Involvement of thyroid hormones in brain development and cancer

The development and maturation of the mammalian brain are regulated by thyroid hormones (THs). Both hypothyroidism and hyperthyroidism cause serious anomalies in the organization and function of the nervous system. Most importantly, brain development is sensitive to TH supply well before the onset of the fetal thyroid function, and thus depends on the trans-placental transfer of maternal THs during pregnancy. Although the mechanism of action of THs mainly involves direct regulation of gene expression (genomic effects), mediated by nuclear receptors (THRs), it is now clear that THs can elicit cell responses also by binding to plasma membrane sites (non-genomic effects). Genomic and non-genomic effects of THs cooperate in modeling chromatin organization and function, thus controlling proliferation, maturation, and metabolism of the nervous system. However, the complex interplay of THs with their targets has also been suggested to impact cancer proliferation as well as metastatic processes. Herein, after discussing the general mechanisms of action of THs and their physiological effects on the nervous system, we will summarize a collection of data showing that thyroid hormone levels might influence cancer proliferation and invasion

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

Synthesis of H1\u2da histone protein, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins, we have been searching for RNA-binding proteins involved in the post-transcriptional regulation of the H1\u2da gene. We recently reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). Herein we show that LPI, as well as PEP-19, can bind H1\u2da RNA. Moreover, in order to improve production of functional LPI/PEP-19, we modified the protocol normally adopted for preparing histidine tagged-proteins from bacteria, by adding an additional purification step. We also found that both LPI and PEP can compete for H1\u2da RNA binding with PIPPin (CSD-C2), another RNA-binding protein previously discovered in our laboratory. Since PEP19/LPI contain a calmodulin binding domain, we finally investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1\u2da RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. This finding suggests that calcium/calmodulin may have a role in controlling H1\u2da mRNA metabolism in the developing brain

Potential roles of extracellular vesicles in brain cell-to-cell communication

Potential roles of extracellular vesicles in brain cell-to-cell communication Extracellular vesicles (EVs) are released into thè extracellular space from both cancer and normal brain cells, and are probably able to modify thè phenotypic properties of receiving cells1. EVs released from astrocytes and neurons contain FGF2 and VEGF2'3 and induce a 'blood-brain barrier' (BBB) phenotype in cultured brain capillary endothelial cells (BCECs, unpublished results), On thè other hand, EVs from G26/24 oligodendroglioma induce apoptosis in neurons and astrocytes4-5. These effects are probably due to Fas Ligand and TRAIL, present in G26/24 vesicles4-5. Moreover, G26/24 EVs contain extracellular matrix remodeling proteases (such as ADAMTS)6, H1.0 histone protein, and H1.0 mRNA7. In particular, we previously hypothesized that G26/24 cells, and tumor cells in generai, can escape differentiation cues, and continue to proliferate by eliminating proteins, such as thè H1° linker histone (and its mRNA)7, which could otherwise block proliferation. To study vesicle release in a System that can better resemble in vivo conditions, astrocytes and BCECs were cultured on poly-L-lactic acid (PLLA) scaffolds and tested for their ability to grow and survive on this three-dimensional structures. We analyzed in parallel thè celi growth in 2D and 3D culture systems and observed thè differences in celi morphology by fluorescence analysis: threedimensional scaffolds have thè ability to guide celi growth, provide support, encourage celi adhesion and proliferation. Astrocytes8 and BCECs (unpublished results) adapted well to these porous matrices, not only remaining on thè surface, but also penetrating inside thè scaffolds. EVs released by astrocytes in these scaffolds are probably exosomes, as suggested by transmission electron microscopy pictures, and by thè presence of intracellular structures resembling multivesicular bodies. This 3D celi culture System could be further enriched to host different brain celi types, in order to set, for example, an in vitro model of BBB, that may be useful for drug delivery studies, and for thè formulation of new therapeutic strategies for thè treatment of neurological diseases. References [1] Schiera, G., Di Liegro, C.M., Di Liegro I. Int J Mol Sci. 2017, 18(12). pii: E2774. [2] Schiera, G., Proia, P., Alberti, C., Mineo, M., Savettieri, G., Di Liegro, I., 2007. J Celi Mol Med. 2007, 111(6), 1384-94. [3] Proia, P., Schiera, G., Mineo, M., Ingrassia, A.M. Santoro, G., Savettieri, G., Di Liegro, I. Int J Mol Med. 2008, 21(1), 63-7. [4] D'Agostino, S., Salamene, M., Di Liegro, I., Vittorelli, ML, Int J Oncol. 2006, 29(5), 1075-85. [5] Lo Cicero, A., Schiera, G., Proia, P., Saladino, P., Savettieri, G., Di Liegro, C.M., Di Liegro, I. Int J Oncol. 2011,39(6): 1353-7. [6] Lo Cicero, A., Majkowska, I., Nagase, H., Di Liegro, I., Troeberg, L., Matrix Biol. 2012, 31(4), 229-33. [7] Schiera, G., Di Liegro, C.M., Saladino, P., Pitti, R., Savettieri, G., Proia, P., Di Liegro, I. Int J Oncol. 2013, 43(6), 1771-6. [8] Carfì Pavia, F., Di Bella, M.A., Brucato, V., Blanda, V., Zummo, F., Vitrano, I., Di Liegro, C.M., Ghersi, G., Di Liegro, I., Schiera, G. Mol Med Rep. 2019 [Epub ahead of print]. [9] Di Bella MA, Zummo F., Carfì Pavia F., Brucato V., Di Liegro I., Schiera G. 2017, In: Microscopy and Imaging Science: practical approaches to applied research and education, pp 260-264. Ed: A. Méndez-Vilas Publisher, Formatex Research Center (Spain), ISBN-13, 978-84-942134-9-6

Neurobiology of performance anxiety:A new approach

The aim of this study is to investigate the neurobiology of stress/emotionality, creating a multidisciplinary assessment model, which can help to provide psychological and physiological responses depending on the genetic background related to sport performances, social closeness and performance anxiety management in team sports. We enrolled 20 female volleyball players aged 13 \ub1 1 years old played in two different teams during a regional championship final. Saliva collection was carried out before and after the match. In order to evaluate the neuroendocrine effectors involved in stress and performance, we analyzed cortisol and progesterone levels through Elisa standard kit as well as HSP70 and amylase activity as stress-induced markers. As concern the psychometric assesment, we administrated he CSAI-2 test, Closeness Generating Procedure and STAI test. Genomic DNA was isolated from saliva cells using a QIAamp saliva kit according to the manufacturer\u2019s protocols. The SNP of the 5-HTTLPR, BDNF, DRD4 were analyzed. The results of the T-test performed on the total results showed a statistically significant relationship (p < 0.05) in cortisol levels pre and post match, as well between amylase and HP70 according to the genetic background. The analysis performed using just post match samples show a negative correlation between social closeness, cortisol and progesterone levels, with p < 0.010 for progesterone vs social closeness and p < 0.012 for cortisol vs social closeness. About the winner teams and the looser teams, there is a negative correlation between pre match cortisol levels and performance anxiety (p < 0.042)

Enzymatic spermine metabolites induce apoptosis associated with increase of p53, caspase-3 and mir-34a in both neuroblastoma cells, SJNKP and the N-Myc-amplified form IMR5

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down-or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis

Studio longitudinale sul benessere e le attitudini degli Studenti di Medicina e Chirurgia. Primi risultati della fase 1: Le caratteristiche degli studenti selezionati

We present the first results of a longitudinal study for the recognition and assessment of non-cognitive aspects of the candidates access to medicine course, and throughout the course. This study considered 8 CLMs equally distributed throughout the country and 980 students enrolled in the first year, in the academic year 2013-2014. Preliminary results obtained from the analysis of the questionnaire used in the research show that students expect a profession characterized by the aid for patients and socially useful, while they do not expect a low pay as well as a work activity risky for health. Students are motivated to the academic course mainly by the desire to care for others, while variously opportunistic aspects are much less relevant. Students show high capacity of self-regulation, a high level of empathy in its both aspects of propensity to engagement with others in their difficult moments, as well as of tendency to spontaneously take the perspective of others, while they are able to maintain a goal directed behavior even in the presence of suffering others. These students are basically satisfied, they show a confident and positive attitude towards life and a substantial psychological health. However, it is possible to identify a sub-group of students showing signals of psychological fragility who must be carefully monitored: their profiles will be analyzed in more detail, through in-depth interviews scheduled for the third/fourth year as part of faculties counselling services

Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. * 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. * 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression

Obstacles and opportunities in the functionalanalysis of extracellular vesicle RNA – an ISEVposition paper

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data

Biogenesis and Dynamics of Mitochondria during the Cell Cycle: Significance of 3′UTRs

Nowadays, we are facing a renaissance of mitochondria in cancer biology. However, our knowledge of the basic cell biology and on the timing and mechanisms that control the biosynthesis of mitochondrial constituents during progression through the cell cycle of mammalian cells remain largely unknown. Herein, we document the in vivo changes on mitochondrial morphology and dynamics that accompany cellular mitosis, and illustrate the following key points of the biogenesis of mitochondria during progression of liver cells through the cycle: (i) the replication of nuclear and mitochondrial genomes is synchronized during cellular proliferation, (ii) the accretion of OXPHOS proteins is asynchronously regulated during proliferation being the synthesis of β-F1-ATPase and Hsp60 carried out also at G2/M and, (iii) the biosynthesis of cardiolipin is achieved during the S phase, although full development of the mitochondrial membrane potential (ΔΨm) is attained at G2/M. Furthermore, we demonstrate using reporter constructs that the mechanism regulating the accretion of β-F1-ATPase during cellular proliferation is controlled at the level of mRNA translation by the 3′UTR of the transcript. The 3′UTR-driven synthesis of the protein at G2/M is essential for conferring to the daughter cells the original phenotype of the parental cell. Our findings suggest that alterations on this process may promote deregulated β-F1-ATPase expression in human cancer

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications