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La generazione di ricercatori che si è formata nel corso degli ultimi quindici anni nel campo dell’architettura degli interni ha vissuto una stagione molto particolare. Da un lato, a causa del ridimensionamento delle risorse destinate all’università, si è spesso trovata di fronte all’impossibilità di progettare percorsi di ricerca di medio e lungo periodo. Dall’altro, avendo vissuto la crisi del settore universitario come una condizione quasi permanente, ha dovuto continuamente indagare nuove strade e diversi strumenti operativi per farvi fronte. Finendo, così, per individuare percorsi e profili innovativi di ricerca che, pur aderendo maggiormente alle necessità del reale, in molti casi sono ancora in attesa di valorizzazione. Questo volume raccoglie i contributi di giovani studiosi del settore, attivi in Italia o all’estero, e di chi conduce studi sui temi delle discipline degli interni, per provare a mappare gli orientamenti emergenti nel campo, su e attraverso il progetto. Il suo obiettivo è quello di fare emergere convergenze inaspettate, linee di ricerca promettenti e profili ancora poco valorizzati, nonché di restituire all’esterno la vitalità di una comunità che raramente trova occasioni di incontro e di confronto interno.

Background. Modification of gene expression profile, a first step in adaptation to exercise, leads to changes in the level of those molecules associated with skeletal muscle activity and energy metabolism, e.g., myokines, as well as those involved in their transcriptional regulation, e.g., microRNA. This study was aimed to investigate the influence of strenuous exercise on circulating microRNAs and their possible association with myokines response. Methods. Pre-competition and post-competition plasma samples were collected from 14 male athletes participating in a vertical run (+1000 m gain, 3600 m length). Circulating total (t-miRNA) and extracellular vesicles-associated (EV-miRNA) miRNAs were extracted from pooled plasma. Nanoparticle tracking analysis was performed to investigate pre- and post-competition EV concentration and size distribution. A panel of 179 miRNAs was assayed by qPCR and analyzed by Exiqon GenEx v6. Relative expression was calculated by the 2-ΔΔCT method. t-miRNA and EV-miRNAs whose level was ≥5-fold up- or down-regulated were validated in each single subject. Target prediction on MirWalk 3.0, Gene-Ontology and pathway enrichment analysis on Panther were performed to define the potential biological role of the identified miRNAs. A panel of 14 myokines was assayed in each sample by a multiplex immunoassay. Results. In whole plasma, 5 miRNAs were upregulated and 2 miRNAs were downregulated; in the EV fraction, 5 miRNAs were upregulated and 3 miRNAs were downregulated. Nanoparticle tracking analysis revealed a similar EV size distribution in pre- and post-competition samples and a decreased concentration in post-competition samples related to pre-competition samples. Gene-Ontology and pathway enrichment analysis revealed that the identified t-miRNAs and EV-miRNAs were potentially involved in metabolism regulation in response to exercise. Correlation between fold-change of post-competition relative to pre-competition plasma level of both t-miRNAs and EV-miRNAs and myokines further confirm these results. Conclusion. This study provides an example of the systemic response to acute endurance exercise, in wich circulating miRNAs play a pivotal role. Trial registration: ClinicalTrials.gov, (SportMarker NCT03386981), retrospectively registered on 4th January 2018.

Introduction: Circulating microRNAs (miRNAs) are emerging as potential biomarkers. However, the lack of preanalytical and analytical standardization limits their use. The aim of this study was to determine the expression of different miRNAs in plasma according to different collection and storage conditions. Materials and methods: Venous blood from 10 volunteers was collected in tubes spray-coated with dipotassium salt of ethylendiaminetetraacetic acid, either with (plasma-preparation tube, PPT) or without (K2EDTA) gel separator. Platelet-poor plasma (PPP) was also obtained from K2EDTA plasma. After storage under different conditions, miRNA-enriched total RNA was isolated from plasma and reverse transcribed. A panel of 179 miRNAs was assayed by quantitative polymerase chain reaction and the results were analysed by GenEx software. Detectability and stability of miRNAs were determined. Results: The number of undetected miRNAs was: 18, 24, and 22 in PPT; 83, 43, and 20 in K2EDTA; and 76, 106, and 104 in PPP samples, for plasma immediately frozen at - 80°C and plasma stored for 24h at room temperature or 4°C, respectively. Circulating miRNA expression in PPT samples was not affected by storage delay or temperature, while the percentage of up- and down-regulated miRNA in K2EDTA and PPP samples ranged from 2%, and 1% to 7%, and 5%, respectively. Conclusions: Sample matrix, temperature and delay in storage strongly influence the expression level of plasma miRNAs. Our results indicate PPT tubes as the most suitable matrix to improve total miRNA detectability and stability, independently of temperature.

(1) Background: In literature it is reported that 20&ndash, 30% of psoriatic patients evolve to psoriatic arthritis over time. Currently, no specific biochemical markers can either predict progression to psoriatic arthritis or response to therapies. This study aimed to identify osteoimmunological markers applicable to clinical practice, giving a quantitative tool for evaluating pathological status and, eventually, to provide prognostic support in diagnosis. (2) Methods: Soluble (serum) bone and cartilage markers were quantified in 50 patients with only psoriasis, 50 psoriatic patients with psoriatic arthritis, and 20 healthy controls by means of multiplex and enzyme-linked immunoassays. (3) Results: Differences in the concentrations of matrix metalloproteases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), receptor activator of nuclear factor kappa-B- ligand (RANK-L), procollagen type I N propeptide (PINP), C-terminal telopeptide of type I collagen (CTx-I), dickkopf-related protein 1 (DKK1), and sclerostin (SOST) distinguished healthy controls from psoriasis and psoriatic arthritis patients. We found that MMP2, MMP12, MMP13, TIMP2, and TIMP4 distinguished psoriasis from psoriatic arthritis patients undergoing a systemic treatment, with a good diagnostic accuracy (Area under the ROC Curve (AUC) &gt, 0.7). Then, chitinase-3-like protein 1 (CHI3L1) and MMP10 distinguished psoriasis from psoriatic arthritis not undergoing systemic therapy and, in the presence of onychopathy, MMP8 levels were higher in psoriasis than in psoriatic arthritis. However, in these latter cases, the diagnostic accuracy of the identified biomarkers was low (0.5 &lt, AUC &lt, 0.7). (4) Conclusions. By highlighting never exploited differences, the wide osteoimmunological biomarkers panel provides a novel clue to the development of diagnostic paths in psoriasis and psoriasis-associated arthropathic disease.

Summary: The integration of vascular structures into in vitro cultured tissues provides realistic models of complex tissue-vascular interactions. Despite the incidence and impact of muscle-wasting disorders, advanced in vitro systems are still far from recapitulating the environmental complexity of skeletal muscle. Our model comprises differentiated human muscle fibers enveloped by a sheath of human muscle-derived fibroblasts and supported by a vascular network with mural-like cells. Here, we demonstrate the induction of muscle-specific endothelium and the self-organization of endomysial muscle fibroblasts mediated by endothelial cells. We use this model to mimic the fibrotic environment characterizing muscular dystrophies and to highlight key signatures of fibrosis that are neglected or underestimated in traditional 2D monocultures. Overall, this vascularized meso-scale cellular construct finely recapitulates the human skeletal muscle environment and provides an advanced solution for in vitro studies of muscle physiology and pathology. : Bersini et al. demonstrate the generation of a mesoscale model of the human muscle environment and prove its application for the study of fibrosis. This engineered muscle environment promotes the organ-specific differentiation of endothelial cells and the self-assembly of myofibers spontaneously wrapped by a continuous endomysium-like structure. Keywords: endothelial specificity, 3D vascularized muscle model, fibrosis, muscle environment

Mutations in the SCN1A gene causing either loss or gain of function have been frequently found in patients affected by genetic epilepsy with febrile seizures plus (GEFS+) or Dravet syndrome (also named severe myoclonic epilepsy in infancy SMEI). By mutation screening of the SCN1A gene, we identified for the first time a case of two missense mutations in cis (p.[Arg1525Gln;Thr297Ile]) in all affected individuals of an Italian family showing GEFS+ and idiopathic generalized epilepsy (IGE). The p.Arg1525Gln mutation was not previously reported yet and was predicted to be pathological by prediction tools, whereas the p.Thr297Ile was already identified in patients showing SMEI. Functional studies revealed that the Nav1.1 channels harboring both mutations were characterized by a significant shift in the activation curve towards more positive potentials. Our data demonstrate that the p.Arg1525Gln represents a novel mutation in the SCN1A gene altering the channel properties in the co-presence of the p.Thr297Ile.

The aim of this study was to identify the relationship between metabolic effort, muscular damage/activity indices, and urinary amino acids profile over the course of a strenuous prolonged endurance activity, as a cycling stage race is, in order to identify possible fatigue markers. Nine professional cyclists belonging to a single team, competing in the Giro d'Italia cycling stage race, were anthropometrically characterized and sampled for blood and urine the day before the race started, and on days 12 and 23 of the race. Diet was kept the same over the race, and power output and energy expenditure were recorded. Sera were assayed for muscle markers (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase activities, and blood urea nitrogen), and creatinine, all corrected for plasma volume changes. Urines were profiled for amino acid concentrations, normalized on creatinine excretion. Renal function, in terms of glomerular filtration rate, was monitored by MDRD equation corrected on body surface area. Creatine kinase activity and blood urea were increased during the race as did serum creatinine while kidney function remained stable. Among the amino acids, taurine, glycine, cysteine, leucine, carnosine, 1-methyl histidine, and 3-methyl histidine showed a net decreased, while homocysteine was increased. Taurine and the dipeptide carnosine (β-alanyl-L-histidine) were significantly correlated with the muscle activity markers and the indices of effort. In conclusion, the metabolic profile is modified strikingly due to the effort. Urinary taurine and carnosine seem useful tools to evaluate the muscle damage and possibly the fatigue status on a long-term basis.KEYWORDS:Cycling stage race; Endurance; Fatigue; Muscular activity; Urinary amino acids

PURPOSE: Aim of this study was to investigate RANKL and osteoprotegerin plasma concentrations in patients affected by disc herniation, the most common epiphenomenon of disc degenerative diseases, and in a matched cohort of healthy subjects and whether the expression of these markers was associated to a polymorphism of the vitamin D receptor gene. METHODS: For this case-control study, 110 consecutive cases affected by lumbar disc herniation (confirmed by MRI) and 110 healthy age- and sex-matched controls were enrolled. Subjects affected by any other pathology were excluded. RANKL and osteoprotegerin were measured in plasma by immunoassays. The difference in these markers between cases and controls was assessed by t test. The correlation between osteoimmunological markers concentrations, anthropometrical variables, and the expression of the pathology was statistically assessed (Pearson's test) along with the association (Fisher's exact test) with the vitamin D receptor gene genotype, determined elsewhere. RESULTS: Despite comparable osteoprotegerin concentrations, cases, altogether or grouped for gender, express lower RANKL and, consequently, RANKL-to-osteoprotegerin ratio. While in cases RANKL and osteoprotegerin concentrations were independent from age and BMI, in controls they increased with age. Disc herniation was strongly associated with RANKL and the presence of the F allele of the VDR gene. CONCLUSIONS: Whether vertebral bone changes precede or follow cartilage deterioration in intervertebral disc degeneration is not known. Our results suggest a reduced bone turnover rate, associated to a specific genetic background, in patients affected by lumbar disc herniation which could be one of the favoring factors for disc degeneration. KEYWORDS: Disc herniation; OPG; RANKL; VDR polymorphisms

Evidence suggests that endurance and even recreational cycling may stimulate bone resorption; however, little is known about cartilage response to endurance cycling exercise. We investigated effort-dependent changes in bone turnover and cartilage biomarkers in blood and urine samples from elite cyclists during a 3-week stage race. Whole blood and urine samples were collected the day before the start of the race, at mid and end-race for serum and urinary CTx-I, NTx-I, PINP, COMP (only in serum), and CTx-II analysis by enzyme-linked immunosorbent assay. The values were corrected for plasma volume or creatinine excretion, respectively, and correlated with power output (corrected for body weight) and net energy expenditure. Bone marker concentrations in both serum and urine were slightly but significantly decreased. Among the cartilage degradation markers, only CTx-II was decreased, while COMP remained unchanged. The changes in bone and cartilage turnover indexes were correlated with the indexes of physical effort and energy consumption. Strenuous physical effort, in the absence of mechanical loading, slows bone metabolism and only minimally affects cartilage turnover. Since changes in plasma and urine volume, which normally occur in exercising athletes, can mask these effects, biomarker concentrations need to be corrected for shifts in plasma volume and urinary creatinine for correct interpretation of the data.

Objective Adalimumab has proven effective in psoriasis; however, secondary failure may result from the drug9s immunogenicity. Prevalence data on the immunogenicity of biologicals, and of adalimumab in particular, are highly variable. We investigated the prevalence of anti-adalimumab antibodies and the association with clinical indexes and tumour necrosis factor α (TNFα) serum levels in psoriatic patients. Design Case–control, longitudinal. Setting Single centre. Participants Patient groups: I (n=20) receiving biological therapies after switching from adalimumab; II (n=30) ongoing adalimumab therapy; III (n=30) novel adalimumab therapy; IV (n=15) biological therapies other than adalimumab. Healthy subjects : (group V; n=15) never treated with immunosuppressants or biologicals. Interventions All groups were tested at enrolment. Group II was also tested at 12 months, and group III at 1, 3, and 6 months. Primary and secondary outcome measures Standard clinical evaluations (Psoriasis Area Severity Index (PASI)), blood samples and two-site ELISA-based measurement of serum adalimumab trough levels, anti-adalimumab antibodies and TNFα. Results The false-positive rate was 23% for adalimumab detection and 22% for anti-adalimumab antibodies in patients naive to adalimumab. Spurious positivity for anti-adalimumab antibodies (one-time-point positivity in group III during follow-up) accounted for 33% of the total. The prevalence of anti-drug antibodies was highest (87%) in group I patients. No correlations were found between the presence of anti-adalimumab antibodies or adalimumab levels and changes in PASI scores. Conclusions High variability of results, high prevalence of false-positives and lack of association between anti-adalimumab antibodies and TNFα level/PASI score limit this assay9s usefulness. Accurate clinical evaluation is key to early identification of treatment failures.

Nocturnal frontal lobe epilepsy has been historically considered a channelopathy caused by mutations in subunits of the neuronal nicotinic acetylcholine receptor or in a recently reported potassium channel. However, these mutations account for only a minority of patients, and the existence of at least a new locus for the disease has been demonstrated. In 2005, we detected two nucleotide variations in the promoter of the CRH gene coding for the corticotropin releasing hormone in 7 patients. These variations cosegregated with the disease and were demonstrated to alter the cellular levels of this hormone. Here, we report the identification in an Italian affected family of a novel missense mutation (hpreproCRH p.Pro30Arg) located in the region of the CRH coding for the protein pro-sequence. The mutation was detected in heterozygosity in the two affected individuals. In vitro assays demonstrated that this mutation results in reduced levels of protein secretion in the short time thus suggesting that mutated people could present an altered capability to respond immediately to stress agents.

Idiopathic epilepsies are common and devastating neurological disorders in which genetic background and physiopathological mechanisms underlying the clinical phenotype are not fully characterized yet. These diseases are assumed to have a strong genetic component, being monogenic or oligo/polygenic with different recurrence risks in the same family. However, even in monogenic epilepsy, additional genes and environmental factors may modulate its expression, thus resulting in incomplete penetrance and variable phenotype. Ethiology, phenotypic manifestations and prognosis are indeed highly heterogeneous. Idiopathic epilepsies represent about 30-40% of all epilepsies in childhood and 20% in adults. Most of them are complex diseases: patients may shift from one phenotype to another during their lifetime and parents affected by one form may have children suffering from another epileptic syndrome. The identification of genes responsible for distinct epilepsy syndromes or influencing the risk for epilepsy has important implications, for both research and clinical purposes. In this work we studied the genetic bases of two different epilepsies: nocturnal frontal lobe epilepsy (NFLE/ADNFLE) and febrile seizures (FS/GEFS+). In the case of the NFLE/ADNFLE phenotype, we performed a mutational screening of known genes, including CRH and its promoter, in a sample of both sporadic and familial patients. The study allowed the identification of: an already known mutation in the CHRNA4 gene (p.Ser284Leu) originated de novo in one NFLE patient; three unknown variants in the CRH promoter in both sporadic and familial patients which we demonstrated to not cosegregate with the disease; one unknown missense mutation in the coding portion of the CRH gene in one ADNFLE patient. By functional in vitro analysis we demonstrated that the missense mutation causes impairment in the production and release of the CRH hormone. This impairment could be related to an altered capability of patients to respond quickly to stress agents. Finally, by analyzing candidate genes encoding the orexin system we demonstrated an unlikely role of this system in the pathogenesis of ADNFLE: none of the patients has mutations in the three genes. In the study of FS/GEFS+ phenotype, the role of the SCN1A gene was evaluated. Several intronic and exonic polymorphisms were detected. In the case of unknown intronic variants, an in silico analysis revealed that these variations do not introduce or remove any splicing sites. Interestingly, we found in a patient two missense mutations. These two variants co-segregated with the pathology being present in all affected individuals and in two obligate carriers. Owing to the location of both mutations in important regions of the sodium channel, we are now testing the hypothesis of their causative role in the pathogenesis of this family’s disease. The study will allow the evaluation of the effect of these mutations (considered either singly or in conjunction with the other) on the activation/inactivation properties of the sodium channel in the presence/absence of the β-1 accessory subunit.

BACKGROUND:: Nocturnal frontal lobe epilepsy (NFLE) is an idiopathic partial epilepsy characterized by a wide spectrum of stereotyped motor manifestations, mostly occurring during non rapid eye movements sleep. NFLE is underdiagnosed since semiological similarities make it difficult to distinguish NFLE from parasomnias. In 1994, authors reported families with NFLE inherited as an autosomal dominant trait and they introduced the term of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). A family history of possible NFLE is found in about 25% of cases. The genetic bases of the disease have been detected in a minority of cases. Mutations causing a gain of function of the neuronal nicotinic acetylcholine receptors were reported in 3 different subunits. REVIEW SUMMARY:: This review discusses the clinical aspects of NFLE and the diagnostic procedures. Furthermore, the genetic aspects are outlined. The main differentiating features characterizing NFLE are: (a) several attacks per night at any time during the night; (b) brief duration of the attacks; (c) stereotyped motor pattern. Nocturnal video-polysomnography is crucial for the diagnosis. Neurological examination in NFLE/ADNFLE is normal. About 30% of NFLE cases are resistant to antiepileptic drugs. Concerning the genetics, putative susceptibility nucleotide variations affecting the promoter of the CRH gene and altering the corticotrophin-releasing hormone levels have been reported in some NFLE patients. CONCLUSIONS:: Distinguishing NFLE seizures from paroxysmal nonepileptic sleep disorders is often difficult and sometimes impossible on clinical grounds alone. Nocturnal video-polysomnography is mandatory. Further genetic studies could help the diagnosis and treatment in NFLE patients.