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Antonio Alessio Leonardi,1– 3 Rosalia Battaglia,4 Dario Morganti,1,2 Maria Josè Lo Faro,1,3 Barbara Fazio,2 Chiara De Pascali,5 Luca Francioso,5 Gerardo Palazzo,6,7 Antonia Mallardi,8 Michele Purrello,4 Francesco Priolo,1 Paolo Musumeci,1,* Cinzia Di Pietro,4,* Alessia Irrera2,* 1Dipartimento di Fisica e Astronomia, Università di Catania, Catania, 95123, Italy; 2CNR-IPCF, Istituto per i Processi Chimico-Fisici, Messina, 98158, Italy; 3CNR-IMM UoS Catania, Istituto per la Microelettronica e Microsistemi, Catania, 95123, Italy; 4Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy; 5CNR-IMM, Institute for Microelectronics and Microsystems, Via Monteroni, University Campus, Lecce, 73100, Italy; 6Chemistry Department, University of Bari ‘Aldo Moro’, Bari, 70125, Italy; 7CSGI, Center for Colloid and Surface Science c/o Chemistry Department, Bari, 70125, Italy; 8CNR-IPCF, Institute for Chemical-Physical Processes, c/o Chemistry Department, Bari, 70125, Italy*These authors contributed equally to this workCorrespondence: Paolo Musumeci; Cinzia Di Pietro Email paolo.musumeci@ct.infn.it; dipietro@unict.itIntroduction: Small extracellular vesicles (sEVs), thanks to their cargo, are involved in cellular communication and play important roles in cell proliferation, growth, differentiation, apoptosis, stemness and embryo development. Their contribution to human pathology has been widely demonstrated and they are emerging as strategic biomarkers of cancer, neurodegenerative and cardiovascular diseases, and as potential targets for therapeutic intervention. However, the use of sEVs for medical applications is still limited due to the selectivity and sensitivity limits of the commonly applied approaches.Methods: Novel sensing solutions based on nanomaterials are arising as strategic tools able to surpass traditional sensor limits. Among these, Si nanowires (Si NWs), realized with cost-effective industrially compatible metal-assisted chemical etching, are perfect candidates for sEV detection.Results: In this paper, the realization of a selective sensor able to isolate, concentrate and quantify specific vesicle populations, from minimal volumes of biofluid, is presented. In particular, this Si NW platform has a detection limit of about 2× 105 sEVs/mL and was tested with follicular fluid and blastocoel samples. Moreover, the possibility to detach the selectively isolated sEVs allowing further analyses with other approaches was demonstrated by SEM analysis and several PCRs performed on the RNA content of the detached sEVs.Discussion: This platform overcomes the limit of detection of traditional methods and, most importantly, preserves the biological content of sEVs, opening the route toward a reliable liquid biopsy analysis.Keywords: silicon nanowires, biosensor, small extracellular vesicles, label-free, luminescence

In this study, for the first time, we demonstrated the presence of microRNAs and extracellular vesicles in human blastocoel fluid. The bioinformatic and comparative analyses identified the biological function of blastocoel fluid microRNAs and suggested a potential role inside the human blastocyst. We found 89 microRNAs, expressed at different levels, able to regulate critical signaling pathways controlling embryo development, such as pluripotency, cell reprogramming, epigenetic modifications, intercellular communication, cell adhesion and cell fate. Blastocoel fluid microRNAs reflect the miRNome of embryonic cells and their presence, associated with the discovery of extracellular vesicles, inside blastocoel fluid, strongly suggests their important role in mediating cell communication among blastocyst cells. Their characterization is important to better understand the earliest stages of embryogenesis and the complex circuits regulating pluripotency. Moreover, blastocoel fluid microRNA profiles could be influenced by blastocyst quality, therefore, microRNAs might be used to assess embryo potential in IVF cycles.

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.

The Human Genome Project led to the discovery that about 80% of our DNA is transcribed in RNA molecules. Only 2% of the human genome is translated into proteins, the rest mostly produces molecules called non-coding RNAs, which are a heterogeneous class of RNAs involved in different steps of gene regulation. They have been classified, according to their length, into small non-coding RNAs and long non-coding RNAs, or to their function, into housekeeping non-coding RNAs and regulatory non-coding RNAs. Their involvement has been widely demonstrated in all cellular processes, as well as their dysregulation in human pathologies. In this review, we discuss the function of non-coding RNAs in endometrial physiology, analysing their involvement in embryo implantation. Moreover, we explore their role in endometrial pathologies such as endometrial cancer, endometriosis and chronic endometritis.

Tissue engineering (by culturing cells on appropriate scaffolds, and using bioreactors to drive the correct bone structure formation) is an attractive alternative to bone grafting or implantation of bone substitutes. Osteogenesis is a biological process that involves many molecular intracellular pathways organized to optimize bone modeling. The use of bioreactor systems and especially the perfusion bioreactor, provides both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In this mini-review all the characteristics for the production of an appropriate bone construct are analyzed: the stem cell source, scaffolds useful for the seeding of pre-osteoblastic cells and the effects of fluid flow on differentiation and proliferation of bone precursor cells. By automating and standardizing tissue manufacture in controlled closed systems, engineered tissues may reduce the gap between the process of bone formation in vitro and subsequent graft of bone substitutes in vivo.

Purpose Bacterial adhesion and colonization play a crucial function in the pathogenesis of peri-implant tissue infection, which is considered the main cause of fixture loss. The aim of this study is to evaluate the differences in bacterial adhesion between a machined titanium surface, a double acid etched surface (Osseotite®) and an Osseotite surface with Nanometer-scale Discrete Crystalline Deposition (DCD™) of calcium phosphate (CaP)(Nanotite®). Methods Surface roughness properties of each sample were determined by a laser profilometer and scanning electron microscopy (SEM) observation. Bacterial adhesion on machined, Osseotite®, and Nanotite® discs were performed using the following bacterial strains: Streptococcus mutans CCUG 35176, Streptococcus sanguis CCUG 17826, Streptococcus salivarius CCUG 11878, Actinobacillus actinomycetecomitans CCUG 37002, Porphyromonas gingivalis CCUG 2521. The assessment of bacterial adhesion was performed by comparing two methods: Total Viable Count (TVC) estimation and Confocal Laser Scanning Microscopic (CSLM) studies. Results The surface roughness parameter increased as follows: machined®®. The attachment of all bacterial strains performed by both methods showed a significant reduction on Osseotite® and even higher on Nanotite® in comparison to machined surfaces (p® and Nanotite® for A. actinomycetecomitans, S. mutans and S. sanguis than for P. gingivalis and S. salivarius strains. Conclusions Nanotite® samples showed the lowest amount of bacterial contamination in comparison to the smoother machined and rougher Osseotite® surfaces.