DEVELOPMENT OF A NOVEL METHOD FOR AMNIOTIC FLUID STEM CELL STORAGE

Background - Current procedures for collection of human Amniotic Fluid Stem Cells (hAFSCs) imply that amniotic fluid cells were cultured in flask for two weeks, than can be devoted to research purpose. However, hAFSCs could be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of the study was to verify if a direct freezing of amniotic fluid cells is able to maintain and / or improve the potential of the sub-population of stem cells. Methods - We compared the potential of the hAFSCs depending on the moment in which they are frozen, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis, and differentiation potential of C and D samples were compared. Results - hAFSCs isolated from D samples expressed MSC markers until later passages, had a good proliferation rate, and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, the direct freezing induce a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. Conclusions - This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration affecting stem cell properties. This technique might be a reasonable approach in terms of costs and for the process of accreditation in GMP for a stem cell bank

Novel GC-rich DNA-binding compound produced by a genetically engineered mutant of the mithramycin producer Streptomyces argillaceus exhibits improved transcriptional repressor activity: implications for cancer therapy

The aureolic acid antibiotic mithramycin (MTM) binds selectively to GC-rich DNA sequences and blocks preferentially binding of proteins, like Sp1 transcription factors, to GC-rich elements in gene promoters. Genetic approaches can be applied to alter the MTM biosynthetic pathway in the producing microorganism and obtain new products with improved pharmacological properties. Here, we report on a new analog, MTM SDK, obtained by targeted gene inactivation of the ketoreductase MtmW catalyzing the last step in MTM biosynthesis. SDK exhibited greater activity as transcriptional inhibitor compared to MTM. SDK was a potent inhibitor of Sp1-dependent reporter activity and interfered minimally with reporters of other transcription factors, indicating that it retained a high degree of selectivity toward GC-rich DNA-binding transcription factors. RT–PCR and microarray analysis showed that SDK repressed transcription of multiple genes implicated in critical aspects of cancer development and progression, including cell cycle, apoptosis, migration, invasion and angiogenesis, consistent with the pleiotropic role of Sp1 family transcription factors. SDK inhibited proliferation and was a potent inducer of apoptosis in ovarian cancer cells while it had minimal effects on viability of normal cells. The new MTM derivative SDK could be an effective agent for treatment of cancer and other diseases with abnormal expression or activity of GC-rich DNA-binding transcription factors

Complement Involvement in Renal Transplantation

The complement system is involved in several renal diseases and in renal transplantation (RTx). The authors review the complement cascade and its involvement in innate and adaptive immunity in the field of RTx. The complement cascade is involved in several steps of RTx: ischaemia—reperfusion injury (IRI), T cell-mediated acute rejection (TMR), antibody-mediated rejection (ABMR), and progressive kidney injury and fibrosis. The high frequency of complement involvement in RTx is the subject of several studies because complement could be a relevant target in treating the aforementioned conditions. There is an increasing number of ongoing clinical trials aimed at verifying the efficacy and safety of many drug candidates. The anti-C5 monoclonal antibody is already approved to prevent and treat ABMR and is the subject of trials investigating the treatment of other conditions such as IRI, TMR, and progressive fibrosis. Other molecular targets, such as C1, C3, C5a, and C5a receptor, are the subject of international trials and could prove to be effective in the near future

How alternative food networks work in a metropolitan area? An analysis of Solidarity Purchase Groups in Northern Italy

Our paper focuses on Solidarity Purchase Group (SPG) participants located in a highly urbanized area, with the aim to investigate the main motivations underlining their participation in a SPG and provide a characterization of them. To this end, we carried out a survey of 795 participants involved in 125 SPGs in the metropolitan area of Milan (Italy). Taking advantage of a questionnaire with 39 questions, we run a factor analysis and a two-step cluster analysis to identify different profiles of SPG participants. Our results show that the system of values animating metropolitan SPG practitioners does not fully conform to that traditionally attributed to an alternative food network (AFN). In fact, considerations linked to food safety and healthiness prevail on altruistic motives such as environmental sustainability and solidarity toward small producers. Furthermore, metropolitan SPGs do not consider particularly desirable periurban and local food products. Observing the SPGs from this perspective, it emerges as such initiatives can flourish also in those places where the lack of connection with the surrounding territory is counterbalanced by the high motivation to buy products from trusted suppliers who are able to guarantee genuine and safe products, not necessarily located nearby

Amplification of the pericentromeric region of chromosome 1 in a newly established colon carcinoma cell line

The LRWZ cell line was established from an ascitic effusion of a colon adenocarcinoma. We studied the karyotype of LRWZ cells using G-banding and chromosome painting. The cell line is near triploid and is characterized by several chromosome rearrangements and pronounced intermetaphase variation. Chromosome painting probes revealed numerous labeled regions on different chromosomes, indicating that several translocations occurred during the evolution of the cell population. The 10 recurrent marker chromosomes identified (M1-M10) were derived from complex rearrangements involving up to three different chromosomes. M2 is a particularly interesting marker that originated from the amplification of the pericentromeric region of chromosome 1 and has a peculiar organization comprising five copies of the region included between 1p21 and 1q21 and is surprisingly stable: it is present in all the metaphases analyzed, has telomeric DNA at both termini, and contains one active and four inactivated centromeres. To provide insights into the molecular mechanisms that generated M2, we performed fluorescence in situ hybridization experiments using a panel of probes mapping near the centromere of chromosome 1 and three probes for different satellite sequences; the formation of chromosome M2 required the intervention of several rearrangements including unequal exchange, chromatid breakage followed by fusion of the sister chromatids, and loss of centromeric heterochromatin

Cdc25 inhibitors in melanoma cells

Cell division cycle 25 (Cdc25) proteins are dual specificity phosphatases involved in the progression of the cell cycle. In particular, mammalian cells express three isoforms of Cdc25, called Cdc25A, -B and -C: Cdc25A mainly controls G1/S progression, whereas Cdc25B and Cdc25C predominantly activate G2/M transition. Moreover, over-expression of Cdc25A and B activities has been frequently observed in a wide variety of human tumors with poor prognosis. Thus, the design and study of small molecules endowed with an inhibitory activity towards Cdc25 proteins represent a promising strategy for the development of new anti-cancer therapies. A previous work from this group described the properties of a set of small molecules, which were able to inhibit Cdc25 functions (Lavecchia et al. J Med Chem 55, 2012, 4142–4158). Among them, compound 11, possessing a quinonic structure, acted as an irreversible inhibitor of Cdc25B and showed a strong antiproliferative action on some cancer cell lines. To expand the structure-activity relationship study and to explore potential new structure analogues of 11, we performed a multiple ligand-based chemoinformatics approach against full ZINC database (~35 million purchasable compounds) and NCI Open database (~260 000 compounds). 25 structures identified from these searches (hereafter called AL1–AL25) were analyzed, and selected compounds were tested in vitro to assess the Cdc25B inhibition activity. In particular, compounds AL11, -12, -13, -14, -15, -16, -23, -24 and -25 maintained a percentage of Cdc25B inhibition comparable to that exerted by compound 11, and some of them were even more powerful. The effect of these active analogues was also evaluated on melanoma cell lines (A2058 and SAN), because of the limited information on the effect of Cdc25 inhibitors in this cellular system. In both melanoma cells, after 48-h treatment with 100 mM of each inhibitor, a significant reduction of cell growth was observed only with AL11, whereas the other active analogues, as well as compound 11, were ineffective. Furthermore, cytofluorimetric analysis and measurements of the activity of caspase-9 and -3 indicated that AL11 exerted a pro-apoptotic effect on melanoma cells. In addition, the cellular treatment with AL11 produced an alteration of the cellular redox state and a reduction of the mitochondrial membrane potential. Studies are in progress to clarify the mechanisms involved in the cytotoxic potential of AL11 and other related molecules in melanoma cell lines, with the aim at the refinement of the inhibitory action of the studied compounds

Cellular and Molecular Consequences of Peroxisome Proliferator-Activated Receptor-γ Activation in Ovarian Cancer Cells

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-activated transcription factor. In addition to its canonical role in lipid and glucose metabolism, PPAR-γ controls cell proliferation, death, and differentiation in several tissues. Here we have examined the expression of PPAR-γ in ovarian tumors and the cellular and molecular consequences of its activation in ovarian cancer cells. PPAR-γ was expressed in a large number of epithelial ovarian tumors and cell lines. The PPAR-γ ligand ciglitazone inhibited the growth and clonogenic survival of ovarian cancer cells, inducing cell cycle arrest and cell death. Growth inhibition by ciglitazone was reversed by the PPAR-γ antagonist GW9662, indicating the involvement of PPAR-γ-dependent mechanisms. Microarray-based gene profiling revealed complex changes in the transcriptional program of ovarian cancer cells on treatment with ciglitazone and identified multiple pathways that may contribute to PPAR-γ ligands' antitumor activity. Genes upregulated by ciglitazone were predominantly associated with metabolic, differentiation, and tumor-suppressor pathways, whereas downregulated genes were involved in cell proliferation, cell cycle, cell organization, and steroid biosynthesis. Collectively, our data indicate that PPAR-γ activation by selective agonists is a valid strategy for ovarian cancer therapy and prevention, and should be tested alone and in combination with other anticancer drugs