A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Novel invadopodia components revealed by differential proteomic analysis

When highly invasive cancer cells are cultured on an extracellular matrix substrate, they extend proteolytically active membrane protrusions, termed invadopodia, from their ventral surface into the underlying matrix. Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete.To accelerate component discovery, we resorted to a proteomics approach by applying DIfference Gel Electrophoresis (DIGE) to compare invadopodia-enriched sub-cellular fractions with cytosol and cell body membrane fractions and the whole cell lysate. The fractionation procedure was validated through step-by-step monitoring of the enrichment in typical actin-related invadopodia-associated proteins. After statistical analysis, 129 protein spots were selected for peptide mass fingerprinting analysis; of these 76 were successfully identified and found to correspond to 58 proteins belonging to different functional classes including aerobic glycolysis and other metabolic pathways, protein synthesis, degradation and folding, cytoskeletal components and membrane-associated proteins.Finally, validation of a number of identified proteins was carried out by a combination of immuno-blotting on cell fractions and immunofluorescence localization at invadopodia. These results reveal newly identified components of invadopodia and open further avenues to the molecular study of invasive growth behavior of cancer cells.     

Long-term population dynamics in a Mediterranean aquatic snake

A review of several long-term studies has recently suggested that snakes might be declining in large parts of the world. Additional data from other long-term studies are therefore urgently needed in order to assess the generalities of such suggested declines. Based on a 20-year study, we analyzed demographic data on adult dice snakes (Natrix tessellata) studied in central Italy between 1985 and 2004. Both male and female dice snakes were relatively long-lived, with no significant differences in longevity between the sexes. Individual males and females were observed over a maximum of 10 and 14 years, respectively. However, the among-year recapture rates between the year the snakes were initially captured and the subsequent year (i.e., year 1 to year 2) was significantly lower (45%) than the among-year recapture rates during subsequent years (74%; i.e., year 2 to year 3), suggesting that a large proportion of the snakes at first capture were in fact not resident within our study area, and hence many snakes were migrating in and out of our 2-km stream study site, with no inter-sexual difference in dispersal rates. Sex ratio was virtually equal if we consider the study period as a whole. Significant annual fluctuations were, however, observed through the study. In 1985–1990, 1993–1995, 1998 and 1999 the sex ratio was male-biased, whereas in 2000–2004 female-biased. In terms of both survival and recapture probabilities, model selection showed that Akaike’s information criterion favored the model incorporating body size, with the model incorporating year having an intermediate likelihood, and the model with sex included being the most disfavored. Total population number estimates suggest an average 86 adult individuals along the 2 km of stream with only minor annual variations. However, a significant decrease in the number of males occurred during the last 6 years of our study. Thus, further monitoring of this population is warranted in light of the decline of snake populations reported recently.     

Dinosaur Tracks as Paleogeographic Constraints: New Scenarios for the Cretaceous Geography of the Periadriatic Region

A really unexpected finding of sauropod and theropod footprints in southern Latium raises to four the number of the trampled levels recognized in central and southern Italy. After the recent findings in Latest Jurassic and Early, mid and Late Cretaceous carbonate platform deposits of the Periadriatic region, dinosaur footprints seem to provide very important paleogeographic constraints for reconstructing the geodynamic history of the Mediterranean area. The presence of a varied ichnoassociation makes acceptance of the current paleogeographic models concerning the relative and absolute position of the Laziale-Abruzzese-Campano and of Apulian-Dinaric domains during the Late Cretaceous more and more problematic. Dinosaur footprints, combined with other paleontological data, demonstrate that these areas were never completely pulled apart by deep seaways, while frequent or continuous links between them, and to southern and northern mainlands, probably persisted. These data also allowed us to improve our understanding of the timing of the Mesozoic plate motion in this segment of the Western Tethys.     

Immunological characteristics associated with the protective efficacy of antibodies to ricin

A/B toxins, produced by bacteria and plants, are among the deadliest molecules known. The B chain binds the cell, whereas the A chain exerts the toxic effect. Both anti-A chain and anti-B chain Abs can neutralize toxins in vivo and in vitro. B chain Abs block binding of the toxin to the cell. It is not known how anti-A chain Abs function. Working with ricin toxin, we demonstrate that immunization with A chain induces greater protection than immunization with B chain. A panel of mAbs, binding to A chain, B chain, or both chains, has been produced and characterized. Immunologic characteristics evaluated include isotype, relative avidity, and epitope specificity. The ability to inhibit ricin enzymatic or cell binding activity was studied, as was the ability to block ricin-mediated cellular cytotoxicity on human and murine cell lines. Finally, the in vivo protective efficacy of the Abs in mice was studied. The Ab providing the greatest in vivo protective efficacy was directed against the A chain. It had the greatest relative avidity and the greatest ability to block enzymatic function and neutralize cytotoxicity. Interestingly, we also obtained an anti-A chain Ab that bound with high avidity, blocked enzymatic activity, did not neutralize cytotoxicity, and actually enhanced the in vivo toxicity of ricin. Anti-A chain Abs with moderate avidity had no in vivo effect, nor did any anti-B chain Abs.