Novel Optical Methodology Unveils the Impact of a Polymeric Pour-Point Depressant on the Phase Morphology of Waxy Crude Oils.

Crude oil, also known as petroleum, plays a crucial role in global economies, politics, and technological advancements due to its widespread applications in industrial organic chemistry. Despite environmental concerns, the dwindling supply of easily accessible oil reservoirs necessitates the exploration of unconventional resources, such as heavy and extra-heavy oils. These oils, characterized by high viscosity and complex composition, pose challenges in extraction, transportation, and refinement. With decreasing temperatures, heavy oils undergo phase changes, with transitions from Newtonian to non-Newtonian fluid behavior, leading to difficulties in transportation. Alternative methods, such as the use of polymeric pour-point depressants, help mitigate flowability issues by preventing wax precipitation. Understanding the properties of waxy crude oil, such as the wax appearance temperature (WAT), is crucial for effective mitigation strategies. The objective of this research is to determine the WATs of different types of waxy crude oils through a comparative analysis using advanced techniques such as cross-polar microscopy (CPM), standard rheology, and differential scanning calorimetry (DSC). Disparities in WAT identified through different analytical methods highlight the potential of microscopy to enhance our understanding of complex fluid dynamics in real time in order to proactively identify and address crystallization issues in oilfields.

i-Rheo-optical assay: Measuring the viscoelastic properties of multicellular spheroids.

This study introduces a novel mechanobiology assay, named "i-Rheo-optical assay", that integrates rheology with optical microscopy for analysing the viscoelastic properties of multicellular spheroids. These spheroids serve as three-dimensional models resembling tissue structures. The innovative technique enables real-time observation and quantification of morphological responses to applied stress using a cost-effective microscope coverslip for constant compression force application. By bridging a knowledge gap in biophysical research, which has predominantly focused on the elastic properties while only minimally exploring the viscoelastic nature in multicellular systems, the i-Rheo-optical assay emerges as an effective tool. It facilitates the measurement of broadband viscoelastic compressional moduli in spheroids, here derived from cancer (PANC-1) and non-tumoral (NIH/3T3) cell lines during compression tests. This approach plays a crucial role in elucidating the mechanical properties of spheroids and holds potential for identifying biomarkers to discriminate between healthy tissues and their pathological counterparts. Offering comprehensive insights into the biomechanical behaviour of biological systems, i-Rheo-optical assay marks a significant advancement in tissue engineering, cancer research, and therapeutic development.

The role of air relative humidity on the wettability of Pseudomonas fluorescens AR11 biofilms.

Biofilms are complex porous materials formed by microorganisms, polysaccharides, proteins, eDNA, inorganic matter, and water. They are ubiquitous in various environmental niches and are known to grow at solid-liquid, solid-air and air-liquid interfaces, often causing problems in several industrial and sanitary fields. Their removal is a challenge in many applications and numerous studies have been conducted to identify promising chemical species as cleaning agents. While these substances target specific components of biofilm structure, the role of water content in biofilm, and how it can influence wettability and detergent absorption have been quite neglected in the literature. Estimating water content in biofilm is a challenging task due to its heterogeneity in morphology and chemical composition. In this study, we controlled water content in Pseudomonas fluorescens AR 11 biofilms grown on submerged glass slides by regulating environmental relative humidity after drying. Interfacial properties of biofilm were investigated by measuring wetting of water and soybean oil. The morphology of biofilm structure was evaluated using Confocal Laser Scanning Microscopy and Scanning Electron Microscopy. The results showed that biofilm water content has a significant and measurable effect on its wettability, leading to the hypothesis that a preliminary control of water content can play a crucial role in biofilm removal process.

Electrospun Chitosan-Based Nanofibrous Coating for the Local and Sustained Release of Vancomycin.

As the population ages, the number of vascular surgery procedures performed increases. Older adults often have multiple comorbidities, such as diabetes and hypertension, that increase the risk of complications from vascular surgery including vascular graft infection (VGI). VGI is a serious complication with significant morbidity, mortality, and healthcare costs. Here, we aimed to develop a nanofibrous chitosan-based coating for vascular grafts loaded with different concentrations of the vancomycin antibiotic vancomycin (VAN). Blending chitosan with poly(vinyl alcohol) or poly(ethylene oxide) copolymers improved solubility and ease of spinning. Thermal gravimetric analysis and Fourier transform infrared spectroscopy confirmed the presence of VAN in the nanofibrous membranes. Kinetics of VAN release from the nanofibrous mats were evaluated using high-performance liquid chromatography, showing a burst followed by sustained release over 24 h. To achieve longer sustained release, a poly(lactic-co-glycolic acid) coating was applied, resulting in extended release of up to 7 days. Biocompatibility assessment using human umbilical vein endothelial cells demonstrated successful attachment and viability of the nanofiber patches. Our study provides insights into the development of a drug delivery system for vascular grafts aimed at preventing infection during implantation, highlighting the potential of electrospinning as a promising technique in the field of vascular surgery.

Designing Advanced Drug Delivery Systems: Core-Shell Alginate Particles through Electro-Fluid Dynamic Atomization.

Innovations in drug delivery systems are crucial for enhancing therapeutic efficiency. Our research presents a novel approach based on using electro-fluid dynamic atomization (EFDA) to fabricate core-shell monophasic particles (CSMp) from sodium alginate blends of varying molecular weights. This study explores the morphological characteristics of these particles in relation to material properties and process conditions, highlighting their potential in drug delivery applications. A key aspect of our work is the development of a mathematical model that simulates the release kinetics of small molecules, specifically sodium diclofenac. By assessing the diffusion properties of different molecules and gel formulations through transport and rheological models, we have created a predictive tool for evaluating the efficiency of these particles in drug delivery. Our findings underscore two critical, independent parameters for optimizing drug release: the external shell thickness and the diffusivity ratios within the dual layers. This allows for precise control over the timing and intensity of the release profile. This study advances our understanding of EFDA in the fabrication of CSMp and offers promising avenues for enhancing drug delivery systems by tailoring release profiles through particle characteristic manipulation.

Hybrid Cellular Automata Modeling Reveals the Effects of Glucose Gradients on Tumour Spheroid Growth.

PURPOSE: In recent years, mathematical models have become instrumental in cancer research, offering insights into tumor growth dynamics, and guiding the development of pharmacological strategies. These models, encompassing diverse biological and physical processes, are increasingly used in clinical settings, showing remarkable predictive precision for individual patient outcomes and therapeutic responses. METHODS: Motivated by these advancements, our study introduces an innovative in silico model for simulating tumor growth and invasiveness. The automated hybrid cell emulates critical tumor cell characteristics, including rapid proliferation, heightened motility, reduced cell adhesion, and increased responsiveness to chemotactic signals. This model explores the potential evolution of 3D tumor spheroids by manipulating biological parameters and microenvironment factors, focusing on nutrient availability. RESULTS: Our comprehensive global and local sensitivity analysis reveals that tumor growth primarily depends on cell duplication speed and cell-to-cell adhesion, rather than external chemical gradients. Conversely, tumor invasiveness is predominantly driven by chemotaxis. These insights illuminate tumor development mechanisms, providing vital guidance for effective strategies against tumor progression. Our proposed model is a valuable tool for advancing cancer biology research and exploring potential therapeutic interventions.

Exploring Cell Migration Mechanisms in Cancer: From Wound Healing Assays to Cellular Automata Models.

PURPOSE: Cell migration is a critical driver of metastatic tumor spread, contributing significantly to cancer-related mortality. Yet, our understanding of the underlying mechanisms remains incomplete. METHODS: In this study, a wound healing assay was employed to investigate cancer cell migratory behavior, with the aim of utilizing migration as a biomarker for invasiveness. To gain a comprehensive understanding of this complex system, we developed a computational model based on cellular automata (CA) and rigorously calibrated and validated it using in vitro data, including both tumoral and non-tumoral cell lines. Harnessing this CA-based framework, extensive numerical experiments were conducted and supported by local and global sensitivity analyses in order to identify the key biological parameters governing this process. RESULTS: Our analyses led to the formulation of a power law equation derived from just a few input parameters that accurately describes the governing mechanism of wound healing. This groundbreaking research provides a powerful tool for the pharmaceutical industry. In fact, this approach proves invaluable for the discovery of novel compounds aimed at disrupting cell migration, assessing the efficacy of prospective drugs designed to impede cancer invasion, and evaluating the immune system's responses.

A modeling-based approach to optimize COVID-19 vaccine dosing schedules for improved protection.

While the development of different vaccines slowed the dissemination of SARS-CoV-2, the occurrence of breakthrough infections has continued to fuel the COVID-19 pandemic. To secure at least partial protection in the majority of the population through 1 dose of a COVID-19 vaccine, delayed administration of boosters has been implemented in many countries. However, waning immunity and emergence of new variants of SARS-CoV-2 suggest that such measures may induce breakthrough infections due to intermittent lapses in protection. Optimizing vaccine dosing schedules to ensure prolonged continuity in protection could thus help control the pandemic. We developed a mechanistic model of immune response to vaccines as an in silico tool for dosing schedule optimization. The model was calibrated with clinical data sets of acquired immunity to COVID-19 mRNA vaccines in healthy and immunocompromised participants and showed robust validation by accurately predicting neutralizing antibody kinetics in response to multiple doses of COVID-19 mRNA vaccines. Importantly, by estimating population vulnerability to breakthrough infections, we predicted tailored vaccination dosing schedules to minimize breakthrough infections, especially for immunocompromised individuals. We identified that the optimal vaccination schedules vary from CDC-recommended dosing, suggesting that the model is a valuable tool to optimize vaccine efficacy outcomes during future outbreaks.

Forced Wetting Properties of Bacteria-Laden Droplets Experiencing Initial Evaporation.

Microbial adhesion and spreading on surfaces are crucial aspects in environmental and industrial settings being also the early stage of complex surface-attached microbial communities known as biofilms. In this work, Pseudomonas fluorescens-laden droplets on hydrophilic substrates (glass coupons) are allowed to partially evaporate before running wetting measurements, to study the effect of evaporation on their interfacial behavior during spillover or splashing. Forced wetting is investigated by imposing controlled centrifugal forces, using a novel rotatory device (Kerberos). At a defined evaporation time, results for the critical tangential force required for the inception of sliding are presented. Microbe-laden droplets exhibit different wetting/spreading properties as a function of the imposed evaporation times. It is found that evaporation is slowed down in bacterial droplets with respect to nutrient medium ones. After sufficient drying times, bacteria accumulate at droplet edges, affecting the droplet shape and thus depinning during forced wetting tests. Droplet rear part does not pin during the rotation test, while only the front part advances and spreads along the force direction. Quantitative results obtained from the well-known Furmidge's equation reveal that force for sliding inception increases as evaporation time increases. This study can be of support for control of biofilm contamination and removal and possible design of antimicrobial/antibiofouling surfaces.

Collective rotational motion of freely expanding T84 epithelial cell colonies.

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.

Migration of surface-associated microbial communities in spaceflight habitats.

Astronauts are spending longer periods locked up in ships or stations for scientific and exploration spatial missions. The International Space Station (ISS) has been inhabited continuously for more than 20 years and the duration of space stays by crews could lengthen with the objectives of human presence on the moon and Mars. If the environment of these space habitats is designed for the comfort of astronauts, it is also conducive to other forms of life such as embarked microorganisms. The latter, most often associated with surfaces in the form of biofilm, have been implicated in significant degradation of the functionality of pieces of equipment in space habitats. The most recent research suggests that microgravity could increase the persistence, resistance and virulence of pathogenic microorganisms detected in these communities, endangering the health of astronauts and potentially jeopardizing long-duration manned missions. In this review, we describe the mechanisms and dynamics of installation and propagation of these microbial communities associated with surfaces (spatial migration), as well as long-term processes of adaptation and evolution in these extreme environments (phenotypic and genetic migration), with special reference to human health. We also discuss the means of control envisaged to allow a lasting cohabitation between these vibrant microscopic passengers and the astronauts.

Nanoparticle Coatings on Glass Surfaces to Prevent Pseudomonas fluorescens AR 11 Biofilm Formation.

Microbial colonization of surfaces is a sanitary and industrial issue for many applications, leading to product contamination and human infections. When microorganisms closely interact with a surface, they start to produce an exo-polysaccaridic matrix to adhere to and protect themselves from adverse environmental conditions. This type of structure is called a biofilm. The aim of our work is to investigate novel technologies able to prevent biofilm formation by surface coatings. We coated glass surfaces with melanin-ZnO2, melanin-TiO2, and TiO2 hybrid nanoparticles. The functionalization was performed using cold plasma to activate glass-substrate-coated surfaces, that were characterized by performing water and soybean oil wetting tests. A quantitative characterization of the antibiofilm properties was done using Pseudomonas fluorescens AR 11 as a model organism. Biofilm morphologies were observed using confocal laser scanning microscopy and image analysis techniques were used to obtain quantitative morphological parameters. The results highlight the efficacy of the proposed surface coating to prevent biofilm formation. Melanin-TiO2 proved to be the most efficient among the particles investigated. Our results can be a valuable support for future implementation of the technique proposed here in an extended range of applications that may include further testing on other strains and other support materials.

Compressional stress stiffening & softening of soft hydrogels - how to avoid artefacts in their rheological characterisation.

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.

A modeling-based approach to optimize COVID-19 vaccine dosing schedules for improved protection.

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, delayed administration 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.

5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway.

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.

Studying Activated Fibroblast Phenotypes and Fibrosis-Linked Mechanosensing Using 3D Biomimetic Models.

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 properties of dehydrated biofilms under different growth conditions.

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 through a dedicated image analysis technique. A detailed analysis of biofilm formation and morphology/topography is also provided here.

Wetting of Dehydrated Hydrophilic Pseudomonas fluorescens Biofilms under the Action of External Body Forces.

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.

Quantitative methods to detect phospholipids at the oil-water interface.

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.