Daniele Bellavia

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16(INK4a) gene in human colorectal carcinoma (Caco-2) cells.

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25 mg fresh pulp equivalents/mL, and 115±15 μM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16(INK4a) gene promoter, reactivation of the silenced mRNA expression and accumulation of p16(INK4a), a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells.

Chromosomal localization and molecular characterization of three different 5S ribosomal DNA clusters in the sea urchin Paracentrotus lividus

In this paper the chromosomal localization and molecular cloning and characterization of three 5S rDNA clusters of 700 bp (base pairs), 900 bp, and 950 bp in the sea urchin Paracentrotus lividus are reported. Southern blot hybridization demonstrated the existence of three 5S rDNA repeats of differing length in the P. lividus genome. Fluorescence in situ hybridization analysis, performed in parallel on both haploid and diploid metaphases and interphase nuclei using different 5S rDNA units as probes, localized these 5S rDNA clusters in 3 different pairs of P. lividus chromosomes. This is the first complete gene mapping not only in a sea urchin but also in the phylum of echinoderms as a whole.

Interleukin 3- receptor targeted exosomes inhibit in vitro and in vivo chronic myelogenous Leukemia cell growth

Despite Imatinib (IM), a selective inhibitor of Bcr-Abl, having led to improved prognosis in Chronic Myeloid Leukemia (CML) patients, acquired resistance and long-term adverse effects is still being encountered. There is, therefore, urgent need to develop alternative strategies to overcome drug resistance. According to the molecules expressed on their surface, exosomes can target specific cells. Exosomes can also be loaded with a variety of molecules, thereby acting as a vehicle for the delivery of therapeutic agents. In this study, we engineered HEK293T cells to express the exosomal protein Lamp2b, fused to a fragment of Interleukin 3 (IL3). The IL3 receptor (IL3-R) is overexpressed in CML blasts compared to normal hematopoietic cells and thus is able to act as a receptor target in a cancer drug delivery system. Here we show that IL3L exosomes, loaded with Imatinib or with BCR-ABL siRNA, are able to target CML cells and inhibit in vitro and in vivo cancer cell growth.

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.

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 (700 bp) cluster and the other which is expressed at every stage and encoded by longer clusters (900 and 950 bp). 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.

Exploration of the Sea Urchin Coelomic Fluid via Combinatorial Peptide Ligand Libraries

The urchin Paracentrotus lividus has been characterized via previous capture and enhancement of low-abundance proteins with combinatorial peptide ligand libraries (CPLL, ProteoMiner). Whereas in the control only 26 unique gene products could be identified, 82 species could be detected after CPLL treatment. Due to the overwhelming presence of two major proteins—the toposome (a highly glycosylated, modified calcium-binding, iron-less transferrin) and the major yolk proteins, belonging to the class of cell adhesion proteins—which constituted about 70% of the proteome of this biological fluid and strongly interfered with the capture of the minority proteome, no additional proteins could be detected. Yet, at present, this constitutes the most thorough investigation of the proteome of this biological fluid.

Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product.

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

In this paper 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.

A homemade device for linear sucrose gradients

We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials--a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained.

Circulating biomarkers in osteosarcoma: new translational tools for diagnosis and treatment

Osteosarcoma (OS) is a rare primary malignant bone tumour arising from primitive bone-forming mesenchymal cells, with high incidence in children and young adults, accounting for approximately 60% of all malignant bone tumours. Currently, long-term disease-free survival can be achieved by surgical treatment plus chemotherapy in approximately 60% of patients with localized extremity disease, and in 20–30% of patients with metastatic lung or bone disease. Diagnosis of primary lesions and recurrences is achieved by using radiological investigations and standard tissue biopsy, the latter being costly, painful and hardly repeatable for patients. Therefore, despite some recent advances, novel biomarkers for OS diagnosis, prediction of response to therapy, disease progression and chemoresistance, are urgently needed. Biological fluids such as blood represent a rich source of non-invasive cancer biomarkers, which allow to understand what is really happening inside the tumour, either at diagnosis or during disease progression. In this regard, liquid biopsy potentially represents an alternative and non-invasive method to detect tumour onset, progression and response to therapy. In this review, we will summarize the state of the art in this novel area, illustrating recent studies on OS. Although the data reported in literature seem preliminary, liquid biopsy represents a promising tool with the potential to be rapidly translated in the clinical practice.