optical determination of the first normal stress difference of the matrix fluid can be obtained. Furthermore, it is shown here that the interfacial tension of the fluid pair can be readily obtained by comparison between the theoretical predictions and the optical data taken in both the “side” and “top” views (i.e. along the vorticity and velocity gradient direction, respectively) of the deformed drop.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.

Red blood cell deformation in microconfined flow

In this work, we report on a systematic fluidodynamic investigation of red blood cell (RBC) suspensions flowing in microcapillaries with diameters comparable to the cell size in vitro. By using high-speed video microscopy and image analysis, we provide the first simultaneous determination of both cell velocity and shape parameters related to RBC membrane deformability over an extended range of pressure drops, and the first quantitative comparison with theoretical results from the literature. Good agreement was found with the predicted axisymmetric shapes tending towards an apparent bullet-like asymptotic configuration at increasing cell velocity. A potential application of this work is in the design of flow-based devices to study the pathological conditions affecting cell deformability, thus allowing us to overcome the limits of classical static methods, such as micropipette aspiration, which are not suitable for handling a large number of cells.

Phase inversion emulsification: Current understanding and applications

This review is addressed to the phase inversion process, which is not only a common, low-energy route to make stable emulsions for a variety of industrial products spanning from food to pharmaceuticals, but can also be an undesired effect in some applications, such as crude oil transportation in pipelines. Two main ways to induce phase inversion are described in the literature, i.e., phase inversion composition (PIC or catastrophic) and phase inversion temperature (PIT or transitional). In the former, starting from one phase (oil or water) with surfactants, the other phase is more or less gradually added until it reverts to the continuous phase. In PIT, phase inversion is driven by a temperature change without varying system composition. Given its industrial relevance and scientific challenge, phase inversion has been the subject of a number of papers in the literature, including extensive reviews. Due to the variety of applications and the complexity of the problem, most of the publications have been focused either on the phase behavior or the interfacial properties or the mixing process of the two phases. Although all these aspects are quite important in studying phase inversion and much progress has been done on this topic, a comprehensive picture is still lacking. In particular, the general mechanisms governing the inversion phenomenon have not been completely elucidated and quantitative predictions of the phase inversion point are limited to specific systems and experimental conditions. Here, we review the different approaches on phase inversion and highlight some related applications, including future and emerging perspectives.

Drop shape dynamics under shear-flow reversal

The shape evolution of a liquid drop immersed in an immiscible liquid is studied under transient flow conditions. The drop is Newtonian and buoyancy free; the external liquid is Newtonian and subjected to shear flow reversal. Three model systems, polydimethylsiloxane/polyisobutylene, polybutene/silicone oil, and silicone oil/polybutene, all Newtonian under the experimental conditions investigated, have been selected to have a range of viscosity ratios. The three drop axes and the drop orientation within the shear plane are independently measured. The results are compared with the predictions of a phenomenological model. The agreement between experimental results and theory is good. Peculiar behavior of the orientation angle has been observed and correctly predicted. The results are also used to explain some rheological features typical of immiscible polymer blends.

High-throughput screening-compatible single-step protocol to differentiate embryonic stem cells in neurons.

Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, gamma-aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by timelapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.