ROLE OF RNA BINDING PROTEIN IN THE NERVE CELL DIFFERENTIATION

Synthesis of H1˚ and H3.3 histone proteins, 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 (RBPs), we have been searching for RBPs involved in the post-transcriptional regulation of the H1˚ and H3.3 genes. Previously, we 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). We showed that LPI, as well as PEP-19, can bind H1˚ RNA. Since PEP19 and LPI contain a calmodulin binding domain, we also investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1˚ RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. Pep-19/calmodulin high affinity binding has been demonstrated by Biolayer interferometry (BLI). This finding suggests that calcium/calmodulin may have a role in controlling H1˚ mRNA metabolism in the developing brain. 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. Furthermore, we found that both LPI and PEP-19 can compete for H1˚ RNA binding with PIPPin (also known as CSD-C2), another RBP previously discovered in our laboratory. PIPPin/CSD-C2 binds with high specificity to the mRNAs encoding H1° and H3.3 histone variants, undergoes thyroid hormone-dependent SUMOylation, and has been recently demonstrated to interact with other RBPs. PIPPin belongs to the CSD-containing class of RBPs, also called Y-box proteins, that play a key role in controlling the recruitment of mRNAs to the translational machinery, in response to environmental cues, both in development and in differentiated cells. Another aspect of this study was to confirm histone mRNAs-PIPPin interactions and to describe binding properties through streptavidin-biotin conjugation method, by BLI. We report the data obtained in the case of H3.3 and H1° mRNA-PIPPin interactions, and the specific affinity constant for these bindings. In order to identify RNA portions involved in binding, we used different RNA probes for H3.3 and H1°. In summary, we were able to confirm that PIPPin binds H3.3 and H1° mRNA with very high affinity. Searching for other RBPs, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of CSD-C2. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70, whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70

Oligodendroglioma cells shed microvesicles which contain TRAIL as well as molecular chaperones and induce cell death in astrocytes.

Microvesicles (MVs) shed from G26/24 oligodendroglioma cells were previously reported to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and eventually neuronal apoptosis, when added to primary cultures of rat cortical neurons. These effects were reduced but not abolished by functional monoclonal antibodies against Fas-L. In order to investigate whether MVs contain other factors able to induce cell death, we tested them for TRAIL and found clear evidence of its presence in the vesicles. This finding suggests the possibility that Fas-L and TRAIL cooperate in inducing brain cell death. Aimed at understanding the route through which the vesicles deliver their messages to the target cells, we labeled oligodendroglioma cells with radioactive methionine and then added the labeled vesicles shed from tumor cells to unlabeled astrocytes in culture. Here we report that labeled proteins were delivered to the test cells. In order to investigate whether astrocytes, like neurons, are sensitive to oligodendroglioma-derived vesicles, MVs were prepared from media conditioned by G26/24 oligodendroglioma cells and added to primary cultures of rat cortical astrocytes. These cells were clearly more resistant than neurons to microvesicle-induced damage: a high dose (40 µg) of shed MVs induced cell death in only about 40% of astrocytes. Finally, we demonstrated that Hsp70 is specifically enriched in MVs which also contain, even if at lower level, the Hsc70 constitutive chaperone

Graphene Oxide Carboxymethylcellulose Nanocomposite for Dressing Materials

Sore, infected wounds are a major clinical issue, and there is thus an urgent need for novel biomaterials as multifunctional constituents for dressings. A set of biocomposites was prepared by solvent casting using different concentrations of carboxymethylcellulose (CMC) and exfoliated graphene oxide (Exf-GO) as a filler. Exf-GO was first obtained by the strong oxidation and exfoliation of graphite. The structural, morphological and mechanical properties of the composites (CMCx/Exf-GO) were evaluated, and the obtained composites were homogenous, transparent and brownish in color. The results confirmed that Exf-GO may be homogeneously dispersed in CMC. It was found that the composite has an inhibitory activity against the Gram-positive Staphylococcus aureus, but not against Gram-negative Pseudomonas aeruginosa. At the same time, it does not exhibit any cytotoxic effect on normal fibroblasts

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

Replication-independent expression of H1˚ and H3.3 histone variants is probably regulated by different RNA-binding proteins

DNA in eukaryotes is wrapped around core histones to form nucleosomes, the basic units of chromatin. The linker histones H1 bind DNA where it enters and leaves the nucleosome, thus stabilizing higher order structures. Chromatin is a dynamic complex, modulated by different processes such as DNA-methylation, post-translational modifications of histones, and incorporation of specific histone variants. Throughout rat brain development, expression of H1° and H3.3 histone variants is mainly regulated at the post-transcriptional level. These proteins are of interest for their possible involvement in the replication-independent chromatin remodelling induced by extracellular stimuli. We previously cloned two cDNAs encoding, respectively, PIPPin (or CSD-C2), a brain-enriched protein able to bind the 3’end of both H1° and H3.3 mRNAs, and LPI (longer isoform of PEP-19). Both PEP-19 and LPI are brain-specific. By western blot, we found that PIPPin expression in PC12 cells is enhanced by NGF-induced differentiation. We investigated the RNA-binding properties of the three proteins using their 6 histidine-tagged recombinant fusions and found that they all bind H1° and H3.3 RNAs. Since PEP-19 and LPI are camstatins, we also analyzed whether calmodulin could interfere with RNA-binding, and found that calmodulin competes with H1° RNA binding to both proteins, while it is not able to bind RNA on its own. This finding suggests that, in the brain, PEP19 and LPI could induce histone mRNA translation in response to calcium. By using biotinylated H1°/H3.3 RNA as fishing molecules, we isolated by affinity chromatography a group of proteins which were analyzed by mass spectrometry. Among them some heterogeneous nuclear ribonucleoproteins (HnRNP K, A1, A2/B1) and the Hsc70 chaperone. We are currently studying the interactions among these proteins by co-immunoprecipitation assays. Castiglia D. et al. (Biochem Biophys Res Commun 218: 390-41996) Scaturro M. et al. (1998) J Biol Chem 273: 22788-91 Nastasi T. et al. (1999) J Biol Chem 274: 24087-93 Sala A et al. (2007) Int J Mol Med 19: 501-9 Saladino P. et al. (2012) Int J Mol Med 29:141-

Identification of nuclear proteins which interact with H1° mRNA.

In developing rat brain the synthesis of H1° histone is mainly regulated at posttranscriptional level and probably depends on RNA-binding proteins (RBPs) (1). We previously identified RBPs apparently specific for this messenger (2) and cloned two novel proteins by screening an expression cDNA library by binding to radiolabeled RNA (3-10). Here we report the use of biotinylated H1° RNA as bait to isolate by chromatography nuclear proteins which interact with H1° mRNA. We identified by mass spectrometry abundant RBPs and molecular chaperones. By western blot we also evidenced, among the RNA-bound proteins, the cold shock domain-containing protein 2 (CSD-C2, also know as PIPPin), a brain-enriched RNA-binding protein cloned in our laboratory. Co-immunoprecipitation assays were performed to confirm interactions. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1 and with the Y box-binding protein 1 (YB1). We also got evidences that CSD-C2 interacts with Hsc70. In conclusion, we characterized a set of interactions which suggest the existence of a ribonucleoprotein particle, that might control H1º histone expression in maturing brain

Identification in the rat brain of a set of nuclear proteins interacting with H1° mRNA

Synthesis of H1° histone, in the developing rat brain, is also regulated at post-transcriptional level. Regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs); therefore, we searched for H1° mRNA-interacting proteins. With this aim, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of cold shock domain-containing protein 2 (CSD-C2, also known as PIPPin), a brain-enriched RBP previously described in our laboratory. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70. Overall, we have contributed to the molecular characterization of a ribonucleoprotein particle possibly controlling H1° histone expression in the brain