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Genomic epidemiology and phylogeographic reconstruction of West Nile virus 2 in Italy from 2011 to 2023
No full textSince its introduction to Europe in 2004, West Nile Virus Lineage 2 (WNV-2) has become endemic, with Italy reporting the highest number of cases each season. In 2022, WNV infections in Italy exceeded those recorded during the major 2018 outbreak—the largest ever reported in Europe. This study investigates the genomic epidemiology of WNV during the 2022 and 2023 transmission seasons.
We analyzed 123 environmental samples from wild birds and mosquito pools collected between May and October 2022–2023 in northwestern Italy. All but one sample belonged to Lineage 2; lineage 1 was detected in two bird samples, with one showing co-infection. A total of 98 complete genomes were sequenced.
Phylogeographic reconstruction indicated the origin of the main European clade in Hungary in 2004, with introduction into Italy between 2009 and 2010. Most Italian genomes clustered within a single highly supported subclade, with one sampled in the Marche region in 2011 as the outgroup. Continuous phylogeographic analysis suggested the Italian WNV-2 clade originated in 2009 in the area between Emilia-Romagna and Lombardy, followed by east-west spread during 2022–2023.
Several mutations were identified, including F49L in the NS2A gene linked to neuronal tropism, and M184V in the NS4B gene, associated with increased pathogenicity.
Our results highlight how integrated genomic surveillance of WNV, combining whole genome sequencing and phylogenetic analyses to environmental samples, can support One Health approaches for early detection and risk assessment of arboviral transmission
Will climate change affect nutrient, micronutrient and bioactive bioavailability?
No full textClimate change is projected to profoundly affect global food systems, directly altering food availability and composition and, as a result, nutritional outcomes. Modifications to the composition and properties of food matrices may, in turn, influence the digestion, absorption, and metabolism of food compounds. This can lead to changes in the bioavailability of macronutrients, micronutrients, and bioactive compounds. Additionally, strategies implemented to mitigate climate change, such as transitioning to green food processing methods or modifying diets, may also affect the content and bioavailability of (micro)nutrients in foods. In this review, we will discuss, for the first time, the direct and indirect relationships between climate change and the bioavailability of selected food compounds: proteins, fat-soluble micronutrients, minerals, phenolic compounds, and glucosinolates
ECLETTISMO MUSICALE E INDUSTRIA VIDEOLUDICA: TRAIETTORIE GENERAZIONALI E TRANSNAZIONALI
No full textL’eclettismo musicale si configura come un elemento capace di intrecciarsi con tantissimi aspetti della produzione di musica per videogiochi, mettendone a nudo dinamiche tecnologiche, industriali, transmediali e culturali capaci di permettere una più profonda comprensione di alcune peculiarità stilistiche del repertorio di riferimento. Servendosi di un approccio neo-materialista integrato da concetti di matrice semiologica e fenomenologica e da strumenti propri delle digital humanities, il percorso di ricerca articolato nel presente lavoro affronta il tema dell’eclettismo ludomusicale (in particolare timbrico, e nella variante “verticale apolide”) seguendo due linee direttive: una temporale e l’altra spaziale. Nel primo caso, viene posta enfasi sulle affordance delle tecnologie di produzione musicale, a cavallo tra popular music e musica per audiovisivi, e sul loro peso nel promuovere (o scoraggiare) soluzioni eclettiche. In questo contesto, vengono messi a fuoco alcuni fattori che possono aver cristallizzato alcune pratiche singolari di arrangiamento a partire dal sostrato tecnologico implicato, ma anche alcuni cambiamenti radicali nella produzione ludmusicale che hanno sortito l’effetto opposto, spingendo la musica per videogiochi in direzione di soluzioni più convenzionali, a stretto contatto con la produzione cinematografica. La seconda linea direttiva si sviluppa invece mediante il confronto della produzione ludomusicale statunitense e nipponica, con particolare approfondimento di quest’ultima, per via della centralità del Giappone nella storia dei videogiochi ma anche della particolare posizione geopolitica e culturale del Paese stesso nei confronti del mondo occidentale. Vengono in particolare messe in luce pratiche radicate in un’industria dell’intrattenimento di tipo transmediale da un lato (americano) e polimediale dall’altro (giapponese), analizzando come i rapporti tra videogiochi, cinema hollywoodiano (sul fronte occidentale) e anime (sul fronte orientale) possano incidere sulle fattezze di approcci eclettici differenziati. Entrambe le linee direttive sono corredate da casi di studio che mettono in luce possibili risultati di traiettorie generazionali e transnazionali che influiscono sulle fattezze dell’eclettismo riscontrabile nella colonna sonora dei prodotti coinvolti. Diventa in questo modo possibile osservare dinamiche di mutamento dei linguaggi eclettici a seconda che si influisca sulle tecnologie ludomusicali coinvolte da un lato, e sul radicamento di opere e franchise in Oriente o in Occidente dall’altro. In conclusione, il lavoro di ricerca fornisce strumenti concettuali potenzialmente utili all’analisi dell’eclettismo all’interno di repertori diversificati, mettendo al contempo in evidenza tensioni culturali, rapporti produttivi e sinergie industriali nell’ambito della produzione videoludica e cinematografica, usando il filo rosso dell’eclettismo musicale come chiave di volta.Musical eclecticism emerges as an element capable of intertwining with numerous aspects of music production for video games, showcasing technological, industrial, transmedia, and cultural dynamics that enable a deeper understanding of certain stylistic peculiarities of the reference repertoire. Drawing on a neo-materialist approach integrated with concepts of semiotic and phenomenological origin, as well as tools from the digital humanities, this research work addresses the theme of ludomusical eclecticism (particularly timbral, and in its “vertical stateless” variant) along two guiding lines: one temporal and the other spatial. In the first case, emphasis is placed on the affordances of music production technologies –situated at the intersection of popular music and music for audiovisual media – and on their role in promoting (or discouraging) eclectic solutions. Within this framework, the study focuses on certain factors that may have crystallized specific arrangement practices suggested by the underlying technological configuration, as well as on some radical changes in ludomusical production that have produced the opposite effect, pushing video game music toward more conventional solutions, closely connected with film production. The second guiding line follows a path of comparison between American and Japanese ludomusical production, with particular attention devoted to the latter, due both to Japan’s central role in the history of video games and to the country’s distinctive geopolitical and cultural position in relation to the Western world. In particular, the work highlights practices rooted in an entertainment industry that is focused on the concept of transmediality on the one hand (the American one) and one that is focused on polymediality on the other (the Japanese one), analyzing how the relationships between video games, Hollywood cinema (on the Western front), and anime (on the Eastern front) may shape diverse forms of eclectic approaches. Both guiding lines are supported by case studies that illuminate possible outcomes of generational and transnational trajectories affecting the characteristics of eclecticism employed by the soundtracks of the examined products. This allows to observe mutational processes in eclectic aesthetics depending, on the one hand, on the modification of the ludomusical technologies involved, and, on the other, on the migration of works and franchises from the East to the West and vice versa. In conclusion, the research work provides conceptual tools that could be deemed useful for analyzing eclecticism across diverse repertoires, while simultaneously highlighting cultural tensions, production relationships, and industrial synergies within video game and cinematic production, using musical eclecticism as the unifying thread and interpretative key
Central and peripheral excitability in restless limbs syndrome
No full textRestless limbs syndrome (RLS) is a neurological disorder characterized by an uncontrollable urge to move the limbs. Although it affects up to 10% of the general population, its underlying mechanisms remain poorly understood. Neurophysiological excitability testing may help elucidate mechanisms related to sensorimotor integration, axonal ion channel dysfunction and impaired neural inhibition. This study aimed to assess both CNS and PNS function by examining cortical, spinal and peripheral nerve excitability within the same individuals for the first time. To investigate potential widespread excitability changes in RLS, we specifically analysed hand muscles, offering new insights into the extent of neural involvement beyond the lower limbs. The study included 56 RLS patients, divided into treated and untreated groups, along with 32 healthy controls. Notably, none of the patients experienced symptoms in their hands. Cortical excitability was assessed via threshold-tracking transcranial magnetic stimulation (TMS) to evaluate intracortical inhibition and facilitation. Sensory-motor integration was measured via long-latency reflexes (LLRs), while spinal cord excitability was assessed using F-waves, H-reflexes and RIII-reflexes. Axonal excitability was examined using the extended TRONDNF protocol. TMS revealed a significant reduction in short-interval intracortical inhibition (SICI) in patients, particularly at inter-stimulus intervals (ISIs) of 2.5 and 3 ms. When averaging across ISIs from 1 to 7 ms, patients on medication exhibited significantly less inhibition compared to healthy controls. Long-interval intracortical inhibition (LICI) was also reduced at ISIs of 150 and 200 ms, while facilitation parameters remained within normal ranges. Patients exhibited increased amplitude of the second component of the LLR recorded from the abductor pollicis brevis, whereas RIII reflex measurements showed no significant differences. Axonal excitability testing revealed a graded increase in hyperpolarization-activated currents in patients with more severe symptoms. The observed reductions in SICI and LICI suggest impaired intracortical inhibition in the M1 hand area, offering indirect evidence of cortical dysfunction in regions clinically unaffected by the disease. The increased LLR amplitudes further indicate altered sensorimotor integration at the cortical level, whereas the absence of significant changes in RIII reflexes suggests that segmental spinal dysfunction within the pain pathway of the upper limbs is unlikely. Finally, axonal excitability findings point to a potential role of hyperpolarization-activated currents in either contributing to or predisposing individuals to RLS symptoms.De Grado et al. report that patients with restless limbs syndrome exhibit altered cortical excitability, impaired sensorimotor integration and abnormal nerve excitability even in clinically unaffected body regions. These findings suggest widespread cortical dysfunction and implicate ion channel abnormalities as potential contributors to the pathophysiology of this common disorder
CHARACTERIZATION AND VALORISATION OF MOUNTAIN PLANT GENETIC RESOURCES
No full textNowadays, there has been a significant global loss of agrobiodiversity, with an estimated 75% decline. Plant agrobiodiversity encompasses wild relatives, landraces, and modern cultivars of agricultural and food interest. Landraces, in particular, are traditional, locally adapted crop varieties that can hold immense value in terms of agronomic traits, phytochemical-nutritional profiles, and resilience to climate change. Many of them exhibit unique nutritional and phytochemical characteristics, especially in terms of secondary metabolites, antioxidants, and micronutrients, making them highly relevant for crop improvement, functional food development, and human health. However, only a limited number of these varieties are currently studied and conserved either in situ (on-farm) or ex situ (in germplasm banks) leaving many at risk of genetic erosion.
To address these concerns, the European Union (EU) has implemented strategies such as the EU Biodiversity Strategy 2020, the National Recovery and Resilience Plan (PNRR), and the 2030 Agenda for Sustainable Development, all of which emphasize the need for innovative solutions to safeguard agrobiodiversity. These policies encourage the conservation and valorization of traditional cultivars, while promoting circular economy models to counteract genetic erosion in agriculture. In line with these directives, Italy has established the National Register of Agrobiodiversity, an innovative policy tool designed to protect local genetic resources of plant, animal, or microbial origin that are threatened by extinction or erosion.
Italy is rich in agrobiodiversity; in 2020 more than 1600 traditional local cultivars were registered and most of them cultivated in the mountain and submountain areas. Thought, many of these varieties are little or not at all scientifically characterized and, due to their characteristics (biological, ecological and phytochemical), can constitute important resources for the sustainable development of mountain areas. In particular, they could be considered raw materials for the creation of innovative agro-food systems.
The research presented in this thesis focused on the scientific characterization and valorization of four plant genetic resources: the “Copafam” bean, “Carciofo di Malegno” and “Grano Siberiano Valtellinese” landraces and the Italian saffron.
This work is the result of the research activities realized at UNIMONT, the University of Milan’s Hub located in Edolo (BS) dedicated to mountain research, in collaboration with the Department of Agricultural and Environmental Sciences-Production, Landscape and Agroenergy (DISAA) and at the Department of Food, Environmental and Nutritional Sciences (DeFENS) of the University of Milan and at the Department of Nutrition and Food Science of the University of Madrid.
The four case studies presented in this thesis demonstrate the potential of mountain landraces and high-value crops as strategic resources for agrobiodiversity conservation and valorization. The “Copafam” bean (P. coccineus), a traditional landrace from the Brescia Pre-Alps, was characterized by high dietary fiber, the richest phenolic content, strong antioxidant activity, and low levels of antinutritional factors, confirming its potential as a functional food ingredient. Multivariate analyses highlighted its unique profile, while technological trials demonstrated its suitability for bakery products.
Valorization of processing by-products focused on cooking water, which proved to be a source of proteins and phenolics. Protein recovery reached 47% at pH 6, while phenolic retention was markedly higher, up to 85% in enriched systems. Moreover, 97% of externally added rutin was encapsulated, confirming the potential of alginate hydrogels for developing polyphenol-enriched functional ingredients.
In parallel, “Copafam” bean pods was explored, which are typically discarded after seed harvest. Pods, initially rich in insoluble fiber, were effectively transformed through high hydrostatic pressure (HHP) combined with enzymatic hydrolysis, leading to the release of soluble fiber and prebiotic oligosaccharides (raffinose, verbascose, cellobiose). HHP treatments also enhanced the availability of phenolic compounds, with moderate pressures (200 MPa) improving antioxidant potential and higher pressures (400 MPa) releasing bound hydroxycinnamic acids.
Overall, “Copafam” demonstrated value both as a food crop and through its by-products, supporting sustainable valorisation pathways for mountain agrobiodiversity.
The “Carciofo di Malegno” was morphologically different from commercial cultivars; it resulted belong to the “spinosi” group. Phytochemical characterization revealed appreciable levels of chlorogenic acid, cynarine, luteolin, and apigenin, comparable to those of commercial varieties, with edible capitula showing the highest concentrations. Interestingly, stems and outer bracts, usually discarded as waste, were also found to contain relevant amounts of these compounds, highlighting opportunities for circular economy valorization. Ecological niche modeling predicted an upward altitudinal shift in cultivation suitability under future climate scenarios, raising concerns for in situ conservation in the traditional lowland areas but also identifying new opportunities for mountain cultivation. On the valorization side, the landrace was submitted for inclusion in the Italian National Register of Agrobiodiversity, securing its legal recognition and protection. In parallel, participatory innovation initiatives, such as the development of leaf-based spirit prototypes in collaboration with local stakeholders and their successful sensory evaluation, demonstrated the potential of this landrace to generate added value through new agri-food products. These results underline the dual cultural and economic importance of the “Carciofo di Malegno”, showing how scientific characterization, conservation tools, and community-driven valorisation can converge to strengthen biodiversity conservation and support rural development in mountain regions.
The “Grano Siberiano Valtellinese” showed a distinctive nutritional and phytochemical profile, with high protein, fiber, and quercetin content. Extraction studies confirmed that hydroalcoholic solvents maximized flavonol recovery, particularly quercetin and rutin, while aqueous systems favored polar phenolic acids due to rutinosidase activity. Antioxidant assays (Folin, FRAP, ORAC) reinforced the strong bioactive potential of this landrace. Valorization of its by-product (straw) demonstrated that high hydrostatic pressure combined with enzymatic hydrolysis enhanced the release of soluble fibers and bound phenolic acids, improving antioxidant activity. These findings highlight the potential of this neglected crop both as a functional food and as a source of value-added ingredients in circular economy models.
Finally, Italian saffron quality analysis confirmed an overall excellent quality according to ISO 3632 classification (93% of samples were found to be of the first quality category). Non-destructive approaches, such as NIR spectroscopy combined with chemometric models, also proved highly effective in predicting quality parameters, offering valuable tools for rapid and sustainable monitoring. Ecological niche modelling projected a future upward shift of suitable cultivation areas, indicating that saffron will increasingly become an exclusive crop of mountain regions. For the valorization aspects, a new first category ISO 3632 subclassification (“premium”, “superior”, “high-quality”) was proposed to better reward excellence. Valorization was further supported by participatory networks, producer engagement, and educational initiatives, reinforcing both competitiveness and sustainability of this niche mountain crop.
These results highlight how scientific characterization, advanced analytical tools, and participatory valorization strategies can transform underutilized landraces and crops into key drivers for sustainable rural development, functional food innovation, and the long-term conservation of mountain agrobiodiversity
DECODING THE GENETIC LANDSCAPE OF CONGENITAL HYPOGONADOTROPIC HYPOGONADISM: FROM PATIENT ALLELIC VARIANTS TO FUNCTIONAL INSIGHTS IN ZEBRAFISH
No full textCongenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by incomplete or absent puberty and infertility, due to deficient secretion or action of gonadotropin-releasing hormone (GnRH). Although more than 60 causative genes have been described, the genetic heterogeneity of CHH remains only partially understood and many patients still lack a molecular diagnosis. Among the novel candidates, WFS1 and TBX3 have recently emerged as potential candidate genes for isolated CHH. The central research question of this thesis is whether heterozygous variants in WFS1 and TBX3, identified in CHH patients, can disrupt GnRH neuronal development, migration, or regulatory pathways, contributing to the disease pathogenesis.
In our cohort of CHH patients, genetic analysis revealed heterozygous WFS1 variants identified by whole-exome sequencing (WES) and TBX3 variants identified by next-generation sequencing (NGS) panel. To evaluate the functional impact of these variants, we performed site-directed mutagenesis followed by in vitro characterization in cellular models. Specifically, WFS1 wild-type and mutant constructs were evaluated in COS7 cells, while TBX3 constructs were tested in HEK293 cells. Functional assays included Western blotting, immunofluorescence, quantitative real-time PCR, and luminometric assays to investigate protein localization, expression stability, turnover and transcriptional activity.
To validate these findings in vivo, we used the Tg(GnRH3:EGFP) zebrafish line and generated knockdown (KD) model via morpholino of wfs1b and tbx3a/b. This strategy allows direct visualization of GnRH3 neurons development, proliferation and migration steps.
Knockdown of wfs1b in Tg (GnRH3:EGFP) zebrafish embryos disrupted GnRH3 neuronal architecture and downregulated isl1a, a key transcription factor involved in axon guidance. Our findings establish a mechanistic link between WFS1-mediated ER homeostasis, isl1a expression, and GnRH neuron development.
In parallel, tbx3 knockdown revealed defective GnRH3 neurons and kisspeptin neuronal development, associated with a marked downregulation of the Wnt signaling pathway. These results suggest that TBX3 variants may affect both migration and differentiation of GnRH neurons.
In conclusion, this thesis provides novel functional evidence that heterozygous variants in WFS1 and TBX3 converge on the disruption of GnRH and kisspeptin neuronal networks, through distinct but complementary mechanisms: WFS1 via ISL1-related pathways and axon guidance, and TBX3 through Wnt signaling and kisspeptin regulation. These findings enlarge the genetic and mechanistic landscape of CHH, propose zebrafish as a versatile model for functional validation of candidate genes and open new perspectives for targeted therapeutic approaches aiming to restore GnRH neuron function
Through the Calf’s Eye: Exploring Infrared Thermography to Uncover Pair-Housed Calves’ Affective States
No full textPair housing has been proven to improve calves’ welfare during the pre-weaning phase, potentially promoting positive emotions. Based on the emotional valence hypothesis, according to which positive emotions are mainly processed in the left brain hemisphere, infrared thermography (IRT) may detect changes in eye temperature, particularly thermal asymmetries. This study aimed to evaluate whether pair-housed calves presented ocular thermal asymmetry, compared to individually housed calves during the pre-weaning phase. Fifty-six Friesian female calves from two commercial dairy farms were enrolled and housed either individually or in pairs from birth until 8 weeks of age. IRT images of the lacrimal caruncle of both eyes were collected at 7, 21, 35, and 56 days of age. A linear mixed model tested the effects of housing, farm, year, and timepoint. No significant effect of pair housing on ocular asymmetry emerged, while absolute eye temperatures were significantly higher in pair-housed calves. Moreover, eye temperature declined over time, suggesting reduced arousal and habituation with age. Although the lateralization hypothesis was not confirmed, the study offers insights into IRT for assessing calf emotions and supports further investigation in positive contexts to better explore links between housing, emotional valence, and brain activity
Combination of laboratory automation and next-generation sequencing for the diagnosis of osteoarticular infections: a technical note
No full textOsteoarticular infections (OAIs) present a diagnostic challenge, particularly when conventional cultures fail to identify the causative pathogen. We developed and implemented a sequential diagnostic workflow for intraoperative orthopaedic tissue samples, integrating laboratory automation (LA), broth enrichment, and next-generation sequencing (NGS). Between September 2023 and March 2025, 702 samples from 117 patients with suspected OAIs were processed. LA identified pathogens in 42% of cases. Among LA-negative patients, broth enrichment and NGS yielded additional identifications in 23% and 11.5% of cases, respectively. Our workflow achieved a diagnostic result within 10 days while improving detection rates, particularly in culture-negative cases. Our experience demonstrates that combining advanced microbiology tools is feasible in the context of suspected OAIs. A multidisciplinary evaluation of results remains essential for optimal patient management
