DNA-methylation dependent regulation of embryo-specific 5S ribosomal DNA cluster transcription in adult tissues of sea urchin Paracentrotus lividus

We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus and recently, demonstrated the presence of high heterogeneity in functional 5S rRNA. In this paper, we show some important distinctive data on 5S rRNA transcription for this organism. Using Single Strand Conformation Polymorphism (SSCP) analysis, we demonstrate the existence of two classes of 5S rRNA, one which is embryo-specific and encoded by the smallest (700bp) cluster and the other which is expressed at every stage and encoded by longer clusters (900 and 950bp). We also demonstrate that the embryo-specific class of 5S rRNA is expressed in oocytes and embryonic stages and is silenced in adult tissue and that this phenomenon appears to be due exclusively to DNA methylation, as indicated by sensitivity to 5-azacytidine, unlike Xenopus where this mechanism is necessary but not sufficient to maintain the silenced status

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach,we studied the polymorphisms andmethylation of sites contained in the 5′ flanking region of themetabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associatedwith the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins

MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

Mesenchymal stromal cells (hMSCs) display a pleiotropic function in bone regeneration. The signaling involved in osteoblast commitment is still not completely understood, and that determines the failure of current therapies being used. In our recent studies, we identified two miRNAs as regulators of hMSCs osteoblast differentiation driving hypoxia signaling and cytoskeletal reorganization. Other signalings involved in this process are epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) signalings through the regulation of Yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression. In the current study, we investigated the role of miR-33a family as a (i) modulator of YAP/TAZ expression and (ii) a regulator of EGFR signaling during osteoblast commitments. Starting from the observation on hMSCs and primary osteoblast cell lines (Nh-Ost) in which EMT genes and miR-33a displayed a specific expression, we performed a gain and loss of function study with miR-33a-5p and 3p on hMSCs cells and Nh-Ost. After 24 h of transfections, we evaluated the modulation of EMT and osteoblast genes expression by qRT-PCR, Western blot, and Osteoimage assays. Through bioinformatic analysis, we identified YAP as the putative target of miR-33a-3p. Its role was investigated by gain and loss of function studies with miR-33a-3p on hMSCs; qRT-PCR and Western blot analyses were also carried out. Finally, the possible role of EGFR signaling in YAP/TAZ modulation by miR-33a-3p expression was evaluated. Human MSCs were treated with EGF-2 and EGFR inhibitor for different time points, and qRT-PCR and Western blot analyses were performed. The above-mentioned methods revealed a balance between miR-33a-5p and miR-33a-3p expression during hMSCs osteoblast differentiation. The human MSCs phenotype was maintained by miR-33a-5p, while the maintenance of the osteoblast phenotype in the Nh-Ost cell model was permitted by miR-33a-3p expression, which regulated YAP/TAZ through the modulation of EGFR signaling. The inhibition of EGFR blocked the effects of miR-33a-3p on YAP/TAZ modulation, favoring the maintenance of hMSCs in a committed phenotype. A new possible personalized therapeutic approach to bone regeneration was discussed, which might be mediated by customizing delivery of miR-33a in simultaneously targeting EGFR and YAP signaling with combined use of drugs

Mung Bean nuclease mapping of RNAs 3' end

A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases

Chapter Il Piccolo Masaccio e le Terre Nuove. Creativity and Computer Graphics for Museum Edutainment

Since its opening, the Museum of the New Towns, housed in the Palazzo di Arnolfo in San Giovanni Valdarno, has dedicated a particular attention to the relationship with its audiences. In this context, the video “Il piccolo Masaccio e le Terre Nuove” has the purpose of bringing children and young people, in particular, closer to the museum main themes. The video presents a series of very different techniques, such as live shots, taken also by drone, Computer Graphics, 2D drawings executed with a tablet, drawings sketched with traditional techniques, such as India ink and watercolours, and digital videos taken from Google Earth

Sharing Circulating Micro-RNAs between Osteoporosis and Sarcopenia: A Systematic Review

Background: Osteosarcopenia, a combination of osteopenia/osteoporosis and sarcopenia,is a common condition among older adults. While numerous studies and meta-analyses have beenconducted on osteoporosis biomarkers, biomarker utility in osteosarcopenia still lacks evidence. Here,we carried out a systematic review to explore and analyze the potential clinical of circulating microR-NAs (miRs) shared between osteoporosis/osteopenia and sarcopenia. Methods: We performed asystematic review on PubMed, Scopus, and Embase for differentially expressed miRs (p-value < 0.05)in (i) osteoporosis and (ii) sarcopenia. Following screening for title and abstract and deduplication,83 studies on osteoporosis and 11 on sarcopenia were identified for full-text screening. Full-textscreening identified 54 studies on osteoporosis, 4 on sarcopenia, and 1 on both osteoporosis andsarcopenia. Results: A total of 69 miRs were identified for osteoporosis and 14 for sarcopenia. Therewere 9 shared miRs, with evidence of dysregulation (up- or down-regulation), in both osteoporo-sis and sarcopenia: miR-23a-3p, miR-29a, miR-93, miR-133a and b, miR-155, miR-206, miR-208,miR-222, and miR-328, with functions and targets implicated in the pathogenesis of osteosarcopenia.However, there was little agreement in the results across studies and insufficient data for miRsin sarcopenia, and only three miRs, miR-155, miR-206, and miR-328, showed the same directionof dysregulation (down-regulation) in both osteoporosis and sarcopenia. Additionally, for mostidentified miRs there has been no replication by more than one study, and this is particularly true forall miRs analyzed in sarcopenia. The study quality was typically rated intermediate/high risk of bias.The large heterogeneity of the studies made it impossible to perform a meta-analysis. Conclusions:The findings of this review are particularly novel, as miRs have not yet been explored in the context ofosteosarcopenia. The dysregulation of miRs identified in this review may provide important clues tobetter understand the pathogenesis of osteosarcopenia, while also laying the foundations for furtherstudies to lead to effective screening, monitoring, or treatment strategies (PDF) Sharing Circulating Micro-RNAs between Osteoporosis and Sarcopenia: A Systematic Review. Available from: https://www.researchgate.net/publication/368667300_Sharing_Circulating_Micro-RNAs_between_Osteoporosis_and_Sarcopenia_A_Systematic_Review [accessed Feb 26 2023]

How miR-31-5p and miR-33a-5p Regulates SP1/CX43 Expression in Osteoarthritis Disease: Preliminary Insights

Osteoarthritis (OA) is a degenerative bone disease that involved micro and macro-environment of joints. To date, there are no radical curative treatments for OA and novel therapies are mandatory. Recent evidence suggests the role of miRNAs in OA progression. In our previous studies, we demonstrated the role of miR-31-5p and miR-33a families in different bone regeneration signaling. Here, we investigated the role of miR-31-5p and miR-33a-5p in OA progression. A different expression of miR-31-5p and miR-33a-5p into osteoblasts and chondrocytes isolated from joint tissues of OA patients classified in based on different Kellgren and Lawrence (KL) grading was highlighted; and through a bioinformatic approach the common miRNAs target Specificity proteins (Sp1) were identified. Sp1 regulates the expression of gap junction protein Connexin43 (Cx43), which in OA drives the modification of (i) osteoblasts and chondrocytes genes expression, (ii) joint inflammation cytokines releases and (iii) cell functions. Concerning this, thanks to gain and loss of function studies, the possible role of Sp1 as a modulator of CX43 expression through miR-31-5p and miR-33a-5p action was also evaluated. Finally, we hypothesize that both miRNAs cooperate to modulate the expression of SP1 in osteoblasts and chondrocytes and interfering, consequently, with CX43 expression, and they might be further investigated as new possible biomarkers for OA

Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1\u3b1 Axis

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1\u3b1/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1\u3b1 by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1\u3b1 (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors

Evidence for a novel cytoplasmic processing event in ribosome maturation in the sea urchin Paracentrotus lividus

In this work, we demonstrate the existence of a cytoplasmic processing step, never before described, involving both the pre-ribosomal subunits in the sea urchin Paracentrotus lividus. Northern-blot hybridization, primer extension, S1 mapping experiments and in situ hybridizations allowed us to demonstrate that cytoplasmic processed particles are successively re-imported into the nucleus, where maturation of their RNAs is completed prior to being exported to the cytoplasm. Our findings lead to the proposal of a new model of ribosome maturation and shuttling. Moreover, preliminary data from our laboratory suggest that the maturation pathway we propose in P. lividus may not be unique to the sea urchin, but a common feature in eukaryotes