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Key Points In addition to the classical clathrin-dependent mechanisms of endocytosis, there are several pathways that do not use a clathrin coat and are, therefore, referred to as clathrin-independent (CI) mechanisms. CI mechanisms of uptake have gained much attention with the realization that they have important roles in the regulation of cell growth and development as well as important implications in the study of certain diseases and pathogens. Two well-known CI mechanisms are the caveolar pathway and fluid-phase endocytosis. However, there is much debate concerning the number of distinct CI mechanisms that exist, the best cargo molecules for the study of a particular pathway, and the underlying protein machinery that regulates these pathways. To organize the extensive literature on CI endocytosis for the purpose of arriving at a mechanistic understanding, this Review classifies CI mechanisms as follows: first, on whether or not they are dynamin dependent and, second, according to the involvement of the small GTPases CDC42, RhoA or ARF6. Protein-based mechanisms (for example, ubiquitylation) and lipid-based mechanisms (for example, nanoscale clustering of lipid-tethered proteins) may both function in the selection of cargo for CI endocytosis. The mechanism of budding in the dynamin-independent pathways remains elusive; however, recent theoretical studies provide testable ideas in this area. Classically, endocytosis involves the formation of clathrin-coated carriers that bud from the plasma membrane by dynamin-dependent mechanisms. Recently, several clathrin-independent endocytic pathways have been identified, which represent the main pathway of entry into cells for a diverse array of cargoes, including receptors, lipids and pathogens. There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.

Bone substitutes are being increasingly used in surgery as over two millions bone grafting procedures are performed worldwide per year. Autografts still represent the gold standard for bone substitution, though the morbidity and the inherent limited availability are the main limitations. Allografts, i.e. banked bone, are osteoconductive and weakly osteoinductive, though there are still concerns about the residual infective risks, costs and donor availability issues. As an alternative, xenograft substitutes are cheap, but their use provided contrasting results, so far. Ceramic-based synthetic bone substitutes are alternatively based on hydroxyapatite (HA) and tricalcium phosphates, and are widely used in the clinical practice. Indeed, despite being completely resorbable and weaker than cortical bone, they have exhaustively proved to be effective. Biomimetic HAs are the evolution of traditional HA and contains ions (carbonates, Si, Sr, Fl, Mg) that mimic natural HA (biomimetic HA). Injectable cements represent another evolution, enabling mininvasive techniques. Bone morphogenetic proteins (namely BMP2 and 7) are the only bone inducing growth factors approved for human use in spine surgery and for the treatment of tibial nonunion. Demineralized bone matrix and platelet rich plasma did not prove to be effective and their use as bone substitutes remains controversial. Experimental cell-based approaches are considered the best suitable emerging strategies in several regenerative medicine application, including bone regeneration. In some cases, cells have been used as bioactive vehicles delivering osteoinductive genes locally to achieve bone regeneration. In particular, mesenchymal stem cells have been widely exploited for this purpose, being multipotent cells capable of efficient osteogenic potential. Here we intend to review and update the alternative available techniques used for bone fusion, along with some hints on the advancements achieved through the experimental research in this field.

The onset of Multifragmentation phenomenon is investigated at low excitation energies. A detailed study on the origin of Intermediate Mass Fragment (IMF, Z>=3) produced in central collisions in the 58 Ni+ 40 Ca reaction at 25 AMeV is presented. The experimental campaign was performed with CHIMERA multi-detector at INFN Laboratori Nazionali del Sud in Catania (Italy). The multiple identification techniques of the 4p apparatus, together with low detection thresholds, enable the performance of a careful selection of Fusion-evaporation residues, Multifragmentation sources and their decay products. Comparisons with dynamical approach based on Boltzmann-Langevin-One-Body (BLOB) model predictions coupled with sequential emission code, were used as useful tools to depict and understand the characteristics of fragments emitted from an equilibrated compound nucleus or formed simultaneously in the multifragmenting source. A comparison with the preliminary results obtained for fragment production in central collisions of the same system, 58 Ni+ 40 Ca, at higher incident energies E beam =35AMeV, allows to study and characterize the evolution of multifragmentation phenomenon at the lower end of Fermi energies.

With the advent of the new radioactive beam facilities it is necessary to develop neutron detection systems integrated with charged particle ones. The integration of the neutron signal, especially in using neutron rich beams, becomes a mandatory requirement in order to study the property of the nuclear matter in extreme conditions. For this reason new detectors using new materials have to be built. In this contribution, some new results about the efficiency estimation and the cross talk problem studied through GEANT4 simulations, related to the NArCoS project, will be described with the aim to design a new detector of both good energy and angular resolution. The detection of neutrons and charged particles in the same elementary detection cell is envisaged.

The interplay between diet, host genetics, microbiota, and immune system has a key role in the pathogenesis of inflammatory bowel disease (IBD). Although the causal pathophysiological mechanisms remain unknown, numerous dietary nutrients have been shown to regulate gut mucosal immune function, being effective in influencing innate or adaptive immunity. Here, we proved that transient receptor potential melastatin 8 (TRPM8), a non-selective cation channel, mediates LPS- evoked Ca 2+ influx in macrophages leading to their activation. Additionally, we showed that TRPM8 is selectively blocked by the dietary flavonoid luteolin, which induced a pro-tolerogenic phenotype in pro-inflammatory macrophages. Accordingly, genetic deletion of Trpm8 in macrophages caused a deficit in the activation of pro-inflammatory metabolic and transcriptional reprogramming, leading to reduced production of key pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. The TRPM8 anti-inflammatory effect was found to be dependent on lactate which in turn induces IL-10 gene expression. Adoptive transfer of TRPM8-deficient bone marrow in wild-type mice improved intestinal inflammation in a model of colitis. Accordingly, oral administration of luteolin protected mice against colitis through an impairment in the innate immune response. Our study reveals the potential of targeting TRPM8 through specific nutrient interventions to regulate immune function in sub-clinical scenarios or to treat inflammatory diseases, primarily driven by chronic immune responses, such as IBD.

Arginine and lysine, frequently appearing as a pair on histones, have been proven to carry diverse modifications and execute various epigenetic regulatory functions. However, the most context‐specific and transient effectors of these marks, while significant, have evaded study as detection methods have thus far not reached a standard to capture these ephemeral events. Herein, a pair of complementary photo‐arginine/δ‐photo‐lysine (R‐dz/K‐dz) probes is developed and involve these into histone peptide, nucleosome, and chromatin substrates to capture and explore the interactomes of Arg and Lys hPTMs. By means of these developed tools, this study identifies that H3R2me2a can recruit MutS protein homolog 6 (MSH6), otherwise repelDouble PHD fingers 2 (DPF2), Retinoblastoma binding protein 4/7 (RBBP4/7). And it is disclosed that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 (one component of cBAF) and the nucleosome. In addition, the novel pairs of H4K5 PTMs and respective readers are highlighted, namely H4K5me‐Lethal(3)malignant brain tumor‐like protein 2 (L3MBTL2), H4K5me2‐L3MBTL2, and H4K5acK8ac‐YEATS domain‐containing protein 4 (YEATS4). These powerful tools pave the way for future investigation of related epigenetic mechanisms including but not limited to hPTMs. Photo‐arginine/δ‐photo‐lysine (R‐dz/K‐dz) probes are developed and involved into histone peptide, nucleosome, and chromatin to capture novel interactomes of Arg and Lys hPTMs. This work discloses that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 and the nucleosome. In addition, this work highlights novel pairs of H4K5 PTMs and respective readers.

The study of clustering states in neutron-rich nuclei is an important subject of research in the field of nuclear physics, steadily growing in interest in the international scientific community. In this context, break-up reactions play an important role for the characterization of exotic states in radioactive light nuclei, like neutron halos around stable cores, α-clustering structures or exotic clusters. The CLIR (Clusters in Light Ion Reactions) experiment was performed at INFN - Laboratori Nazionali del Sud (LNS), aiming at the investigation of such states in light radioactive nuclei, by producing a radioactive beam at the FRIBs facility. Reaction products were detected by the CHIMERA multidetector, coupled with four telescopes of the FARCOS array. Calibrations of the tagging system and of the FARCOS telescopes have been performed, for which accurate procedures have been carried out. In this paper, results on the analysis will be presented. Moreover, a brief review on the new fragment separator FRAISE, currently under construction at LNS, will be given.

Plants defend against pathogens such as fungi by initiating coordinated structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx and calcium oscillations that drive defense gene expression, yet the mechanisms by which these signals encode stressor intensity and propagate systematically remain unclear. Here, we present a microfluidic system to characterize intracellular calcium dynamics in protonemal colonies of the moss Physcomitrium patens (Hedw.) upon precise and reversible exposure to fungal chitin oligosaccharides. Epifluorescent imaging of cells expressing the calcium indicator GCaMP6f revealed a rapid, coordinated calcium response to chitin addition, followed by stereotyped oscillations that subsided quickly upon stimulus removal. We implemented an unbiased image segmentation algorithm using pixel-based k-means clustering to automatically locate regions with specific oscillatory signatures. Calcium dynamics were distinct across adjacent cells, distinguishable by cell type, and significantly modulated by circadian rhythm, adaptation time within the device, and stimulus timing. Cytosolic calcium oscillations, which rose and fell symmetrically within about 60 s, occurred spontaneously during the subjective night and following short adaptation periods. Chitin elicited strong oscillations with increased frequency, amplitude, and duration, and repeated pulses entrained regular, colony-wide oscillations at the stimulation interval. This study complements prior investigations of whole plant and growth tip dynamics and provides a quantitative framework to study calcium signaling in plants, including mechanisms of signal propagation and the role of oscillation frequency on gene expression.

Although obsessive-compulsive disorder (OCD) has been found to be the 10th leading cause of disability of all medical conditions in the industrialized world, comparatively little is known about psychosocial functioning and quality of life (QOL) in OCD, particularly with regard to their relationship with symptom severity. Quality of life and psychosocial function of 197 adults were assessed as part of a larger intake interview for a 5-year prospective study of OCD course. Two self-report measures (the Quality of Life Enjoyment and the Medical Outcomes Survey 36-Item Short-Form Health Survey) and 2 clinician-rated measures (the Range of Impaired Functioning Tool and the Social and Occupational Functioning Assessment Scale), each with established reliability and validity, were administered. Symptom severity was assessed with the Yale-Brown Obsessive-Compulsive Scale (YBOCS). Quality of life was significantly impaired compared with published community norms with large effect sizes found for all domains assessed. The correlations ( r) between all QOL measures and the YBOCS total score were significant, ranging from 0.40 to 0.77. Correlations between the YBOCS obsessions subscore and QOL measures were higher than those found between the YBOCS compulsions subscore and QOL. Insight as measured by the Brown Assessment of Beliefs Scale was significantly correlated with 5 of the 7 measures, although more modestly than the YBOCS correlations ( r = 0.22 to 0.37). Subjects with a YBOCS score of 20 or higher had significant decline in QOL compared with those subjects with YBOCS scores lower than 20. Severity of obsessions and depressive symptoms, as well as marital status, were significant predictors of impairment in QOL. These findings indicate that all aspects of QOL are markedly affected in individuals with OCD and are associated with OCD severity (particularly obsessional severity) and depression severity. Exploratory results suggest that QOL and psychosocial functioning begins to be more significantly affected at YBOCS scores higher than 20. This score might be considered as a threshold criterion for OCD for Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.

In the last 15 years the FRIBs@LNS facility has successfully produced Radioactive Ion Beams using the In-Flight technique. We report on the current status and future perspectives opened by FRAISE, a new fragment separator that will be build in connection with the upgrade of Superconducting Cyclotron of the INFN-LNS laboratories.