An Atlas of Altered Expression of Deubiquitinating Enzymes in Human Cancer

BACKGROUND: Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the "normal" samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition

Differentiation of Mesenchymal Stem Cells towards an insulin-releasing phenotype after co-culture with Pancreatic Islets

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001

Positive effect of Mesenchymal Stem Cells therapeutic administration on chronic Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001

Control of Meiotic and Mitotic Progression by the F Box Protein \u3b2-Trcp1 In Vivo

SCF ubiquitin ligases, composed of three major sub- units, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp -Trcp1 in mice. -Trcp1/ males show reduced fer- tility correlating with an accumulation of methaphase I spermatocytes. -Trcp1/ MEFs display a lengthened mitosis, centrosome overduplication, multipolar meta- phase spindles, and misaligned chromosomes. Fur- thermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic -Trcp1/ MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of -Trcp1. In contrast, stabilization of -catenin and IB, two previously re- ported -Trcp1 substrates, does not occur in the ab- sence of -Trcp1 and instead requires the additional silencing of -Trcp2 by siRNA. Thus, -Trcp1 regulates the timely order of meiotic and mitotic events

Degradation of Cdc25A by \u3b2-TrCP during S phase and in response to DNA damage

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A1\u20133. These events stimulate the ubiquitin-mediated pro- teolysis of Cdc25A1,4,5 and contribute to delaying cell-cycle progression, thereby preventing genomic instability1\u20137. Here we report that b-TrCP is the F-box protein that targets phosphory- lated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of b-TrCP1 and b-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that b-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of b-TrCP expression results in radioresistant DNA synthesis in response to DNA damage\u2014a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that b-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation

Planck 2015 results: XXV. Diffuse low-frequency Galactic foregrounds

We discuss the Galactic foreground emission between 20 and 100 GHz based on observations by Planck and WMAP. The total intensity in this part of the spectrum is dominated by free-free and spinning dust emission, whereas the polarized intensity is dominated by synchrotron emission. The Commander component-separation tool has been used to separate the various astrophysical processes in total intensity. Comparison with radio recombination line templates verifies the recovery of the free-free emission along the Galactic plane. Comparison of the high-latitude H\u3b1 emission with our free-free map shows residuals that correlate with dust optical depth, consistent with a fraction (\ue2\u2030 30%) of H\u3b1 having been scattered by high-latitude dust. We highlight a number of diffuse spinning dust morphological features at high latitude. There is substantial spatial variation in the spinning dust spectrum, with the emission peak (in I\u3bd) ranging from below 20 GHz to more than 50 GHz. There is a strong tendency for the spinning dust component near many prominent H ii regions to have a higher peak frequency, suggesting that this increase in peak frequency is associated with dust in the photo-dissociation regions around the nebulae. The emissivity of spinning dust in these diffuse regions is of the same order as previous detections in the literature. Over the entire sky, the Commander solution finds more anomalous microwave emission (AME) than the WMAP component maps, at the expense of synchrotron and free-free emission. This can be explained by the difficulty in separating multiple broadband components with a limited number of frequency maps. Future surveys, particularly at 5-20 GHz, will greatly improve the separation by constraining the synchrotron spectrum. We combine Planck and WMAP data to make the highest signal-to-noise ratio maps yet of the intensity of the all-sky polarized synchrotron emission at frequencies above a few GHz. Most of the high-latitude polarized emission is associated with distinct large-scale loops and spurs, and we re-discuss their structure. We argue that nearly all the emission at 40deg > l >-90deg is part of the Loop I structure, and show that the emission extends much further in to the southern Galactic hemisphere than previously recognised, giving Loop I an ovoid rather than circular outline. However, it does not continue as far as the "Fermi bubble/microwave haze", making it less probable that these are part of the same structure. We identify a number of new faint features in the polarized sky, including a dearth of polarized synchrotron emission directly correlated with a narrow, roughly 20deg long filament seen in H\u3b1 at high Galactic latitude. Finally, we look for evidence of polarized AME, however many AME regions are significantly contaminated by polarized synchrotron emission, and we find a 2\u3c3 upper limit of 1.6% in the Perseus region

A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets

The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs) with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an "insulin-releasing" phenotype. Two distinct mechanisms mediated these effects: i) the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii) MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility

Viability assessment of MSC-coated pancreatic islets.

<p>The viability of pancreatic islets coated with MSCs was assessed by using the vital fluorescent dye calcein, which evidenced in green only viable cells. Upper panel: pancreatic islets cultured alone stained with calcein after 2, 3 and 4 weeks of culture. Lower panel: pancreatic islets directly co-cultured with DiI red-stained MSCs which appear as yellow spots after 2, 3 and 4 weeks of culture. Arrows indicate pancreatic islets in which calcein did not spread uniformly. Bar 150 µm. b) Percentage of pancreatic islets' survival, expressed as mean ± SD. Calcein-positive pancreatic islets were counted and the survival percentage was calculated up to 4 weeks of culture.</p