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Circadian rhythms have been shown in the subthalamic nucleus (STN) in Parkinsons disease (PD), but only a few studies have focused on the globus pallidus internus (GPi). This retrospective study investigates GPi circadian rhythms in a large cohort of subjects with PD (130 recordings from 93 subjects) with GPi activity chronically recorded in their home environment. We found a significant change in GPi activity between daytime and nighttime in most subjects (82.4%), with a reduction in GPi activity at nighttime in 56.2% of recordings and an increase in activity in 26.2%. GPi activity in higher frequency bands ( > 20 Hz) was more likely to decrease at night and in patients taking extended-release levodopa medication. Our results suggest that circadian fluctuations in the GPi vary across individuals and that increased power at night might be due to the reemergence of pathological neural activity. These findings should be considered to ensure successful implementation of adaptive neurostimulation paradigms in the real-world.

Abstract Background The COVID-19 pandemic has led to the popularization of information and communication technology in medical education. This study aimed to compare the objective structured clinical examination (OSCE) and the virtual objective structured clinical examination (vOSCE), based on expert opinion, as tools for assessing the competencies needed under medical curriculum guidelines in Brazil. Methods In this multicenter study, the suitability levels of the OSCE and vOSCE for assessing the competencies needed under the Brazilian National Curriculum Guidelines (DCNs) were compared. The DCNs encompass five groups of domains, namely, health education; general; health management; public health needs; and individual health needs. Sixteen teaching experts served as judges. They responded to a questionnaire with the provided Likert scores indicating the ability of the OSCE and vOSCE to assess each type of competency. Virtual meetings were held via the Delphi method and a focus group-based approach to enable the experts to reach a consensus. Results A total of 200 items included in the guidelines (100 for the OSCE and 100 for the vOSCE) were evaluated across a total of 3,200 responses. Analysis via the Likert scale revealed a high proportion of positive evaluations for the use of the vOSCE, with kappa values ​​ranging from 0.4 to 0.72. The values ​​of the absolute agreement (positive view) between examiners and equivalence with the kappa statistic for the adoption of the vOSCE ranged from 0.38 to 0.72. Via the use of virtual meetings (Delphi and focus groups), consensus was reached regarding the capacity of the OSCE and vOSCE to evaluate 97% and 92% of these items, respectively. Conclusion The vOSCE can be employed to assess relevant competencies. However, it faces limitations regarding 8% (8/100) of the items, and some items (3%) cannot be assessed via either method. The difference in results between the two assessment methodologies (OSCE and vOSCE) is 5%. It is necessary to invest in the creation of instruments that can be used to apply the vOSCE and to conduct cost‒benefit analysis of its broader application in health education institutions. We conclude that the vOSCE is an effective tool for assessing most competencies of medical professionals and students required under Brazilian guidelines.

New Italian data on the distribution of some of the Annex I Habitats are reported in this contribution. Specifically, 16 records are presented including 9 new occurrences in Natura 2000 sites, and 27 new cells are added in the EEA 10 km × 10 km reference grid. The new data refer to the Italian administrative regions of Abruzzo, Apulia, Latium, Marche, Lombardy, Piedmont, Sardinia, Sicily, Tuscany, and Veneto.

Abstract Neutrophils (PMNs) are key players of innate immune responses through the release of cytoplasmic granule content and the formation of neutrophil extracellular traps (NETs). RNASET2 is an acidic ribonuclease, recently proposed as an alarmin signal associated with inflammatory responses. Here we show that, along the neutrophil maturation cascade, RNASET2 is expressed in segmented and mature PMNs. In human PMNs, RNASET2 colocalized with primary and tertiary granules and was found to be associated with NETs following PMA or Nigericin stimulation. Similarly, activation of PMNs by soluble immune complexes, a hallmark of several autoimmune diseases, also induced RNASET2-associated NETs. Genome-wide association studies recently identified RNASET2 among a cluster of genes associated with increased susceptibility to develop autoimmune diseases, including rheumatoid arthritis (RA). RNASET2 was found expressed by PMNs and macrophages infiltrating inflamed joints in a murine model of RA (K/BxN Serum-Transfer-Induced Arthritis, STIA), by immunostaining. Similar results were found in synovial biopsies of RA patients with active disease. In addition, we demonstrate that RNASET2 circulating levels correlated with the onset and the severity of disease in two mouse models of inflammatory arthritis, STIA and CIA (Collagen-Induced Arthritis) and in serum of RA patients. These results show that PMNs are an important source of RNASET2 and that its circulating levels are associated with RA development suggesting a role for RNASET2 in the pathogenesis of immune-mediated diseases.

Abstract Background The physiological relevance of cell-to-cell communication mediated by small extracellular vesicle-encapsulated microRNAs (sEV-miRNAs) remains debated because of the limiting representativity of specific miRNAs within the extracellular pool. We hypothesize that sEV-miRNA non-canonical function consisting of the stimulation of Toll-like receptor 7 (TLR7) may rely on a global shift of the sEV cargo rather than on the induction of one or few specific miRNAs. Psoriasis represents an ideal model to test such hypothesis as it is driven by overt activation of TLR7-expressing plasmacytoid dendritic cells (pDCs) following keratinocyte damage. Methods To mimic the onset of psoriasis, keratinocytes were treated with a cocktail of psoriatic cytokines or UV-irradiated. SmallRNA sequencing was performed on sEVs released by healthy and UV-treated keratinocytes. sEV-miRNAs were analyzed for nucleotide composition as well as for the presence of putative TLR7-binding triplets. Primary human pDCs where stimulated with sEVs +/- inhibitors of TLR7 (Enpatoran), of sEV release (GW4869 + manumycin) and of TLR7-mediated pDC activation (anti-BDCA-2 antibody). Secretion of type I IFNs and activation of CD8+T cells were used as readouts. qPCR on psoriatic and healthy skin biopsies was conducted to identify induced miRNAs. Results sEV-miRNAs released by damaged keratinocytes revealed a significantly higher content of TLR7-activating sequences than healthy cells. As expected, differential expression analysis confirmed the presence of miRNAs upregulated in psoriatic skin, including miR203a. More importantly, 76.5% of induced miRNAs possessed TLR7-binding features and among these we could detect several previously demonstrated TLR7 ligands. In accordance with this in silico analysis, sEVs from damaged keratinocytes recapitulated key events of psoriatic pathogenesis by triggering pDCs to release type I interferon and activate cytotoxic CD8+T cells in a TLR7- and sEV-dependent manner. Discussion Our results demonstrate that miR203a is just one paradigmatic TLR7-activating miRNA among the hundreds released by UV-irradiated keratinocytes, which altogether trigger pDC activation in psoriatic conditions. This represents the first evidence that cell damage shifts the miRNA content of sEVs towards a TLR7-activating cargo capable to propagate inflammation and immunity, offering strong support to the physiological role of systemic miRNA-based cell-to-cell communication.

Abstract Background Prevention and treatment of metastatic breast cancer (BC) is an unmet clinical need. The retinoic acid derivative fenretinide (FeR) was previously evaluated in Phase I-III clinical trials but, despite its excellent tolerability and antitumor activity in preclinical models, showed limited therapeutic efficacy due to poor bioavailability. We recently generated a new micellar formulation of FeR, Bionanofenretinide (Bio-nFeR) showing enhanced bioavailability, low toxicity, and strong antitumor efficacy on human lung cancer, colorectal cancer, and melanoma xenografts. In the present study, we tested the effect of Bio-nFeR on a preclinical model of metastatic BC. Methods We used BC cell lines for in vitro analyses of cell viability, cell cycle and migratory capacity. For in vivo studies, we used HER2/neu transgenic mice (neuT) as a model of spontaneously metastatic BC. Mice were treated orally with Bio-nFeR and at sacrifice primary and metastatic breast tumors were analyzed by histology and immunohistochemistry. Molecular pathways activated in primary tumors were analyzed by immunoblotting. Stem cell content was assessed by flow cytometry, immunoblotting and functional assays such as colony formation ex vivo and second transplantation assay in immunocompromised mice. Results Bio-nFeR inhibited the proliferation and migration of neuT BC cells in vitro and showed significant efficacy against BC onset in neuT mice. Importantly, Bio-nFeR showed the highest effectiveness against metastatic progression, counteracting both metastasis initiation and expansion. The main mechanism of Bio-nFeR action consists of promoting tumor dormancy through a combined induction of antiproliferative signals and inhibition of the mTOR pathway. Conclusion The high effectiveness of Bio-nFeR in the neuT model of mammary carcinogenesis, coupled with its low toxicity, indicates this formulation as a potential candidate for the treatment of metastatic BC and for the adjuvant therapy of BC patients at high risk of developing metastasis.

Crouzon syndrome is a rare genetic craniofacial malformation caused by heterozygous gain-of-function mutations in the FGFR2 gene. The resulting constitutive activation of the FGFR2 signaling causes the premature osteogenic differentiation of calvarial mesenchymal stromal cells in skull sutures, leading to early suture ossification. Craniectomy is the gold standard treatment, being invasive and burdened by complications. To address these issues, we developed personalized allele-specific (AS) small interfering RNA (siRNA) to knockdown the expression of the FGFR2 mutant allele in Crouzon patient-derived suture cells. The selected therapeutic siRNA mitigated FGFR2 cascade downregulating phosphorylation of FGFR2 (48%) and of its key effector ERK1/2 (77%) as RUNX2 protein levels (34%). This effect was confirmed by the reduced osteogenic commitment and differentiation of treated cells, evidenced by decreased expression of osteogenic marker genes and a 5-fold decrease in mineralized matrix deposition. We developed a highly biocompatible delivery system for siRNAs, based on human recombinant ferritin nanoparticles (NPs), combining cell targeting with improved nucleic acid encapsulation and endosomal escape properties. We demonstrated the ability of these NPs to deliver and release siRNAs within target cells, sustaining their inhibitory and AS effects. Here, we show that ferritin-mediated AS FGFR2 knockdown by siRNA represents a suitable strategy to dampen FGFR2 overactivation in patients’ cells.

Objective: Venous thromboembolism (VTE) is a global disease with significant morbidity and mortality. However, variations in presentation and management of VTE between different continents have not been studied. This retrospective analysis of prospectively collected registry data compares presenting symptoms and treatment modalities of patients with VTE in three continents: Europe, America, and Asia. Methods: The data from the Registro Informatizado de la Enfermedad ThromboEmbólica registry (2011-2021) were reviewed. The Registro Informatizado de la Enfermedad ThromboEmbólica is the largest international registry dedicated to VTE that started in Spain and expanded worldwide. Patient characteristics and treatments were compared between Europe, America, and Asia using standardized differences. Results: There were a total of 61,531 patients with VTE with the majority from Europe (90.3%), followed by Asia (6.3%) and America (3.3%). Patients diagnosed with VTE in America were significantly younger than the other continents. VTE patients in America were more likely to be Black and patients in Asia were more likely to have a lower body mass index when compared with patients from Europe and America. Patients with VTE in Asia presented with significantly more comorbidities including diabetes, coronary artery disease, and ischemic stroke. Also, patients from Asia were more likely to have recent major bleeding and active cancer. Conversely, patients from America had a significantly greater likelihood of having VTE related to travel. Anatomically, European patients were more likely to present with proximal DVT compared with patients from Asia and America. Patients in America were more likely to have low-risk pulmonary embolism (PE) and less likely to have intermediate-risk PE compared with patients from Europe and Asia. There was no difference in the proportion of patients with high-risk PE between continents. There was significant variation in the choice of initial and outpatient anticoagulation; vitamin K antagonists were more commonly used in Europe, whereas direct oral anticoagulants were more commonly used in America, and low-molecular-weight heparins were favored in Asia. Patients in Asia were less likely to receive systemic thrombolytic therapy. In terms of endovascular treatment, pharmacological thrombolysis was used more often for the treatment of PE in Europe compared with Asia and open pulmonary embolectomy was more common among American patients in the registry. Conclusions: There is significant intercontinental variation in VTE presentation and management. Future analyses should evaluate the relationship of these variations with clinical outcomes. Comparative research using a global registry could shed light on VTE biology and treatment.

Undergraduates' first-year experience in engineering education impacts their retention and persistence. Strategic learning strategies (i.e. skill, will, and self-regulation) are important for students' academic achievement and retention. We examined to what extent (1) students' skill mediated the relationship between self-regulation and will and (2) the constructivist practices in the learning environment related to students' self-regulation. Data were collected from 293 engineering undergraduates in a redesigned first-year experience course. Path analysis showed that (1) skill mediated the relationship between self-regulation and will and (2) one dimension of the constructivist learning environment (i.e. sharing ideas with others) was correlated with self-regulation. This study helped faculty and researchers to better understand how to use integrated strategic learning strategies and create a constructivist learning environment for engineering students' academic success.

We studied the vascular flora of the isthmus of Feniglia, a Nature Reserve in southern Tuscany stretching between the Italian Peninsula and Mt. Argentario. Since the nineteenth century, the area has undergone significant environmental modifications due to intensive grazing and deforestation. Later, rehabilitation interventions were carried out, including reforestation and planting of dune species, making it an interesting protected area to study for its botanical aspects. Therefore, we aim to compile the floristic inventory of this distinctive Mediterranean area by integrating old and new data. The checklist comprises 502 specific and subspecific taxa of vascular plants. The life-form spectrum shows a predominance of therophytes, followed by hemicryptophytes. The chorological spectrum highlights the dominance of Mediterranean species, followed by Euromediterranean and Eurasian species. The presence of 15 species of regional importance, 3 species of the Italian red list and 3 Italian endemics (Ornithogalum exscapum Ten., Limonium multiforme Pignatti, Linaria purpurea (L.) Mill.) is noteworthy. Despite the environmental changes undergone in this area, our study reports the presence of remarkable species, including rare ones such as Atriplex littoralis L., Ruppia spiralis L. ex Dumort., and species at their distribution margin in the Italian Peninsula, such as Staphisagria macrosperma Spach, Juncus sorrentinoi Parl. and Maresia nana (DC.) Batt. Additionally the presence of 26 alien species, of which 15 are invasive, 9 naturalized and 2 casual, indicates a relatively low presence of alien species. Notwithstanding the significant environmental changes that the Feniglia isthmus has experienced in the last century and the high tourist pressure during the summer, the presence of the protected area suggested the effectiveness and benefits of such a form of protection for plant diversity.

Mapping habitats on coastal dunes, crucial yet highly vulnerable ecosystems, requires objectivity and repeatability, which are still lacking in the implementation of the Habitats Directive. Although remote sensing offers promising solutions, the effectiveness of distinguishing habitats on coastal dunes from satellite imagery remains uncertain. In this study, we compare crisp and fuzzy classification approaches using WorldView-3 imagery to map coastal dune habitats in two Natural Parks of Tuscany (Italy).Field-collected vegetation data were classified into Annex I habitats of Habitats Directive and EUNIS habitats. Using field data as reference, we performed image classifications with a crisp method (Random Forests) and three fuzzy methods, namely Random Forests, Spectral Angle Mapper and Multiple Endmember Spectral Mixture Analysis. Metrics of overall accuracy and Mantel tests were used to compare the results.EUNIS habitats exhibited the best performance in terms of classification accuracy, likely due to the simpler classification system. We observed a great disparity among habitats, with coastal dune scrubs and white dunes generally achieving the highest accuracy. Fuzzy classifications, despite yielding lower overall accuracy than the crisp classification, provided a more realistic representation of vegetation patterns, highlighting the inherent fuzziness of vegetation in coastal dunes. Despite challenges related to image resolution and habitat heterogeneity, combining satellite imagery with field surveys proved valuable for mapping coastal dune habitats, contributing essential data to the conservation of these fragile ecosystems. We provide a novel and effective tool, which will reduce the economic and physical efforts needed for habitat search and sampling in the field.

European beech (Fagus sylvatica L.) forests can have a high variability in plant species richness and abundance, from monospecific stands to highly species-rich communities. To understand what causes the low plant diversity observed in some beech forests, we analyzed the drivers of plant community completeness in 155 vegetation plots. Data were collected in mature, closed-canopy beech forests in Tuscany, central Italy. Site-specific species pools were estimated based on species co-occurrences. We used Generalized Least Squares linear modeling to assess the effects of anthropogenic and environmental drivers on the community completeness of whole communities and on the set of specialist species of beech forests. We also tested the response of the total cover of the herb layer to the selected predictors and related both the predictive and response variables to species composition in a Non-metric Multidimensional Scaling ordination. The community completeness of whole communities and that of beech forest specialists were negatively affected by total beech cover and positively influenced by slope. Moreover, the community completeness of whole communities was negatively impacted by elevation and positively influenced by disturbance frequency. The cover of the herb layer decreased with increasing beech cover, elevation, and precipitation. High community completeness and high cover of the herb layer were associated with the presence of thermophilic species of mixed deciduous woods in low-elevation beech forests. Our results suggest that a low plant community completeness and a low cover of the herb layer are mainly due to the competition by beech itself when it forms pure forests in its ecological optimum. Such competition is better exerted at upper elevations and in sites with low slopes, where beech litter accumulation is a limiting factor for understory species. Such evidence suggests that species absence in mature beech forests is mainly due to natural drivers and should therefore not be considered an indicator of ecological degradation of the forest.