Search

Hydrogels have been successfully employed as analogues of the extracellular matrix to study biological processes such as cells' migration, growth, adhesion and differentiation. These are governed by many factors, including the mechanical properties of hydrogels; yet, a one-to-one correlation between the viscoelastic properties of gels and cell fate is still missing from literature. In this work we provide experimental evidence supporting a possible explanation for the persistence of this knowledge gap. In particular, we have employed common tissues' surrogates such as polyacrylamide and agarose gels to elucidate a potential pitfall occurring when performing rheological characterisations of soft-materials. The issue is related to (i) the normal force applied to the samples prior to performing the rheological measurements, which may easily drive the outcomes of the investigation outside the materials' linear viscoelastic regime, especially when tests are performed with (ii) geometrical tools having unbefitting dimensions (i.e., too small). We corroborate that biomimetic hydrogels can show either compressional stress softening or stiffening, and we provide a simple solution to quench these undesired phenomena, which would likely lead to potentially misleading conclusions if they were not mitigated by a good practice in performing rheological measurements, as elucidated in this work.

Coordinated rotational motion is an intriguing, yet still elusive mode of collective cell migration, which is relevant in pathological and morphogenetic processes. Most of the studies on this topic have been carried out on epithelial cells plated on micropatterned substrates, where cell motion is confined in regions of well-defined shapes coated with extracellular matrix adhesive proteins. The driver of collective rotation in such conditions has not been clearly elucidated, although it has been speculated that spatial confinement can play an essential role in triggering cell rotation. Here, we study the growth of epithelial cell colonies freely expanding (i.e. with no physical constraints) on the surface of cell culture plates and focus on collective cell rotation in such conditions, a case which has received scarce attention in the literature. One of the main findings of our work is that coordinated cell rotation spontaneously occurs in cell clusters in the free growth regime, thus implying that cell confinement is not necessary to elicit collective rotation as previously suggested. The extent of collective rotation was size and shape dependent: a highly coordinated disc-like rotation was found in small cell clusters with a round shape, while collective rotation was suppressed in large irregular cell clusters generated by merging of different clusters in the course of their growth. The angular motion was persistent in the same direction, although clockwise and anticlockwise rotations were equally likely to occur among different cell clusters. Radial cell velocity was quite low as compared to the angular velocity, in agreement with the free expansion regime where cluster growth is essentially governed by cell proliferation. A clear difference in morphology was observed between cells at the periphery and the ones in the core of the clusters, the former being more elongated and spread out as compared to the latter. Overall, our results, to our knowledge, provide the first quantitative and systematic evidence that coordinated cell rotation does not require a spatial confinement and occurs spontaneously in freely expanding epithelial cell colonies, possibly as a mechanism for the system.

While the development of different vaccines has slowed the dissemination of SARS-CoV-2, the occurrence of breakthrough infections continues to fuel the pandemic. As a strategy to secure at least partial protection, with a single dose of a given COVID-19 vaccine to maximum possible fraction of the population,delayedadministration of subsequent doses (or boosters) has been implemented in many countries. However, waning immunity and emergence of new variants of SARS-CoV-2 suggest that such measures may jeopardize the attainment of herd immunity due to intermittent lapses in protection. Optimizing vaccine dosing schedules could thus make the difference between periodic occurrence of breakthrough infections or effective control of the pandemic. To this end, we have developed a mechanistic mathematical model of adaptive immune response to vaccines and demonstrated its applicability to COVID-19 mRNA vaccines as a proof-of-concept for future outbreaks. The model was thoroughly calibrated against multiple clinical datasets involving immune response to SARS-CoV-2 infection and mRNA vaccines in healthy and immunocompromised subjects (cancer patients undergoing therapy); the model showed robust clinical validation by accurately predicting neutralizing antibody kinetics, a correlate of vaccine-induced protection, in response to multiple doses of mRNA vaccines. Importantly, we estimated population vulnerability to breakthrough infections and predicted tailored vaccination dosing schedules to maximize protection and thus minimize breakthrough infections, based on the immune status of a sub-population. We have identified a critical waiting window for cancer patients (or, immunocompromised subjects) to allow recovery of the immune system (particularly CD4+ T-cells) for effective differentiation of B-cells to produce neutralizing antibodies and thus achieve optimal vaccine efficacy against variants of concern, especially between the first and second doses. Also, we have obtained optimized dosing schedules for subsequent doses in healthy and immunocompromised subjects, which vary from the CDC-recommended schedules, to minimize breakthrough infections. The developed modeling tool is based on generalized adaptive immune response to antigens and can thus be leveraged to guide vaccine dosing schedules during future outbreaks.

Wetting of dehydrated Pseudomonas fluorescens biofilms grown on glass substrates by an external liquid is employed as a means to investigate the complex morphology of these biofilms along with their capability to interact with external fluids. The porous structure left behind after dehydration induces interesting droplet spreading on the external surface and imbibition into pores upon wetting. Static contact angles and volume loss by imbibition measured right upon droplet deposition indicate that biofilms of higher incubation times show a higher porosity and effective hydrophilicity. Furthermore, during subsequent rotation tests, using Kerberos device, these properties dictate a peculiar forced wetting/spreading behavior. As rotation speed increases a long liquid tail forms progressively at the rear part of the droplet, which stays pinned at all times, while only the front part of the droplet depins and spreads. Interestingly, the experimentally determined retention force for the onset of droplet sliding on biofilm external surface is lower than that on pure glass. An effort is made to describe such complex forced wetting phenomena by presenting apparent contact angles, droplet length, droplet shape contours, and edges position as obtained from detailed image analysis.

Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. Methods: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. Results: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. Conclusion: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.

Biofilms are surface-attached microbial organizations, where microbial species are enclosed in an exo-polysaccharides matrix. Its removal/prevention from surfaces represents an important challenge and a relevant issue in many fields. In this light, antimicrobial peptides (AMPs) represent good candidates to conventional antibiotics due to their selectivity towards targets (cell membranes), rapid action and low tendency to induce bacterial resistance. In our project we are investigating novel techniques to realise nanostructured coatings on surfaces, in order to prevent bacterial adhesion and biofilm formation. We are focusing our attention on a well-known antimicrobial peptide (Magainin-2). Two bacteria models have been considered: E.coli and P. fluorescens. Our goal is to optimize AMPs grafting on surfaces by means of treatment methodologies based on Oxygen plasma technologies. In this work we present a methodology to investigate anti-biofilm efficiency of the nano-functionalized surfaces, compared with the case of the raw surfaces. Biofilm morphologies can be measured using Confocal Laser Scanning Microscopy and image analysis techniques to quantify its structure by measuring quantitative morphological parameters.

Biofilms are resilient to environmental conditions and often resistant even to strong disinfectants. It is crucial to investigate their interfacial properties, which can be effectively characterized by wetting analysis. Wetting phenomena on biofilm surfaces have been poorly investigated in literature, in particular a systematic study of wetting on real biofilm-coated substrates including the application of external body forces (forced wetting, i.e.: centrifugal and gravitational forces) is missing. The aim of this work is to study the role of nutrient and shear flow conditions on wetting properties of Pseudomonas fluorescens dehydrated biofilms, grown on glass substrates. An innovative device (Kerberos®), capable to study spreading/sliding behavior under the application of external body forces, is used here for a systematic analysis of wetting/de-wetting liquid droplets on horizontal substrates under the action of tangential forces. Results prove that, under different growth conditions, (i.e., nutrients and imposed flow), biofilms exhibit different wetting properties. At lower nutrient/shear flow conditions, biofilms show spreading/sliding behavior close to that of pure glass. At higher nutrient and shear flow conditions, droplets on biofilms show spreading followed by imbibition soon after deposition, which leads to peculiar droplet depinning during the rotation test. Wetting properties are derived as a function of the rotation speed from both top and side views videoframes though a dedicated image analysis technique. A detailed analysis of biofilm formation and morphology/topography is also provided here.

Tumour invasion is strongly influenced by microenvironment and, among other parameters, chemical stimuli play an important role. An innovative methodology for the quantitative investigation of chemotaxis in vitro by live imaging of morphology of cell spheroids, in 3D collagen gel, is presented here. The assay was performed by using a chemotactic chamber to impose a controlled gradients of nutrients (glucose) on spheroids, mimicking the chemotactic stimuli naturally occurring in the proximity of blood vessels. Different tumoral cell lines (PANC-1 and HT-1080) are compared to non-tumoral ones (NIH/3T3). Morphology response is observed by means a Time-lapse workstation equipped with an incubating system and quantified by image analysis techniques. Description of invasion phenomena was based on an engineering approach, based on transport phenomena concepts. As expected, NIH/3T3 spheroids are characterized by a limited tendency of cells to invade the surrounding tissue, unlike PANC-1 and HT-1080 that show relatively stronger response to gradients., 14 pages, 9 figures

Fibrosis and solid tumor progression are closely related, with both involving pathways associated with chronic wound dysregulation. Fibroblasts contribute to extracellular matrix (ECM) remodeling in these processes, a crucial step in scarring, organ failure, and tumor growth, but little is known about the biophysical evolution of remodeling regulation during the development and progression of matrix-related diseases including fibrosis and cancer. A 3D collagen-based scaffold model is employed here to mimic mechanical changes in normal (2 kPa, soft) versus advanced pathological (12 kPa, stiff) tissues. Activated fibroblasts grown on stiff scaffolds show lower migration and increased cell circularity compared to those on soft scaffolds. This is reflected in gene expression profiles, with cells cultured on stiff scaffolds showing upregulated DNA replication, DNA repair, and chromosome organization gene clusters, and a concomitant loss of ability to remodel and deposit ECM. Soft scaffolds can reproduce biophysically meaningful microenvironments to investigate early stage processes in wound healing and tumor niche formation, while stiff scaffolds can mimic advanced fibrotic and cancer stages. These results establish the need for tunable, affordable 3D scaffolds as platforms for aberrant stroma research and reveal the contribution of physiological and pathological microenvironment biomechanics to gene expression changes in the stromal compartment.

Wetting of dehydrated

Biofilm is a layer of syntrophic microorganisms stick to each other and to the surface. The importance of biofilms is enormous in various industrial applications and human everyday life. The effects of biofilm could be either positive or negative. Positive effects are encountered in industrial processes, bioremediation, and wastewater treatment. Negative effects are more common with the marine industry being one of the sectors, which confronts severe corrosion problems caused by biofouling on the surfaces of equipment and infrastructures. In space industry, microbial contamination and biofouling adversely affect both crew health and mission-related equipment, the latter including hardware, water systems, piping, and electrical tools. The capacity of biofilms to grow in space environment was confirmed already in 1991. One of the most important surface properties of biofilms is wettability, which dictates not only how a liquid spreads over the uneven external surface of biofilms but also how it penetrates into their porous and morphologically complex structure. To investigate wetting and spreading onto biofilms, model materials are often used to simulate different morphological and functional features of biofilms in a controlled way, for example, soft, deformable, soluble, structured, porous materials. Here, we review recent advances in wetting and spreading on porous and soft deformable surface together with biofilms wetting properties and its importance in space industry. We conclude with a discussion of the main directions for future research efforts regarding biofilm wetting.

The complexity of studying a neoplastic disease relies on understanding specialized cell types within the tumor tissue, which can be recruited and ‘corrupted’ to create a dynamic pro-tumorigenic network called the ‘tumor microenvironment’ (TME). Although the TME is of critical importance during initiation and spread of cancer, relatively little is known about its biophysical evolution during tumor development and progression. In this study a 3D collagen type I–based scaffold model cross-linked with 1,4-butanediol diglycidyl ether (BDDGE) was employed to mimic mechanical changes occurring in normal tissue (2 kPa - soft, So) and advanced cancer tissue (12 kPa - stiff, St) and monitor how these biophysical cues affect the stromal tumor compartment. Viability assays, migration patterns and matrix remodeling together with RNA sequencing investigated cancer-associated fibroblasts (CAFs) response to TME stiffness. In the model, CAFs fail to remodel St scaffolds, showing lower migration and increased cell circularity compared to cells grown on So scaffolds. This behavior is reflected in gene expression profiles, showing an upregulation of DNA replication, DNA repair and chromosome organization gene clusters, with a concommitant loss of their ability to remodel and deposit extracellular matrix after culture on St scaffolds. Soft scaffolds can reproduce biophysically-meaningful microenvironements for tumour early stages investigations, while St scaffolds can better mimic mechanical cues occurring in advanced cancer stages. These results not only establish the need for tunable and affordable 3D scaffolds as effective platforms for cancer research but also reveal the contribution of microenvironment biomechanics in regulating gene expression changes in the stromal tumor tissue compartment.

Smartphones are a promising tool as student response systems (SRS) for interactive teaching due to their widespread diffusion. Here, the main purpose is to assess the efficacy of smartphone-based SRS in large classroom settings of undergraduate Thermodynamics, as representative of engineering courses requiring high-level cognitive skills for problem solving. Four sets of multiple-choice questions were presented during the course. Overall, the results refer to 1055 students between control and SRS classes, each corresponding to a3 years period. One of the main results of this work is the strong linear correlation between the average questionnaire score and the final exam grade (R2 = 0.91). A similar correlation, although with a lower value of R2, is already found in the first questionnaire, thus showing the SRS high predictive power of class performance. The results of this study provide guidance for a quantitative use of smartphone-based SRS in teaching basic disciplines. The SRS monitoring capability allows early detection of struggling students, thus paving the way to personalized tutoring and improved student engagement in active learning practices. This approach is especially important in emergency situations, such as the SARS-Cov-2 pandemic, when distance learning is widely adopted, and remote interactive tools are highly needed.

Phospholipids are the main constituents of cell membranes and act as natural stabilizers of milk fat globules. Phospholipids are used in a wide range of applications, e.g. as emulsifiers in cosmetic, pharmaceutical and food products. While processed emulsion droplets are usually stabilized by a monolayer of phospholipids, cell membranes have a phospholipid bilayer structure and milk fat globules are stabilized by a complex phospholipid trilayer membrane. Despite the broad relevance of phospholipids, there are still many scientific challenges in understanding how their behavior at the fluid-fluid interface affects microstructure, stability, and physico-chemical properties of natural and industrial products. Most of these challenges arise from the experimental difficulties related to the investigation of the molecular arrangement of phospholipids in situ at the fluid-fluid interface and the quantification of their partitioning between the bulk phase and the interface, both under static and flow conditions. This task is further complicated by the presence of other surface-active components, such as proteins, that can interact with phospholipids and compete for space at the interface. Here, we review the methodologies available from the literature to detect and quantify phospholipids, focusing on oil-water interfaces, and highlight current limitations and future perspectives.

Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.

Introduction Biofilm consists of a complex self-produced matrix of polysaccharides, DNA and proteins that protects bacteria from the environment including the host immune system and constitutes the main cause of bacterial resistance against antibiotics. Research is then focused on finding alternative antimicrobial substances able to either hamper biofilm formation or to prevent bacterial growth. Recently, we showed that the antimicrobial peptide Temporin-L impairs E.coli growth by inhibiting cell division (Di Somma et al.; 2020; BBA). Here we investigate the effect of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Materials and methods Biofilm formation in static conditions was performed on coverslips and analyzed by the Crystal Violet assay. Biofilm morphology was assessed using imaging techniques. Investigation of biofilms in dynamic conditions was performed in a flow chamber using a microfluidic system and images were recorded by confocal microscopy. Results The P. fluorescens cells were either grown in the presence of TL or incubated with the antimicrobial peptide after biofilm formation both in static and dynamic conditions using different concentrations of the peptide. When TL was added during cell growth, the peptide affected biofilm formation at 25 µM. Confocal microscopy demonstrated that at this concentration P. fluorescens cells were still alive but a clear disruption of the biofilm architecture was observed. These results had to be ascribed to a specific antibiofilm effect of TL. At 100 µM TL antibiofilm activity biofilm thickness was nearly negligible. When P. fluorescens cells were treated with TL following biofilm formation, confocal images demonstrated that the peptide exerted a strong antibiofilm effect leading to cell detachment and disruption the biofilm architecture. Discussion and Conclusions Investigation of TL effect on P. fluorescens showed that when added during bacterial growth this peptide exerted antibiofilm activity at low concentration impairing biofilm formation both in static and dynamic conditions, leaving most of bacterial cells still alive. However, confocal microscopy measurements could not detect the long necklace-like structures observed in E.coli indicating a different mechanism of action of TL on P. fluorescens. Furthermore, when TL was added to a preformed P. fluorescens biofilm, the peptide showed a strong antibiofilm activity both in static and dynamic conditions, suggesting that TL might penetrate biofilm architecture with a still unknown mechanism leading to disruption of P. fluorescens biofilm.

Biofilms prevention and removal are crucial in many industrial and medical applications. Their complex and cohesive structure provides resistance to cleaning even to strong disinfectants. A key factor for their behavior is the wetting properties of their surfaces. The main goal of this work is to study the wetting properties of biofilms produced by bacteria Pseudomonas fluorescens. Biofilms are obtained on glass coupons under well controlled flow conditions, using custom-made flow cell devices. Different nutrient concentration and shear flow conditions are investigated. Biofilm wetting properties are examined under imposed external body forces (forced wetting) through a specialized device, named Kerberos®. Kerberos® is capable of subjecting sessile droplets to varying tilting angles and centrifugal forces while monitoring the variation of the droplet shape in X, Y and Z-directions through three Wi-Fi cameras. Wetting experiments are carried out using water-based solution (dye solution) droplets on biofilm-coated glass coupons. In this work, spreading/sliding behaviour of droplets are investigated only on horizontal substrates (no tilting) under the action of centrifugal forces. Apart from wetting properties, biofilm growth kinetics and surface morphology at different nutrient and shear flow conditions are also assessed. Results show that, according to the different growth conditions, biofilms present different wetting properties. At lower nutrient concentration and shear flow conditions, spreading and sliding behaviour are similar to that observed in glass coupons in the absence of biofilm. At higher nutrient and shear flow conditions, spontaneous wicking of the biofilm occurs the moment of droplet deposition on the biofilm leading to irregular and jagged shapes of droplets, while on the contrary water droplets look like smooth spherical sections on pure glass. The spontaneous wicking affects the droplet initial shape and so the wetting behaviour during the subsequent rotation tests. In each examined condition, biofilms show hydrophilic properties.

Biofilms are bacterial communities embedded in an extracellular matrix, able to adhere to surfaces. A deeper knowledge of the biofilm as a whole will aid the development of efficient methods to control deleterious biofilms (clinical biofilms, biofouling) or to enhance beneficial ones (waste-water treatment, bio-filtration). P. fluorescens has been widely studied, this strain produces bioactive secondary metabolites, and forms biofilms[1]. Several experimental set-ups have been widely used for in vitro biofilm cultivation of P. fluorescens, even if a deep characterization among different culture conditions is still lacking in the literature. This work, based on previous studies[2], is focused on the investigation of growth conditions on biofilm structure and properties. Growth kinetics of P. fluorescens biofilms was characterized in vitro under stagnant and flow-controlled conditions, using a rotating annular bioreactor. Two different supports in borosilicate glass and polycarbonate have been used. Bacterial growth kinetics has been measured through bio-turbidity analysis and TOC/DOC quantification. Biofilm morphology has been quantified through optical microscopy and image analysis by measuring the fraction of support surface covered by biofilm. The wetting properties of the biofilm layers have been investigated by using an innovative device, named Kerberos®, able to control centrifugal and gravitational forces acting on a single droplet placed on a surface[3]. The evolution of the droplet shape and position was measured as function of the imposed stress, to quantify wetting of different biofilm coated samples, following already assessed methodologies[4]. Different chemo-physical environments, investigated by changing growth medium, physical support, and imposed flow stress, induced different growth kinetics, biofilm morphology, and wetting properties. Accurate experimental measurements allowed us to estimate in a quantitative way the influence of investigated parameters on specific morphologic measurements. [1] Brittan S. Scales and others, ‘Microbiology, Genomics, and Clinical Significance of the Pseudomonas Fluorescens Species Complex, an Unappreciated Colonizer of Humans’, Clinical Microbiology Reviews, 27.4 (2014), 927–48 . [2] Federica Recupido and others, ‘The Role of Flow in Bacterial Biofilm Morphology and Wetting Properties’, Colloids and Surfaces B: Biointerfaces, 2020 . [3] Sotiris P. Evgenidis and others, ‘Kerberos: A Three Camera Headed Centrifugal/Tilting Device for Studying Wetting/Dewetting under the Influence of Controlled Body Forces’, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2017 . [4] Inmaculada Ríos-López and others, ‘Effect of Initial Droplet Shape on the Tangential Force Required for Spreading and Sliding along a Solid Surface’, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2018 .

Skin is a complex structured system primarily involved in Transdermal Drug Delivery (TDD). The outer stratum corneum represents the main barrier to the entrance of external molecules. Nowadays, none of the recognized methods, used for the investigation of penetration processes, is able to give a complete overview of the transport mechanisms involved. Standard protocols are based on the use of human or animal skin samples, which are difficult and expensive to obtain. Here, we present a novel experimental setup to investigate TDD by using Confocal Laser Scanning Microscopy and image analysis. The methodology is based on diffusion experiments of fluorescent-labelled fluids (water, oil, and oil-in-water emulsions), in a model matrix. Using an agarose gel as model for a proof of concept, different penetration efficiencies were observed, suggesting an important role of both chemical composition and fluid microstructure on the transport mechanism. An adequate model system should be used to better mimic the stratum corneum morphology and properties. To this aim, several bicontinuous emulsion gels, obtained from an emulsification process, were preliminary formulated and suggested as model systems to mimic the stratum corneum. In this work, we define the key directions for the development of an innovative approach to study the penetration of formulations through the skin.

The Wound Healing (WH) assay is widely used to investigate cell migration in vitro, in order to reach a better understanding of many physiological and pathological phenomena. Several experimental factors, such as uneven cell density among different samples, can affect the reproducibility and reliability of this assay, leading to a discrepancy in the wound closure kinetics among data sets corresponding to the same cell sample. We observed a linear relationship between the wound closure velocity and cell density, and suggested a novel methodological approach, based on transport phenomena concepts, to overcome this source of error on the analysis of the Wound Healing assay. In particular, we propose a simple scaling of the experimental data, based on the interpretation of the wound closure as a diffusion-reaction process. We applied our methodology to the MDA-MB-231 breast cancer cells, whose motility was perturbed by silencing or over-expressing genes involved in the control of cell migration. Our methodological approach leads to a significant improvement in the reproducibility and reliability in the in vitro WH assay.

Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.

Aberrant activation of epithelial-to-mesenchymal transition has been shown to correlate with triple-negative breast cancer (TNBC) progression and metastasis. Thus, the induction of the reverse process might offer promising opportunities to restrain TNBC metastatic spreading and related mortality. Recently, the Annurca apple polyphenol extract (APE) has been highlighted as a multi-faceted agent that selectively kills TNBC cells by ROS generation and sustained JNK activation. Here, by qualitatively and quantitatively monitoring the real-time movements of live cells we provided the first evidence that APE inhibited the migration of MDA-MB-231 and MDA-MB-468 TNBC cells and downregulated metalloproteinase-2 and metalloproteinase-9. In MDA-MB-231 cells APE decreased SMAD-2/3 and p-SMAD-2/3 levels, increased E-cadherin/N-cadherin protein ratio, induced the switch from N-cadherin to E-cadherin expression and greatly reduced vimentin levels. Confocal and scanning electron microscopy imaging of APE-treated MDA-MB-231 cells evidenced a significant cytoskeletal vimentin and filamentous actin reorganization and revealed considerable changes in cell morphology highlighting an evident transition from the mesenchymal to epithelial phenotype with decreased migratory features. Notably, all these events were reverted by N-acetyl-l-cysteine and JNK inhibitor SP600125 furnishing evidence that APE exerted its effects through the activation of ROS/JNK signaling. The overall data highlighted APE as a potential preventing agent for TNBC metastasis.

Hypothesis While the phase behavior of aqueous surfactant solutions is usually described in term of the equilibrium microstructures of lyotropic liquid crystals, the transformations which take place when a phase turns into another one, either by changing the concentration or the temperature, are still to be elucidated. A simultaneous determination of concentration and microstructure is at order to elucidate the phase behavior under changing conditions, such as in a dissolution experiment. Experiments Confocal Raman micro-spectroscopy and time-lapse polarized light microscopy are combined to study the phase transitions taking place in the dissolution of a common anionic surfactant (sodium laurylethersulfate) in water. Findings By comparing Raman concentration profiles and polarized light images, it is found that the aqueous solution, with initial surfactant concentration of 72 wt%, undergoes a sequence of complex microstructural transformations including distortion of the initial lamellar phase, formation of an intermediate striated texture, which can be considered as a precursor of a cubic phase, and a heterogeneous hexagonal phase going through a transition region before turning into a micellar phase. The effects of the sodium counter-ion and of water confinement are also investigated by analyzing the OH-stretching bands.

Biofilms are bacterial communities embedded in an extracellular matrix, able to adhere to surfaces. Different experimental set-ups are widely used for in vitro biofilm cultivation; however, a well-defined comparison among different culture conditions, especially suited to interfacial characterization, is still lacking in the literature. The main objective of this work is to study the role of flow on biofilm formation, morphology and interfacial properties. Three different in vitro setups, corresponding to stagnant, shaking, and laminar flow conditions (custom-made flow cell), are used in this work to grow single strain biofilms of Pseudomonas fluorescens AR 11 on glass coupons. Results show that flow conditions significantly influenced biofilm formation kinetics, affecting mass transfer and cell attachment/detachment processes. Distinct morphological patterns are found under different flow regimes. Static contact angle data do not depend significantly on biofilm growth conditions in the parametric range investigated in this work.

Concentrated aqueous solutions of surfactants, often referred to as pastes, experience complex phase and rheology changes upon dissolution in water, which is a typical step in the production of liquid detergents. During the dilution process, depending on water content, surfactant molecules can arrange in different morphologies, such as lamellar or cubic and hexagonal structures. These phases are characterized by different physico-chemical properties, such as viscosity or diffusivity, which lead to non-simple transport mechanisms during the dissolution process. In this work, we investigate the dissolution of concentrated Sodium Lauryl Ether Sulfate (SLES) pastes in water under quiescent conditions by coupling different experimental techniques. A thorough rheological characterization of the system showed non-monotonic changes of several orders of magnitude in its viscosity and viscoelastic moduli as a function of water content. Time-lapse microscopy allowed us to image the dynamic evolution of the phase changes as water penetrated in a disk-shaped sample (with the same geometry used in rheological tests). Numerical simulation, based on a simple diffusion-based multi-parameter model is shown to describe satisfactorily SLES dissolution data.

Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways that cooperate to restore tissue integrity and homeostasis. To facilitate healing, injuries need to be promptly treated. Recently, the interest in alternatives to antibiotics has been raised given the widespread phenomenon of antibiotic resistance. Among these alternatives, the use of silver appears to be a valid option, so a resurgence in its use has been recently observed. In particular, in contrast to ionic silver, colloidal silver, a suspension of metallic silver particles, shows antibacterial activity displaying less or no toxicity. However, the human health risks associated with exposure to silver nanoparticles (NP) appear to be conflicted, and some studies have suggested that it could be toxic in different cellular contexts. These potentially harmful effects of silver NP depend on various parameters including NP size, which commonly range from 1 to 100 nm. In this study, we analyzed the effect of a colloidal silver preparation composed of very small and homogeneous nanoparticles of 0.62 nm size, smaller than those previously tested. We found no adverse effect on the cell proliferation of HaCaT cells, even at high NP concentration. Time-lapse microscopy and indirect immunofluorescence experiments demonstrated that this preparation of colloidal silver strongly increased cell migration, re-modeled the cytoskeleton, and caused recruitment of E-cadherin at cell-cell junctions of human cultured keratinocytes.

Cancer continues to be among the leading healthcare problems worldwide, and efforts continue not just to find better drugs, but also better drug delivery methods. The need for delivering cytotoxic agents selectively to cancerous cells, for improved safety and efficacy, has triggered the application of nanotechnology in medicine. This effort has provided drug delivery systems that can potentially revolutionize cancer treatment. Nanocarriers, due to their capacity for targeted drug delivery, can shift the balance of cytotoxicity from healthy to cancerous cells. The field of cancer nanomedicine has made significant progress, but challenges remain that impede its clinical translation. Several biophysical barriers to the transport of nanocarriers to the tumor exist, and a much deeper understanding of nano-bio interactions is necessary to change the status quo. Mathematical modeling has been instrumental in improving our understanding of the physicochemical and physiological underpinnings of nanomaterial behavior in biological systems. Here, we present a comprehensive review of literature on mathematical modeling works that have been and are being employed towards a better understanding of nano-bio interactions for improved tumor delivery efficacy.

This study is addressed to the effect of the elasticity of the continuous phase on confined flow of low viscosity emulsions, a topic of interest in several applications. A Newtonian silicone oil was used as the dispersed phase, while three different water-based fluids, a Newtonian and two Boger fluids, were used as the continuous phase. All the fluids have the same viscosity (ca. 0.05 Pa s). Emulsions were prepared by feeding the two phases to a T-junction followed by a membrane. When the continuous phase was a Boger fluid, the average drop size of the so obtained emulsions was higher (as compared to the Newtonian continuous phase) and a bimodal distribution was observed. Such lower mixing efficiency for a Boger continuous phase is in line with the result for an isolated droplet that breakup is hindered by matrix elasticity. High-speed microscopy visualization of the emulsions flowing through a microcapillary allowed one to determine the velocity profiles and the droplet spatial distribution as a function of the shear rate. In all the experiments, the formation of a droplet-rich core and of a dropletdevoid wall region D was observed, with a significant enhancement when the continuous phase was a Boger fluid (where values of D as high as 37% of capillary radius were found). So, confinement here refers to the fact that, although the droplets are much smaller than capillary radius, the droplet-free layer takes up a significant fraction of the capillary radius. The observed nonhomogeneous droplet spatial distribution was not due to a transient entry effect, as shown by imaging the emulsions flow at different sections from the capillary inlet, but can be explained by a balance between collision-induced drop diffusion and wall lift force. The former acts to homogenize droplet spatial distribution, while the latter tends to push droplets away from the wall and is enhanced by matrix elasticity. These effects lead to the accumulation of larger droplets around the center of the channel, while smaller droplets are pushed toward the periphery. The velocity profiles for the Boger continuous phase were flat in the droplet-rich core, thus suggesting that the confined emulsions behave as a Bingham fluid. The corresponding yield stress was determined by data fitting of the velocity profiles and rather large values were so obtained. This result is consistent with the high values, close to the maximum packing for spherical particles in binary dispersions, of the droplet volume fraction in the core, which were estimated by measuring the thickness of the droplet-devoid annular region as a function of the applied flow rate and making a volume balance. Overall, the results of this work bear strong analogies with the confined flow of other concentrated suspensions of deformable particles, such as red blood cells in microcirculation, where the formation of a cell-free layer at the wall, which is referred to as margination, has important pathophysiological implications. These findings suggest that low viscosity emulsions with an elastic matrix under confined flow can be used as a tunable model system of biomedical relevance.

This paper explores the effects of electrification of water sprays on the absorption of sulphur dioxide from exhaust gases. The results of this paper are based on experimental evidences related to the use of electrified (distilled) water in a lab-scale scrubber, coupled with independent determination of droplets morphology and charge. Experiments clearly show that electrification is associated with an appreciable improvement of sulphur dioxide absorption rate, reducing the height of transfer unit, HTU, of the lab-scale unit by at least 40%, with peaks over 80% with low supplementary costs, and it candidates to be a valuable innovation for spray tower absorbers. Water electrification is accompanied by chemical-physics and morphological modifications of the sprayed droplets influencing the conventional absorption rate of soluble gases. The experimental and theoretical evidences indicate the need to analyse the electrified absorption within a new and integrated theoretical framework that we define chemi-electro-hydrodynamics (CEHD).

Multiphase shear flows often show banded structures that affect the global behavior of complex fluids e.g. in microdevices. Here we investigate numerically the banding of emulsions, i.e. the formation of regions of high and low volume fractions, alternated in the vorticity direction and aligned with the flow (shear bands). These bands are associated with a decrease of the effective viscosity of the system. To understand the mechanism of experimentally observed banding, we have performed interface-resolved simulations of the two-fluid system. The experiments were performed starting with a random distribution of droplets, which under the applied shear, evolve in time resulting in a phase separation. To numerically reproduce this process, the banded structures are initialized in a narrow channel confined by two walls moving in opposite directions. We find that the initial banded distribution is stable when droplets are free to merge and unstable when coalescence is prevented. In this case, additionally, the effective viscosity of the system increases, resembling the rheological behavior of suspensions of deformable particles. Droplet coalescence, on the other hand, allows emulsions to reduce the total surface of the system and, hence, the energy dissipation associated with the deformation, which in turn reduces the effective viscosity.

Phospholipids, which are amphiphilic biomolecules composed of a polar head group and two nonpolar fatty acid tails, play a central role in cellular and body functions. The most common phospholipid is phosphatidylcholine (PC), which is also widespread used as a surfactant in colloidal systems, such as oil-in-water (O/W) emulsions, for several applications ranging from food to cosmetics. In these systems, PC tends to arrange at the interface between polar and nonpolar phases. Specific identification of these molecules at the interface is still an ambitious task. In this work, we propose immunofluorescence and confocal microscopy as valuable tools for the localization of PC at the interface of O/W emulsions, where phospholipids are used as surfactants. The protocol required the use of a primary antibody (TEPC-15), specific for choline, and a secondary fluorescently-labelled antibody, which selectively binds to the primary antibody. By using this approach, we were able to visualize choline-based phospholipids in cell membranes, lipid-based emulsions and in PC films and to estimate molecule concentration by image analysis techniques. We also investigated the interference of proteins on the staining procedure. We attributed this interference to possible molecular interactions close to the interface, which were found to depend on protein concentration. This innovative approach can have a relevant impact in a wide range of interfacial engineering applications, such as tuning emulsion microstructure and stability.

Blood is a complex fluid with non-Newtonian characteristics and consists primarily of a concentrated suspension of red blood cells (RBCs) characterized by high deformability and aggregability. The majority of both research and clinical investigations on blood rheology is based on steady shear measurements aimed at determining blood viscosity as a function of shear rate. On the other hand, investigations of blood rheology in the linear viscoelastic regime are sparse in the literature and currently limited. In principle, small amplitude oscillatory flow is best suited to study blood microstructure and rheology under quasi-static conditions, which are relevant in a range of applications, from blood storage to blood aggregability testing. Here, we present the first systematic experimental investigation of blood rheological behavior in the linear viscoelastic regime, by performing oscillatory shear measurements by conventional bulk rheology. The storage (G′) and loss (G″) moduli of whole human blood have been measured over an extended range of frequencies [0.1–30 rad/s]. Data show that G″ predominates over G′ across the entire tested range of frequency. By comparing steady and oscillatory shear viscosity, it was found that the Cox-Merz rule is followed to a good approximation, with higher deviations at small shear rate/frequencies. The effects of RBC volume fraction and of aggregating media (i.e., dextran solution at two different concentrations) have been also investigated. Overall, our results are consistent with the behavior of a weakly attractive suspension, where RBCs form reversible aggregates that can be broken by the action of flow.

Il saggio fornisce aperture concettuali capaci di definire una prospettiva urbanistica "altra", che ricomprenda finalmente il dato sociale, politico e ambientale. Lo scritto riassume sinteticamente parte dei risultati della riflessione collettiva sui contenuti della legge urbanistica Emilia-Romagna n. 24/2017, contenuta nel volume “Consumo di luogo. Neoliberismo nel disegno di legge urbanistica dell’Emilia-Romagna”.