Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation

3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-β1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-β1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).     

The Association between α-Synuclein and α-Tubulin in Brain Synapses

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.     

Nanoindentation-based study of the mechanical behavior of bulk supercrystalline ceramic-organic nanocomposites

Bulk poly-supercrystalline ceramic-organic nanocomposites were produced and characterized with a nanoindentation-based study. These nanocomposites were processed using two different routines, to compare their properties with and without crosslinking the organic ligands interfacing the ceramic nanoparticles. Together with the expected material strengthening induced by crosslinking, a distinct response emerges when using Berkovich and cube-corner indenters. The supercrystalline materials are prone to compaction, cracking and chipping phenomena that become more severe when a sharper tip is employed, implying that a Berkovich indenter is more suitable for the evaluation of elastic modulus and hardness. The cube-corner tip, on the other hand, is employed for the investigation of fracture toughness, comparing two methods and multiple models available from the literature. The fracture toughness outcomes suggest that cracks evolve with a quarter-penny shaped profile at the indent’s corners, and that extrinsic toughening mechanisms, such as plastic-like deformation and crack deflection, play a significant role.     

Microgravity simulation: physical and psychological workload evaluation tests in an underwater environment

As weightlessness is not completely reproducible on Earth, usability evaluation of space systems is often simulated through tests in an aquatic environment. A Neutral Buoyancy Facility test programme was organized in a special pool to simulate Extra-Vehicular Activities on the Columbus module of the future International Space Station with the aim of assessing various aspects of crew interface design.This study was designed to evaluate workload using visibility, accessibility and operability tests. Diving workload was determined through basic physiological measurements, such as pulmonary ventilation and heart rate during underwater operations.As anxiety can influence physiological processes, and consequently also the workload evaluation determined through these parameters, we developed an evaluation methodology to investigate the anxiety level based on a specific questionnaire submitted to all subjects before and after the dives.Heart rate increased in underwater work to a value approximately 50% larger than the value obtained in the resting condition while sitting outside the pool. This increase in heart rate was accompanied by an increase in pulmonary ventilation of 200% larger than the value recorded in the rest condition while sitting outside the water. The extent of these increases was notable in all the test subjects, who varied in age and stature.Recorded values of workload, heart rate and pulmonary ventilation were evaluated on the basis of Christensen's (Arbeitsphysiol. 14 (1950) 251) and Wells’ (J. Appl. Physiol. 10 (1957) 51) classifications. Through this analysis it was possible to determine that the workload, indicated by performance on our neutral buoyancy tests, corresponds to moderate physiological work.For test subjects, anxiety related to underwater performance was light. Among the causes of anxiety all the subjects indicated the lack of confidence with neutral buoyancy tests and a feeling of lack of safety, typical of aquatic environments.We can conclude that context did not produce considerable psychological effects, and consequently that the psychological load did not influence heart rate and pulmonary ventilation values that can therefore be directly related to task workload.     

A Focus on Enterochromaffin Cells among the Enteroendocrine Cells: Localization,Morphology, and Role

The intestinal epithelium plays a key role in managing the relationship with the environment, the internal and external inputs, and their changes. One percent of the gut epithelium is represented by the enteroendocrine cells. Among the enteroendocrine cells, a group of specific cells characterized by the presence of yellow granules, the enterochromaffin cells, has been identified. These granules contain many secretion products. Studies showed that these cells are involved in gastrointestinal inflammatory conditions and hyperalgesia; their number increases in these conditions both in affected and not-affected zones of the gut. Moreover, they are involved in the preservation and modulation of the intestinal function and motility, and they sense metabolic–nutritional alterations. Sometimes, they are confused or mixed with other enteroendocrine cells, and it is difficult to define their activity. However, it is known that they change their functions during diseases; they increased in number, but their involvement is related mainly to some secretion products (serotonin, melatonin, substance P). The mechanisms linked to these alterations are not well investigated. Herein, we provide an up-to-date highlight of the main findings about these cells, from their discovery to today. We emphasized their origin, morphology, and their link with diet to better evaluate their role for preventing or treating metabolic disorders considering that these diseases are currently a public health burden.     

Tetraspanin CD9 Expression Predicts Sentinel Node Status in Patients with Cutaneous Melanoma

The tetraspanin CD9 is considered a metastasis suppressor in many cancers, however its role is highly debated. Currently, little is known about CD9 prognostic value in cutaneous melanoma. Our aim was to analyse CD9 expression in melanocytic nevi and primary cutaneous melanomas through immunohistochemistry and immunofluorescence approaches to determine its correlation with invasiveness and metastatic potential. CD9 displayed homogeneous staining in all melanocytic nevi. In contrast, it showed a complete loss of reactivity in all thin melanomas. Interestingly, CD9 was re-expressed in 46% of intermediate and thick melanomas in small tumor clusters predominantly located at sites of invasion near or inside the blood or lymphatic vessels. The most notable finding is that all CD9 stained melanomas presented sentinel node positivity. Additionally, a direct association between CD9 expression and presence of distant metastasis was reported. Finally, we confirm that CD9 expression is consistent with an early protective role against tumorigenesis, however, our data endorse in melanoma a specific function of CD9 in vascular dissemination during late tumor progression. The presence of CD9 hotspots could be essential for melanoma cell invasion in lymphatic and endothelial vessels. CD9 could be a valid prognostic factor for lymph node metastasis risk.     

Functional Correlates of Striatal Dopamine Transporter Cerebrospinal Fluid Levels in Alzheimer’s Disease: A Preliminary 18F-FDG PET/CT Study

The aim of our study was to investigate regional glucose metabolism with 18F-FDG positron emission tomography/computed tomography in a population of patients with Alzheimer’s disease (AD) in relation to cerebrospinal (CSF) levels of striatal dopamine transporter (DAT). All patients underwent lumbar puncture and received a biomarker-based diagnosis of AD. Differences in regional brain glucose metabolism were assessed by Statistical Parametric Mapping version 12 with the use of age, gender, and MMSE as covariates in the analysis. A positive correlation between CSF DAT levels and glucose metabolism at the level of two brain areas involved in the pathophysiological process of Alzheimer’s disease, the substantia nigra and the posterior cingulate gyrus, has been highlighted. Results indicate that patients with higher CSF DAT levels have a better metabolic pattern in two key zones, suggesting less advanced disease status in patients with more conserved dopaminergic systems.     

Targeting Microglia-Synapse Interactions in Alzheimer’s Disease

In this review, we focus on the emerging roles of microglia in the brain, with particular attention to synaptic plasticity in health and disease. We present evidence that ramified microglia, classically believed to be “resting” (i.e., inactive), are instead strongly implicated in dynamic and plastic processes. Indeed, there is an intimate relationship between microglia and neurons at synapses which modulates activity-dependent functional and structural plasticity through the release of cytokines and growth factors. These roles are indispensable to brain development and cognitive function. Therefore, approaches aimed at maintaining the ramified state of microglia might be critical to ensure normal synaptic plasticity and cognition. On the other hand, inflammatory signals associated with Alzheimer’s disease are able to modify the ramified morphology of microglia, thus leading to synapse loss and dysfunction, as well as cognitive impairment. In this context, we highlight microglial TREM2 and CSF1R as emerging targets for disease-modifying therapy in Alzheimer’s disease (AD) and other neurodegenerative disorders.     

Feasibility of in situ de-agglomeration during powder consolidation

Consolidation of nano-sized powders is a growing area in manufacturing of advanced materials, thanks to the reduced processing times, enhanced mechanical properties and high potential for the introduction of multi-functionality enabled by such reduced particle sizes. Nanopowders, however, are particularly prone to the agglomeration phenomena, and thus to the formation of hierarchical porous structures. The presence of pores differing up to several orders of magnitude in size leads to undesired differential shrinkage and localized grain growth. In order to avoid such issues, strategies for in situ de-agglomeration are proposed here. These optimization strategies are based on the development of an analytical model for shrinkage kinetics and mechanical properties of a hierarchical porous structure, containing both small-size intra-agglomerate pores and large-size inter-agglomerate ones. The modeling approach is an expansion of the continuum theory of sintering to the case of biporous materials presenting nonlinear viscous rheology, as expected for nano-sized crystalline powders. Considering the nonlinear viscous constitutive behavior of the solid phase also allows assessing the influence of the temperature on the microstructural evolution during processing, due to the dependence of the creep characteristic parameter, strain-rate sensitivity, on the thermal history. Material structure optimization strategies, aimed at de-agglomeration or at the design of tailored porous structures, become then possible and are here explored.     

Role of Regular Physical Activity in Neuroprotection against Acute Ischemia

One of the major obstacles that prevents an effective therapeutic intervention against ischemic stroke is the lack of neuroprotective agents able to reduce neuronal damage; this results in frequent evolution towards a long-term disability with limited alternatives available to aid in recovery. Nevertheless, various treatment options have shown clinical efficacy. Neurotrophins such as brain-derived neurotrophic factor (BDNF), widely produced throughout the brain, but also in distant tissues such as the muscle, have demonstrated regenerative properties with the potential to restore damaged neural tissue. Neurotrophins play a significant role in both protection and recovery of function following neurological diseases such as ischemic stroke or traumatic brain injury. Unfortunately, the efficacy of exogenous administration of these neurotrophins is limited by rapid degradation with subsequent poor half-life and a lack of blood–brain-barrier permeability. Regular exercise seems to be a therapeutic approach able to induce the activation of several pathways related to the neurotrophins release. Exercise, furthermore, reduces the infarct volume in the ischemic brain and ameliorates motor function in animal models increasing astrocyte proliferation, inducing angiogenesis and reducing neuronal apoptosis and oxidative stress. One of the most critical issues is to identify the relationship between neurotrophins and myokines, newly discovered skeletal muscle-derived factors released during and after exercise able to exert several biological functions. Various myokines (e.g., Insulin-Like Growth Factor 1, Irisin) have recently shown their ability to protects against neuronal injury in cerebral ischemia models, suggesting that these substances may influence the degree of neuronal damage in part via inhibiting inflammatory signaling pathways. The aim of this narrative review is to examine the main experimental data available to date on the neuroprotective and anti-ischemic role of regular exercise, analyzing also the possible role played by neurotrophins and myokines.