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The release of molecules from damaged tissues stimulates both resident and circulating stem cells to initiate a tissue repair programme. 1 However, during transplantation procedures the therapeutic efficacy of stem cells is compromised by reduced homing capability towards the target site.2,3 Furthermore, cell survival is very low and many studies focused on improving cell viability upon implantation. In this study, we performed in vitrosevere oxidative stress to select some more resistant mouse mesoangioblasts, vessel-associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. We found that the selected subpopulation retains selfrenewal and myogenic differentiation capabilities under physiological growth conditions, showing, also, an enhancement in cell survival and migration capabilities under stress conditions respect to the unselected cells. In fact, following oxidative stress treatment the isolated cell subpopulation showed more resistance, survival and recovery properties. To evaluate whether or not the isolated cell clone showed selective advantages over the parental mesoangioblasts, we carried out in vivo experiments using immunocompromised dystrophic mice. We injected intramuscularly the Tibialis Anterior with both the selected cells and the parental cells. Actually resistant mesoangioblasts displayed markedly enhanced survival and integration capabilities into the host damaged skeletal muscle, displaying more than 70% increase in integration compared to the non-selected mesoangioblast cell population. In conclusion, the positive effects of sorting on mesoangioblast cells suggest that a selection step using oxidative stress preconditioning may provide a novel methodology to select for resistant cells that can be used in regenerative tissue applications to prevent high mortality rates upon transplantation.

The S100 gene family is the largest subfamily of calcium binding proteins of EF-hand type, expressed in tissue and cell-specific manner. S100 proteins act as intracellular regulators and as extracellular signaling. Within cells, S100 have been involved in the regulation of proliferation, differentiation, apoptosis, energy metabolism, inflammation, migration and invasion via interactions with a variety of target proteins. Extracellular S100 proteins act in an autocrine and paracrine manner through the activation of surface receptors that regulate cell proliferation, differentiation, survival and migration. More recently, there is growing interest in the S100 proteins and their relationship with different cancers because of their involvement in a variety of biological events closely related to tumorigenesis and cancer progression1. However, the occurrence, the role and the possible coordination of this group of proteins in breast cancer is still poorly known. We previously describe a large-scale proteomic investigation performed on breast cancer patients for the screening of multiple forms of S100 proteins2,3. Our results have shown that the majority of S100 proteins are preferentially expressed in the tumor mass compared with the normal adjacent tissue and that some S100 protein members were ubiquitously expressed in almost all patients, while others appeared more sporadic among the same group of patients. More interestingly, patients which developed distant metastases showed a general tendency of higher S100 protein expression, compared to the disease-free group. Present study was aimed to assess the gene expression levels of the S100 protein family members utilizing a breast cancer dataset generated on Affymetrix microarrays technologies4. GOBO (Gene expression-based Outcome for Breast cancer Online) is a user-friendly online tool that allows, also, the identification of co-expressed genes and association with outcome in an 1881 breast cancer samples. Other important association with breast cancer outome was carried out by Kaplan Meir-plotter database5. Integrating results obtained by proteomic and trascriptomic analysis of S100 proteins highlight their important involvement in breast cancer progression, and support the idea that S100 proteins are important prognostic factors, related to survival period of tumor patients. However, the specific mechanisms by which S100 proteins affect progression of breast require further study.

The chemical synthesis of novel chemotherapeutical leads is evolving thanks to possibility to design molecules with desired physical-chemical and, thus, biological properties. The imidazolium salts, recently proven effective to inhibit bacterial and/or cancer cell growth, posses an amphiphilic nature that is conferred by the imidazolium cation having a polar head generally coupled with aliphatic side chains. Thus, biological properties of imidazolium salts can be tuned through modifications involving the cation structure and/or the anion nature. By covalently linking two imidazolium rings, di-imidazolium salts were obtainedobtain differing in: i) kind of anions; ii) geometric isomerization of di-imidazolium anions; iii) length of the imidazolium alkyl side chains. Preliminarly, eleven di-imidazolium salts, differing for their anionic counterparts, were assayed for: i) antibacterial property, quantified as the minimal concentration inhibiting at least the 90% of bacterial growth (MIC90) using Escherichia coli and Micrococcus luteus as Gram-negative and Gram-positive tester strains; ii) antitumoral activity measured as the concentration inhibiting the 50% of cell growth (IC50) using SKBR-3 breast cancer cell line. All the assayed salts possesses biological activity showing i) 0.1-0.5 and 25-50 µg/ml as MIC90 values against of M. luteus, and E. coli; respectively, and ii) 30-55 µg/ml as IC50 values. Among the tested di-imidazolium salts, three were chosen to further investigate the relationship between biological efficacy and either length of alkyl side chains or imidazolium ring configurations. Geometric isomerization revealed few or no effect while a positive correlation between alkyl chain length and cell-growth inhibitory efficacy was shown. Although further studies have to be performed to elucidate the molecular mechanisms leading to cell growth arrest, this study provide insights on the attractive possibility of di-imidazolium salt exploitation as chemotherapeutical compounds whose activity can be tuned by modifying structural characteristics.

S100 gene family is the largest subfamily of calcium binding proteins, expressed in tissue and cell-specific manner. Within cells, S100 have been involved in the regulation of proliferation, differentiation, apoptosis, energy metabolism, inflammation, migration and invasion. Extracellular S100 proteins act in an autocrine and paracrine manner and regulate cell proliferation, differentiation, survival and migration. S100 proteins play important roles in the development and progression of tumors due to their multifunctional roles. However, the occurrence, the role and the possible coordination of this group of proteins in breast cancer is still poorly known. We previously describe a large-scale proteomic investigation performed on breast cancer patients for the screening of multiple forms of S100 proteins1,2. Our results have shown that the majority of S100 proteins are preferentially expressed in the tumor mass compared with the normal adjacent tissue and that some S100 protein members were ubiquitously expressed in almost all patients, while others appeared more sporadic among the same group of patients. More interestingly, patients which developed distant metastases showed a general tendency of higher S100 protein expression, compared to the disease-free group. Present study was aimed to assess the gene expression levels of the S100 protein family members utilizing a breast cancer dataset generated on Affymetrix microarrays technologies3. GOBO (Gene expression-based Outcome for Breast cancer Online) is a user-friendly online tool that allows, also, the identification of co-expressed genes and association with outcome in an 1881 breast cancer samples. Other important association with breast cancer outome was carried out by Kaplan Meir-plotter database4. Integrating results obtained by proteomic and trascriptomic analysis of S100 proteins highlight their important involvement in breast cancer progression. Future studies are needed to disclose molecular mechanisms and signaling pathways that define the multiple and specific roles of S100 proteins in breast cancer.

Our studies in the field of printing nanobiology in aqueous solution are proposed to highlight the role of water in the processes of interaction between biomolecules in drug- screening devices fabricated by bioprinting technologies and to emphasize the influence of water evaporation on the diffusion of molecules in droplets of picoliter-scale.

The first step for prevention and treatment of diseases is the accurate diagnosis. However, proper diagnostic technologies are not available in developing countries due to the lack of reliable electrical power, refrigeration and trained personnel. For this reason, there is an urgent need of low cost, rapid assays not requiring any external support. By coupling such technologies to communication infrastructures, healthcare in areas without access to medical personnel would be possible. “Paper” like substrates are ideal for fabricating such devices since they are cheap, easy to degradate after use and compatible with most of existing printing technologies [1]. We had previously shown the possibility to efficiently deposit oligonucleotides by Dip Pen Lithography(DPL) onto glass surfaces [2]. In this work, we deposited oligonucleotides on nylon substrate for the fabrication of biochips usable for detecting the activity of human topoisomerase I. Subsequently, the chip will be modified to detect the Malariacausing Plasmodium parasites through the detection of Plasmodium topoisomerase I activity [3]. We optimized oligonucleotides printing on nylon substrate, obtaining efficient deposition at 10 - 1 uM oligonucleotide concentrations, 70% relative humidity and 30% glycerol v/v. We obtained circular spots with diameter in the range of 30 - 50 microns, with the dimension being a function of dwell time (1s – 20 s). DPL operation needs ultra tiny amounts of DNA (as low as 0.5 uL, 10 - 1 uM concentration) for printing thousands of spots in a single run so reducing material consumption in comparison with standard bioprinting techniques [4]. In a first set of experiments, the printed oligonucleotides was hybridized with a fluorescence-labelled complementary probe to detect and quantify DNA after DPL deposition. In subsequent experiment, the spotted oligonucleotides generate a topoisomerase substrate, which upon reaction with the enzyme will be coupled to a fluorescently labelled oligonucleotide to allow detection of a signal. In conclusion, the combination of DPL and topoisomerase detection onto nylon substrates would be a suitable solution for point-of-care diagnostic chips fabrication.

The ENO1 transcript, which encodes the glycolitic enzyme alpha-enolase, can be translated into a shorter nuclear protein called Myc-promoter Binding Protein-1 (MBP-1) by using an alternative translation start site. MBP-1 acts as a negative regulator of c-Myc, ErbB2 and Cox2 genes (1). Several evidences indicate that MBP-1 acts as a tumor suppressor in breast carcinoma and prostate cancer and its expression results in a reduced invasive ability (2). In our previous studies, we showed that MBP-1 is expressed and easily detectable in normal breast epithelial cells, but a loss of expression occurs in most primary invasive ductal carcinomas (IDC) of the breast. Furthermore, in these tumors MBP-1 expression inversely correlated with the expression levels of the ErbB2 and Ki67 proteins (3). In order to better understand the role of MBP-1 in breast cancer and to correlate its expression to a gene signature with prognostic value, we evaluated the expression of approximately 21,000 genes in primary breast carcinoma by using the Agilent microarray technology. A comparison of the gene expression profiles obtained from MBP-1+ve and MBP-1-ve ErbB2-negative IDCs led to the identification of differentially expressed (DE) genes that may underlie the different clinical behaviors of these two subtypes of breast carcinoma. Owing to the prognostic influence of nuclear MBP-1 expression in a subgroup of tumors from patients with node-negative and ErbB2-negative carcinomas, the combination of immunohistochemical analyses of MBP-1 and proteins encoded by genes we found DE by expression profiling, may prove to be a clinically valuable prognostic variable for breast cancer patients.

Neuroblastoma is the most common extra-cranial solid tumor of childhood, originated from cells of the neural crest. Amplification of N-MYC gene and 1p-deletion are found in more than 30% of patients with advanced stages and they are associated with poor prognosis. An alternative translated product of the ENO1 gene, known as MBP-1 (c-myc promoter binding protein-1), acts as a negative regulator of the c-MYC oncogene, ERBB2 and COX-2 genes, furthermore, ENO1/MBP-1 overexpression in Neuroblastoma cells significantly reduces cell growth and induces apoptosis. Even though there are similarities between the c-MYC and N-MYC oncogenes, there are no evidences that MBP-1 is able to interact with N-MYC promoter to regulate negatively its expression. Reporter plasmids (luciferase), containing various portions of N-MYC promoter, were used in N-MYC-amplified Neuroblastoma cells (LAN-5) transfection experiments with an effector plasmid expressing Flag-MBP-1 protein. These studies have identified a promoter region involved in the negative regulation of N-MYC by MBP-1, and its binding activity, within the N-MYC promoter was confirmed, both in vitro and in vivo, by EMSA and chromatin-immunoprecipitation (ChIP) assays. SiRNA-mediated induction of endogenous MBP-1 expression in LAN-5 cells determines a decrease of N-MYC and a significant increase of p21, BAX and γ-enolasi expression (a marker of neuronal differentiation). Furthermore, migration and cytotoxicity assays indicate that MBP-1 induces apoptosis and reduces the migration of LAN-5 cells. In conclusion, the data presented indicate that MBP-1 acts as a transcriptional repressor of N-MYC gene and its expression induces senescence, apoptosis and differentiation in LAN-5 Neuroblastoma cells.

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and it is the target for the 5-Fluorouracil (5-FU) activity*. Barbour et al. showed that variant structural forms of TS in tumour cell lines confer resistance to fluoropyrimidines. We planned to perform the whole TS gene structure by means of sequencing techniques in human colorectal cancer (CRC) san-rples to try to identify the presence of any possible TS variant form that could be responsible of fluoropyrimidines drug resistance and of the worse prognosis. We performed the TS-DNA gene sequence in 68 CRC from patients of A, B, and C Dukes' stages and different histological grade, but we did not find any mutation in the TS-DNA structure. In the future we intend to widen the TS structure analysis to the metastatic CRCs, because due to their higher genomic instability, they could present a TS variant form responsilile of the fluoropyrimidines drug resistance and the worse prognosis.