Genomic and Non-genomic Action of Neurosteroids in the Peripheral Nervous System

Since the former evidence of biologic actions of neurosteroids in the central nervous system, also the peripheral nervous system (PNS) was reported as a structure affected by these substances. Indeed, neurosteroids are synthesized and active in the PNS, exerting many important actions on the different cell types of this system. PNS is a target for neurosteroids, in their native form or as metabolites. In particular, old and recent evidence indicates that the progesterone metabolite allopregnanolone possesses important functions in the PNS, thus contributing to its physiologic processes. In this review, we will survey the more recent findings on the genomic and non-genomic actions of neurosteroids in nerves, ganglia, and cells forming the PNS, focusing on the mechanisms regulating the peripheral neuron-glial crosstalk. Then, we will refer to the physiopathological significance of the neurosteroid signaling disturbances in the PNS, in to identify new molecular targets for promising pharmacotherapeutic approaches

M2 muscarinic receptor activation inhibits cell proliferation and migration of rat adipose-mesenchymal stem cells

Mesenchymal stem cells (MSCs), also known as stromal mesenchymal stem cells, are multipotent cells, which can be found in many tissues and organs as bone marrow, adipose tissue and other tissues. In particular MSCs derived from Adipose tissue (ADSCs) are the most frequently used in regenerative medicine because they are easy to source, rapidly expandable in culture and excellent differentiation potential into adipocytes, chondrocytes and other cell types. Acetylcholine (ACh), is one of the most important neurotransmitter in central (CNS) and peripheral nervous system (PNS), playing important roles also in non-neural tissue, but its functions in MSCs are still not investigated. Although MSCs express muscarinic receptor subtypes, their role is completely unknown. In present work muscarinic cholinergic effects were characterized in rat ADSCs. Analysis by RT-PCR demonstrates that ADSCs express M1-M4 muscarinic receptor subtypes, whereas M2 is one of the most expressed subtype. For this reason, our attention was focused on M2 subtype. By using the selective M2 agonist Arecaidine Propargyl Ester (APE) we performed cell proliferation and migration assays demonstrating that APE causes cell growth and migration inhibition without affecting cell survival. Our results indicate that ACh via M2 receptors, may contribute to the maintaining of the ADSCs quiescent status. These data are the first evidence that ACh via muscarinic receptors might contribute to control ADSCs physiology

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool “REPAIRv2” (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in dierent cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFPopal and CFTRW1282X, respectively

Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring

The first determination of presence and biodistribution of PFOA in ninety specimens of sea urchin Paracentrotus lividus from two differently contaminated sites along Palermo’s coastline (Sicily) is reported. Analyses were performed on the sea urchins’ coelomic fluids, coelomocytes, gonads or mixed organs, as well as on seawater and Posidonia oceanica leaves samples from the collection sites. PFOA concentration ranged between 1 and 13 ng/L in seawater and between 0 and 794 ng/g in P. oceanica. The analyses carried out on individuals of P. lividus from the least polluted site (A) showed PFOA median values equal to 0 in all the matrices (coelomic fluid, coelomocytes and gonads). Conversely, individuals collected from the most polluted site (B) showed median PFOA concentrations of 21 ng/g in coelomic fluid, 153 ng/g in coelomocytes, and 195 ng/g in gonads. Calculated bioconcentration factors of log10BCF > 3.7 confirmed the very bioaccumulative nature of PFOA. Significant correlations were found between the PFOA concentration of the coelomic fluid versus the total PFOA concentration of the entire sea urchin. PERMANOVA (p = 0.001) end Welch's t-test (p < 0.001) analyses showed a difference between specimens collected from the two sites highlighting the potential application of P. lividus as sentinel species for PFOA biomonitoring

The effect of behind-the-scenes encounters and interactive presentations on the welfare of captive servals (Leptailurus serval)

The serval (Leptailurus serval) is a small African felid that is well represented in zoos and often serves as an animal ambassador in encounter programs with zoo visitors. The impact on serval welfare in relation to such programs has not been investigated to date, and the aim of this study was to assess short-term welfare effects of varying levels of visitor interaction in two captive servals. Weekly blocks of four different treatments were imposed three times on each animal over 12 weeks, and the treatments involved (1) Presentations (serval undertaking a routine training session in a designated presentation space, typically attracting high visitor numbers), (2) Behind-the-scenes (BTS, a close encounter allowing a small group of visitors to interact closely with the cat in its enclosure), (3) Presentations and BTS combined, and (4) No visitor interaction. Serval activity budgets as well as behavioural diversity were created from behaviours observed from Close Circuit Television (CCTV) footage during four daily recording sessions per animal over three consecutive days per treatment, using instantaneous scan sampling every 60 s. Individual faecal samples were collected daily to monitor changes in faecal glucocorticoid metabolite (FGM) concentration. Results indicate that the mean number of scans with stereotypic pacing was significantly reduced (p = 0.01) during Treatments 1 and 3, when cats participated in presentations only, or the two activities combined. Conversely, a significant reduction in behavioural diversity (p 0.05). Given the reduction in stereotypic pacing, these findings suggest that involvement in an encounter program appears to exert an overall positive short-term welfare effect on the individual servals in this study. Although a reduction in behavioural diversity was not considered a negative welfare effect in the short term, potential long-term negative welfare effects resulting from a more frequent encounter program could not be ruled out in the present study

Tumor suppressor Nf2/merlin drives Schwann cell changes following electromagnetic field exposure through Hippo-dependent mechanisms

Previous evidence showed mutations of the neurofibromin type 2 gene (Nf2), encoding the tumor suppressor protein merlin, in sporadic and vestibular schwannomas affecting Schwann cells (SC). Accordingly, efforts have been addressed to identify possible factors, even environmental, that may regulate neurofibromas growth. In this context, we investigated the exposure of SC to an electromagnetic field (EMF), which is an environmental issue modulating biological processes. Here we show that SC exposed to 50 Hz EMFs change their morphology, proliferation, migration and myelinating capability. In these cells merlin is downregulated, leading to activation of two intracellular signaling pathways, ERK/AKT and Hippo. Interestingly, SC change their phenotype toward a proliferative/migrating state, which in principle may be pathologically relevant for schwannoma development

Reevaluating the function of a transcription factor: MBF-1 as a sea urchin chromatin organizer ?

The Zinc-finger MBF-1 factor is involved in the expression of the early histone genes during devel-opment of the sea urchin embryo (1, 2). In spite of being a transcription activator, the DNA-binding domain of MBF-1 shares high sequence similarity with that of the chromatin organizer CTCF of vertebrates and drosophila (3). On the other hand, extensive in silico analysis failed to identify the sea urchin CTCF ortholog (4). This led us to speculate that MBF-1 somehow could have co-opted the function of CTCF during evolution of the echinoderms. Since in vertebrates CTCF binds Hox chromatin, to support our hypothesis, we first identified high-score putative binding sequences for CTCF/MBF-1 within the single sea urchin Hox gene cluster. Moreover, we observed the full evolu-tionary conservation of these binding sites in S. purpuratus and P. lividus species. Worth of men-tion, by chromatin immunoprecipitation (ChIP) assay, we detected the occupancy of MBF-1 on hox11/13-a, -b, and -c regulatory sequences at distinct stages of development. As expected from the binding of an activator, we found that the association of MBF-1 to the cis-regulatory sequences of both hox11/13-a and -b genes relates to the transcriptional status of these genes. Strikingly, we also mapped the physical binding of MBF-1 to hox11/13-c, which is instead not expressed during em-bryogenesis. Altogether, these observations indeed suggest the possibility that MBF-1, besides be-ing a transcription activator, could also function as a general chromatin organizer. To further support this hypothesis, we are planning ChIP-seq experiments to identify the association of MBF-1 to the sea urchin chromatin at a genome-wide level. 1. Di Caro, V. et al. (2007) J. Mol. Bio.,365, 1285-97. 2. Cavalieri,V et al. (2009) Nucleic Acid Res, 37,7407-7415. 3. Heger , P. et al. (2012) PNAS, 109, 17507\u201317512. 4. Cavalieri, V. et al. (2013) Plos Genetics, 9, e1003847