Engineering Cell Instructive Microenvironments for In Vitro Replication of Functional Barrier Organs.

Multicellular organisms exhibit synergistic effects among their components, giving rise to emergent properties crucial for their genesis and overall functionality and survival. Morphogenesis involves and relies upon intricate and biunivocal interactions among cells and their environment, that is, the extracellular matrix (ECM). Cells secrete their own ECM, which in turn, regulates their morphogenetic program by controlling time and space presentation of matricellular signals. The ECM, once considered passive, is now recognized as an informative space where both biochemical and biophysical signals are tightly orchestrated. Replicating this sophisticated and highly interconnected informative media in a synthetic scaffold for tissue engineering is unattainable with current technology and this limits the capability to engineer functional human organs in vitro and in vivo. This review explores current limitations to in vitro organ morphogenesis, emphasizing the interplay of gene regulatory networks, mechanical factors, and tissue microenvironment cues. In vitro efforts to replicate biological processes for barrier organs such as the lung and intestine, are examined. The importance of maintaining cells within their native microenvironmental context is highlighted to accurately replicate organ-specific properties. The review underscores the necessity for microphysiological systems that faithfully reproduce cell-native interactions, for advancing the understanding of developmental disorders and disease progression.

Exploring the evolution of bacterial cellulose precursors and their potential use as cellulose-based building blocks.

Natural polymers have found increased use in a wider range of applications due to their less harmful effects. Notably, bacterial cellulose has gained significant consideration due to its exceptional physical and chemical properties and its substantial biocompatibility, which makes it an attractive candidate for several biomedical applications. This study attempts to thoroughly unravel the microstructure of bacterial cellulose precursors, known as bioflocculants, which to date have been poorly characterised, by employing both electron and optical microscopy techniques. Here, starting from bioflocculants from Symbiotic Culture of Bacteria and Yeast (SCOBY), we proved that their microstructural features, such as porosity percentage, cellulose assembly degree, fibres' density and fraction, change in a spatio-temporal manner during their rising toward the liquid-air interface. Furthermore, our research identified a correlation between electron and optical microscopy parameters, enabling the assessment of bioflocculants' microstructure without necessitating offline sample preparation procedures. The ultimate goal was to determine their potential suitability as a novel cellulose-based building block material with tuneable structural properties. Our investigations substantiate the capability of SCOBY bioflocculants, characterized by distinct microstructures, to successfully assemble within a microfluidic device, thereby generating a cellulose sheet endowed with specific and purposefully designed structural features.

Exploring altered bovine sperm trajectories by sperm tracking in unconfined conditions.

Mammalian sperm motility is getting more relevant due to rising infertility rates worldwide, generating the need to improve conventional analysis and diagnostic approaches. Nowadays, computer assisted sperm analysis (CASA) technologies represent a popular alternative to manual examination which is generally performed by observing sperm motility in very confined geometries. However, under physiological conditions, sperm describe three-dimensional motility patterns which are not well reconstructed by the limited depth of standard acquisition chambers. Therefore, affordable and more versatile alternatives are needed. Here, a motility analysis in unconfined conditions is proposed. In details, the analysis is characterized by a significant longer duration -with respect to conventional systems- with the aim to observe eventually altered motility patterns. Brightfield acquisition in rectangular glass capillaries captured frozen-thawed bovine spermatozoa which were analyzed by means of a self-written tracking routine and classified in sub-populations, based on their curvilinear velocity. To test the versatility of our approach, cypermethrin -a commonly used pesticides- known to be responsible for changes in sperm motility was employed, assessing its effect at three different time-steps. Experimental results showed that such drug induces an increase in sperm velocity and progressiveness as well as circular pattern formation, likely independent of wall interactions. Moreover, this resulted in a redistribution of sperm with the rapid class declining in number with time, but still showing an overall velocity increase. The flexibility of the approach permits parameter modifications with the experimental needs, allowing us to conduct a comprehensive examination of sperm motility. This adaptability facilitated data acquisition which can be computed at different frame rates, extended time periods, and within deeper observation chambers. The suggested approach for sperm analysis exhibits potential as a valuable augmentation to current diagnostic instruments.

Adverse Effect of Metallic Gold and Silver Nanoparticles on Xenopus laevis Embryogenesis.

Exposure to metal nanoparticles is potentially harmful, particularly when occurring during embryogenesis. In this study, we tested the effects of commercial AuNPs and AgNPs, widely used in many fields for their features, on the early development of Xenopus laevis, an anuran amphibian key model species in toxicity testing. Through the Frog Embryo Teratogenesis Assay-Xenopus test (FETAX), we ascertained that both nanoparticles did not influence the survival rate but induced morphological anomalies like modifications of head and branchial arch cartilages, depigmentation of the dorsal area, damage to the intestinal brush border, and heart rate alteration. The expression of genes involved in the early pathways of embryo development was also modified. This study suggests that both types of nanoparticles are toxic though nonlethal, thus indicating that their use requires attention and further study to better clarify their activity in animals and, more importantly, in humans.

Artificial neural network modelling hydrodenticity for optimal design by microfluidics of polymer nanoparticles to apply in magnetic resonance imaging.

The engineering of nanoparticles impacts the control of their nano-bio interactions at each level of the delivery pathway. Therefore, optimal nanoparticle physicochemical properties should be identified to favour on-target interactions and deliver efficiently active compounds to a specific target. To date, traditional batch processes do not guarantee the reproducibility of results and low polydispersity index of the nanostructures, while microfluidics has emerged as cost effectiveness, short-production time approach to control the nanoparticle size and size distribution. Several thermodynamic processes have been implemented in microfluidics, such as nanoprecipitation, ionotropic gelation, self-assembly, etc., to produce nanoparticles in a continuous mode and high throughput way.   In this work, we show how the Artificial Neural Network (ANN) can be adopted to model the impact of microfluidic parameters (namely, flow rates and polymer concentrations) on the size of the nanoparticles. Promising results have been obtained, with the highest model accuracy reaching 98.9 %, thus confirming the proposed approach's potential applicability for an ANN-guided biopolymer nanoparticle design for biomedical applications. Nanostructures with different degrees of complexity are analysed, and a proof-of-concept machine learning approach is proposed to evaluate Hydrodenticity in biopolymer matrices. STATEMENT OF SIGNIFICANCE: Size, shape and surface charge determine nano-bio interactions of nanoparticles and their ability to target diseases. The ideal nanoparticle design avoids off-target interactions and favours on-target interactions. So, tools enabling the identification of the optimal nanoparticle physicochemical properties for delivery to a specific target are required. In this work, we evaluate the use of Artificial Neural Network (ANN) to analyse the role of microfluidic parameters in predicting the optimal size of the different hydrogel nanoparticles and their ability to trigger Hydrodenticity.

Intracellular Localization during Blood-Brain Barrier Crossing Influences Extracellular Release and Uptake of Fluorescent Nanoprobes.

To improve the efficacy of nanoparticles (NPs) and boost their theragnostic potential for brain diseases, it is key to understand the mechanisms controlling blood-brain barrier (BBB) crossing. Here, the capability of 100 nm carboxylated polystyrene NPs, used as a nanoprobe model, to cross the human brain endothelial hCMEC/D3 cell layer, as well as to be consequently internalized by human brain tumor U87 cells, is investigated as a function of NPs' different intracellular localization. We compared NPs confined in the endo-lysosomal compartment, delivered to the cells through endocytosis, with free NPs in the cytoplasm, delivered by the gene gun method. The results indicate that the intracellular behavior of NPs changed as a function of their entrance mechanism. Moreover, by bypassing endo-lysosomal accumulation, free NPs were released from cells more efficiently than endocytosed NPs. Most importantly, once excreted by the endothelial cells, free NPs were released in the cell culture medium as aggregates smaller than endocytosed NPs and, consequently, they entered the human glioblastoma U87 cells more efficiently. These findings prove that intracellular localization influences NPs' long-term fate, improving their cellular release and consequent cellular uptake once in the brain parenchyma. This study represents a step forward in designing nanomaterials that are able to reach the brain effectively.

Hydrogel particles-on-chip (HyPoC): a fluorescence micro-sensor array for IgG immunoassay.

Novel microparticles have generated growing interest in diagnostics for potential sensitivity and specificity in biomolecule detection and for the possibility to be integrated in a micro-system array as a lab-on-chip. Indeed, bead-based technologies integrated in microfluidics could speed up incubation steps, reduce reagent consumption and improve accessibility of diagnostic devices to non-expert users. To limit non-specific interactions with interfering molecules and to exploit the whole particle volume for bioconjugation, hydrogel microparticles, particularly polyethylene glycol-based, have emerged as promising materials to develop high-performing biosensors since their network can be functionalized to concentrate the target and improve detection. However, the limitations in positioning, trapping and mainly fine manipulation of a precise number of particles in microfluidics have largely impaired point-of-care applications. Herein, we developed an on-chip sandwich immunoassay for the detection of human immunoglobulin G in biological fluids. The detection system is based on finely engineered cleavable PEG-based microparticles, functionalized with specific monoclonal antibodies. By changing the particle number, we demonstrated tuneable specificity and sensitivity (down to 3 pM) in serum and urine. Therefore, a controlled number of hydrogel particles have been integrated in a microfluidic device for on-chip detection (HyPoC) allowing for their precise positioning and fluid exchange for incubation, washing and target detection. HyPoC dramatically decreases incubation time from 180 minutes to one minute and reduces washing volumes from 3.5 ml to 90 µL, achieving a limit of detection of 0.07 nM (with a dynamic range of 0.07-1 nM). Thus, the developed approach represents a versatile, fast and easy point-of-care testing platform for immunoassays.

Direct, precise, enzyme-free detection of miR-103-3p in real samples by microgels with highly specific molecular beacons.

Low abundance, small size, and sequence similarities render microRNA (miRNAs) detection challenging, particularly in real samples, where quantifying weakly expressed miRNAs can be arduous due to interference of more abundant molecules. The standard quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires multiple steps, thermal cycles, and costly enzymatic reactions that can negatively affect results. Here we present a direct, precise, enzyme-free assay based on microgels particles conjugating molecular beacons (MB) capable of optically detecting low abundant miRNAs in real samples. We validate the applicability of microgels assay using qRT-PCR as a reference technology. As a relevant case, we chose miR-103-3p, a valuable diagnostic biomarker for breast cancer, both in serum samples and MCF7 cells. As a result, microgels assay quantifies miRNA molecules at room temperature in a single step, 1 h (vs. 4 hrs for qRT-PCR) without complementary DNA synthesis, amplification, or expensive reagents. Microgels assay exhibits femtomolar sensitivity, single nucleotide specificity, and a wide linear range (102-107 fM) (wider than qRT-PCR), with low sample consumption (2 µL) and excellent linearity (R2= 0.98). To test the selectivity of the microgel assay in real samples, MCF7 cells were considered where the pool of 8 other miRNAs were further upregulated with respect to miRNA 103-3p. In such complex environments, microgels assay selectively detects the miRNA target, mainly due to MB advanced stability and specificity as well as high microgel antifouling properties. These results show the reliability of microgels assay to detect miRNAs in real samples.

Insulin Activation Mediated by Uptake Mechanisms: A Comparison of the Behavior between Polymer Nanoparticles and Extracellular Vesicles in 3D Liver Tissues.

In this work, we compare the role of two different uptake mechanisms in the effectiveness of a nanoformulated drug, specifically insulin. Insulin is activated by interacting with insulin receptors exposed on the liver cell membrane that triggers the uptake and storage of glucose. To prove that the uptake mechanism of a delivery system can interfere directly with the effectiveness of the delivered drug, two extremely different delivery systems are tested. In detail, hydrogel-based NPs (cHANPs) and natural lipid vesicles (EVs) encapsulating insulin are used to trigger the activation of this hormone in 3D liver microtissues (µTs) based on their different uptake mechanisms. Results demonstrated that the fusion mechanism of Ins-EVs mediates faster and more pronounced insulin activation with respect to the endocytic mechanism of Ins-cHANPs. Indeed, the fusion causes an increased reduction in glucose concentration in the culture medium EV-treated l-µTs with respect to free insulin-treated tissues. The same effect is not observed for Ins-cHANPs that, taken up by endocytosis, can only equal the reduction in glucose concentration produced by free insulin in 48 h. Overall, these results demonstrate that the effectiveness of nanoformulated drugs depends on the identity they acquire in the biological context (biological identity). Indeed, the nanoparticle (NP) biological identity, such as the uptake mechanism, triggers a unique set of nano-bio-interactions that is ultimately responsible for their fate both in the extracellular and intracellular compartments.

Colorectal Cancer Bioengineered Microtissues as a Model to Replicate Tumor-ECM Crosstalk and Assess Drug Delivery Systems In Vitro.

Current 3D cancer models (in vitro) fail to reproduce complex cancer cell extracellular matrices (ECMs) and the interrelationships occurring (in vivo) in the tumor microenvironment (TME). Herein, we propose 3D in vitro colorectal cancer microtissues (3D CRC µTs), which reproduce the TME more faithfully in vitro. Normal human fibroblasts were seeded onto porous biodegradable gelatin microbeads (GPMs) and were continuously induced to synthesize and assemble their own ECMs (3D Stroma µTs) in a spinner flask bioreactor. Then, human colon cancer cells were dynamically seeded onto the 3D Stroma µTs to achieve the 3D CRC µTs. Morphological characterization of the 3D CRC µTs was performed to assess the presence of different complex macromolecular components that feature in vivo in the ECM. The results showed the 3D CRC µTs recapitulated the TME in terms of ECM remodeling, cell growth, and the activation of normal fibroblasts toward an activated phenotype. Then, the microtissues were assessed as a drug screening platform by evaluating the effect of 5-Fluorouracil (5-FU), curcumin-loaded nanoemulsions (CT-NE-Curc), and the combination of the two. When taken together, the results showed that our microtissues are promising in that they can help clarify complex cancer-ECM interactions and evaluate the efficacy of therapies. Moreover, they may be combined with tissue-on-chip technologies aimed at addressing further studies in cancer progression and drug discovery.

Review on Bioinspired Design of ECM-Mimicking Scaffolds by Computer-Aided Assembly of Cell-Free and Cell Laden Micro-Modules.

Tissue engineering needs bioactive drug delivery scaffolds capable of guiding cell biosynthesis and tissue morphogenesis in three dimensions. Several strategies have been developed to design and fabricate ECM-mimicking scaffolds suitable for directing in vitro cell/scaffold interaction, and controlling tissue morphogenesis in vivo. Among these strategies, emerging computer aided design and manufacturing processes, such as modular tissue unit patterning, promise to provide unprecedented control over the generation of biologically and biomechanically competent tissue analogues. This review discusses recent studies and highlights the role of scaffold microstructural properties and their drug release capability in cell fate control and tissue morphogenesis. Furthermore, the work highlights recent advances in the bottom-up fabrication of porous scaffolds and hybrid constructs through the computer-aided assembly of cell-free and/or cell-laden micro-modules. The advantages, current limitations, and future challenges of these strategies are described and discussed.

PLGA microparticle formulations for tunable delivery of a nano-engineered filamentous bacteriophage-based vaccine: in vitro and in silico-supported approach.

Bacteriophages have attracted great attention in the bioengineering field in diverse research areas from tissue engineering to therapeutic and clinical applications. Recombinant filamentous bacteriophage, carrying multiple copies of foreign peptides on protein capsid has been successfully used in the vaccine delivery setting, even if their plasma instability and degradation have limited their use on the pharmaceutical market. Encapsulation techniques in polymeric materials can be applied to preserve bacteriophage activity, extend its half-life, and finely regulate their release in the target environment. The main goal of this study was to provide tunable formulations of the bacteriophage encapsulated in polymeric microparticles (MPs). We used poly (lactic-co-glycolic-acid) as a biocompatible and biodegradable polymer with ammonium bicarbonate as a porogen to encapsulate bacteriophage expressing OVA (257-264) antigenic peptide. We demonstrate that nano-engineered fdOVA bacteriophages encapsulated in MPs preserve their structure and are immunologically active, inducing a strong immune response towards the delivered peptide. Moreover, MP encapsulation prolongs bacteriophage stability over time also at room temperature. Additionally, in this study, we show the ability of in silico-supported approach to predict and tune the release of bacteriophages. These results lay the framework for a versatile bacteriophage-based vaccine delivery system that could successfully generate robust immune responses in a sustained manner, to be used as a platform against cancer and new emerging diseases. Graphical abstract: Synopsis: administration of recombinant bacteriophage-loaded PLGA microparticles for antigen delivery. PLGA microparticles release the bacteriophages, inducing activation of dendritic cells and enhancing antigen presentation and specific T cell response. Bacteriophage-encapsulated microneedles potentially can be administered into human body and generate robust immune responses.

Levels of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma from patients with amyotrophic lateral sclerosis.

OBJECTIVE: In this context, our study aimed to ascertain whether the esterification of 24-hydroxycholesterol, a process heavily affected by oxidative stress, is altered in ALS. METHODS: The study examined the level of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma of 18 ALS patients by spectroscopic technique as Ultra-high performance liquid chromatography mass spectrometry (UPLC-MS). RESULTS: The level of 24-hydroxycholesteryl esters in cerebrospinal fluid was found to be lower as the brain-blood barrier was damaged. Such a level was positively correlated with the level of esters in plasma. Both cerebrospinal fluid (CSF) level and plasma level were lower in ALS patients (60.05 ± 4.24 % and 54.07 ± 20.37 % respectively) than in controls (79.51 ± 2.47 % and 80.07 ± 10.02 % respectively). CONCLUSIONS: The data suggest that the level 24-hydroxycholesteryl esters might be a new biomarker of ALS and can be measured for monitoring the disease progression.

Integration of micro-CT and histology data for vasculature morpho-functional analysis in tissue regeneration.

The demand for artificial or bioartificial engineered tissues is increasing today in regenerative medicine techniques to replace and restore the physiological function of damaged tissues. Such engineered constructs hold different properties depending on the tissue to be replicated. As for vascularized tissues, complex biocompatible structures, namely scaffolds, play a key role in supporting oxygen and nutrient supply, thus sustaining tissue neoformation and integration with the host. Scaffold architecture significantly impacts its regenerative potential, while preclinical trials are essential to define scaffold-host interactions. In compliance with the 3 R principle, there is a clear need to optimize both the procedures to evaluate scaffold performance and the analysis methodology decreasing the number of animals required to gain consistent data. In parallel, current technologies used in preclinical research generate huge amounts of data that need to be elaborated and interpreted correctly. Therefore, we designed this study to evaluate the results of scaffold integration with the host tissue after implantation in a mouse subcutaneous pocket model. We evaluated the angiogenic response developed by the host and the degree of scaffold integration by using a combined morphometric approach based on both histological and micro-CT analyses. Six-layer scaffolds, made of polycaprolactone (PCL) microspheres, with an ordered structure were produced by thermal sintering. Scaffolds were then implanted in BALB/c mice and retrieved 21 days post-implantation when the animals were deeply anesthetized and perfused with Microfil, a contrast agent for micro-CT. Here, we describe a method to extract quantitative data from micro-CT reconstructions such as (i) total vessel volume; (ii)% of vessel penetration; (iii) distribution of vessel diameters. The general principle of this approach is the refinement of the region of interest (ROI), thus producing a volume of interest (VOI) that matches scaffold volume. This VOI serves as a dataset from which to extract volumetric information. Then VOIs are divided into three identical parts, proximal, median, and distal, to follow the vessel progression into the scaffold, thus obtaining their depth of penetration (DoP). By this methodology, we observed in mean, among the analyzed samples, a vessel invasion for 1,38 mm3 corresponding to the 1,53% of the scaffold volume. We then looked at the diameter distribution being this value a key indicator of vessel maturity, highlighting that 55% of vessels fall into the range from 5,99-53.99 µm while the remaining 45% are distributed into intervals from 54 to 136 µm. In parallel, to evaluate tissue integration in detail, histological and immunofluorescent analyses were performed to look at vessel distribution and collagen synthesis. Histological results strongly correlate with the micro-CT data providing, however, an overview of the ingrowth tissues. In addition, by immunofluorescent analysis we demonstrate that newly formed vessels are mature at the considered time point and tissue collagen deposition is widespread within the scaffolds. Collectively, we propose a new method to track vessel formation by using a multi-modal approach posing the basis for: i) the fabrication of novel scaffolds for Tissue Engineering; ii) the integration of detailed information for a wide range of morphological and functional analyses.

Cell deformability heterogeneity recognition by unsupervised machine learning from in-flow motion parameters.

Cell deformability is a well-established marker of cell states for diagnostic purposes. However, the measurement of a wide range of different deformability levels is still challenging, especially in cancer, where a large heterogeneity of rheological/mechanical properties is present. Therefore, a simple, versatile and cost-effective recognition method for variable rheological/mechanical properties of cells is needed. Here, we introduce a new set of in-flow motion parameters capable of identifying heterogeneity among cell deformability, properly modified by the administration of drugs for cytoskeleton destabilization. Firstly, we measured cell deformability by identification of in-flow motions, rolling (R), tumbling (T), swinging (S) and tank-treading (TT), distinctively associated with cell rheological/mechanical properties. Secondly, from a pool of motion and structural cell parameters, an unsupervised machine learning approach based on principal component analysis (PCA) revealed dominant features: the local cell velocity (VCell/VAvg), the equilibrium position (YEq) and the orientation angle variation (Δφ). These motion parameters clearly defined cell clusters in terms of motion regimes corresponding to specific deformability. Such correlation is verified in a wide range of rheological/mechanical properties from the elastic cells moving like R until the almost viscous cells moving as TT. Thus, our approach shows how simple motion parameters allow cell deformability heterogeneity recognition, directly measuring rheological/mechanical properties.

In vitro effects of combustion generated carbon dots on cellular parameters in healthy and cancerous breast cells.

Carbon nanomaterials are an inventive class of materials with wide applications in state-of-the-art bioimaging and therapeutics. They allow a broad range of tunable and integrated advantages of structural flexibility, chemical and thermal stability, upright electrical conductivity, and the option of scale-up and mass production. In the context of nanomedicine, carbon nanomaterials have been used extensively to mitigate the serious side effects of conventional chemotherapy and also to enable early cancer diagnostics, given their wide range of tunable properties. A class of carbon nanomaterials, called carbon dots (CDs) are small carbon-based nanoparticles and have been a valued discovery due to their photoluminescence, low photobleaching, and high surface area to mass ratio. The process of producing these CDs had so far been a high energy demanding process involving wet chemistry for purification. A one-step tunable production of luminescent CDs from fuel rich combustion reactors was recently presented by our group. In this paper, we explore the effects of these yellow luminescent combustion-generated CDs in MCF7 adenocarcinoma and MCF10a normal breast epithelial cells. We observed that these CDs, also at nontoxic doses, can affect basic cellular functions, such as cell cycle and proliferation; induce substantial changes on the physical parameters of the plasma membrane; and change the overall appearance of a cell in terms of morphology.

Hydrogelation tunability of bioinspired short peptides.

Supramolecular assemblies of short peptides are experiencing a stimulating flowering. Herein, we report a novel class of bioinspired pentapeptides, not bearing Phe, that form hydrogels with fibrillar structures. The inherent sequence comes from the fragment 269-273 of nucleophosmin 1 protein, that is normally involved in liquid-liquid phase separation processes into the nucleolus. By means of rheology, spectroscopy, and scanning microscopy the crucial roles of the extremities in the modulation of the mechanical properties of hydrogels were elucidated. Three of four peptide showed a typical shear-thinning profile and a self-assembly into hierarchical nanostructures fibers and two of them resulted biocompatible in MCF7 cells. The presence of an amide group at C-terminal extremity caused the fastest aggregation and the major content of structured intermediates during gelling process. The tunable mechanical and structural features of this class of hydrogels render derived supramolecular systems versatile and suitable for future biomedical applications.

Single cell classification of macrophage subtypes by label-free cell signatures and machine learning.

Pro-inflammatory (M1) and anti-inflammatory (M2) macrophage phenotypes play a fundamental role in the immune response. The interplay and consequently the classification between these two functional subtypes is significant for many therapeutic applications. Albeit, a fast classification of macrophage phenotypes is challenging. For instance, image-based classification systems need cell staining and coloration, which is usually time- and cost-consuming, such as multiple cell surface markers, transcription factors and cytokine profiles are needed. A simple alternative would be to identify such cell types by using single-cell, label-free and high throughput light scattering pattern analyses combined with a straightforward machine learning-based classification. Here, we compared different machine learning algorithms to classify distinct macrophage phenotypes based on their optical signature obtained from an ad hoc developed wide-angle static light scattering apparatus. As the main result, we were able to identify unpolarized macrophages from M1- and M2-polarized phenotypes and distinguished them from naive monocytes with an average accuracy above 85%. Therefore, we suggest that optical single-cell signatures within a lab-on-a-chip approach along with machine learning could be used as a fast, affordable, non-invasive macrophage phenotyping tool to supersede resource-intensive cell labelling.

Update on the Use of PET/MRI Contrast Agents and Tracers in Brain Oncology: A Systematic Review.

The recent advancements in hybrid positron emission tomography-magnetic resonance imaging systems (PET/MRI) have brought massive value in the investigation of disease processes, in the development of novel treatments, in the monitoring of both therapy response and disease progression, and, not least, in the introduction of new multidisciplinary molecular imaging approaches. While offering potential advantages over PET/CT, the hybrid PET/MRI proved to improve both the image quality and lesion detectability. In particular, it showed to be an effective tool for the study of metabolic information about lesions and pathological conditions affecting the brain, from a better tumor characterization to the analysis of metabolic brain networks. Based on the PRISMA guidelines, this work presents a systematic review on PET/MRI in basic research and clinical differential diagnosis on brain oncology and neurodegenerative disorders. The analysis includes literature works and clinical case studies, with a specific focus on the use of PET tracers and MRI contrast agents, which are usually employed to perform hybrid PET/MRI studies of brain tumors. A systematic literature search for original diagnostic studies is performed using PubMed/MEDLINE, Scopus and Web of Science. Patients, study, and imaging characteristics were extracted from the selected articles. The analysis included acquired data pooling, heterogeneity testing, sensitivity analyses, used tracers, and reported patient outcomes. Our analysis shows that, while PET/MRI for the brain is a promising diagnostic method for early diagnosis, staging and recurrence in patients with brain diseases, a better definition of the role of tracers and imaging agents in both clinical and preclinical hybrid PET/MRI applications is needed and further efforts should be devoted to the standardization of the contrast imaging protocols, also considering the emerging agents and multimodal probes.

A role for nuclear stretching and NPCs changes in the cytoplasmic-nuclear trafficking of YAP: An experimental and numerical modelling approach.

Mechanical forces, acting on eukaryotic cells, are responsible for cell shape, cell proliferation, cell polarity, and cell differentiation thanks to two cells abilities known as mechanosensing and mechanotransduction. Mechanosensing consists of the ability of a cell to sense mechanical cues, while mechanotransduction is the capacity of a cell to respond to these signals by translating mechanical stimuli into biochemical ones. These signals propagate from the extracellular matrix to the nucleus with different well known physical connections, but how the mechanical signals are transduced into biochemical ones remains an open challenge. Recent findings showed that the cell-generated forces affect the translocation of transcription factors (TFs) from the cytoplasm to the nucleus. This mechanism is affected by the features of nuclear pore complexes. Owing to the complex patterns of strains and stresses of the nuclear envelope caused by cytoskeletal forces, it is likely that the morphology of NPC changes as cytoskeleton assemblies' change. This may ultimately affect molecular transport through the nucleus, hence altering cell functions. Among the various TFs, Yes-associated protein (YAP), which is typically involved in cell proliferation, survival, and differentiation, is able to activate specific pathways when entrapped into the cell nucleus. Here, starting from experimental results, we develop a multiscale finite element (FE) model aimed to simulate the macroscopic cell spreading and consequent changes in the cell mechanical behaviour to be related to the NPCs changes and YAP nuclear transport.