First record of planktonic crustaceans in Sardinian reservoirs

Sardinian man-made lakes are reservoirs of species richness, hosting zooplankton taxa from the Mediterranean region and North Africa. To provide a first record of the taxa composition and diversity of zooplankton communities, we sampled 15 reservoirs during 2008–2009, from the north of the island to the south, representative of a range of size, depth, renewal time, and trophy. The survey was complemented by seasonal sampling in one of the largest lakes studied. Water samples collected from surface to bottom provided data on hydrochemistry and trophy. Crustacean dormant stages were inspected from sediments of the richest, and most diverse, Lake Sos Canales. RDA suggested that productivity, water depth, renewal time and altitude were the main variables related to taxa composition. The ubiquitous Copidodiaptomus numidicus, and its persistence in the water column, resulted from the production of subitaneous eggs throughout the year, an adaptive strategy in perennial water bodies. Genetic analyses of DNA sequences of the diagnostic gene ND5 placed the Sardinian Daphnia pulex in the North American group. Moreover, the ND5 sequence found in Sardinia was identical with that of an asexual hybrid clone between the American D. pulex and American D. pulicaria that replaced native D. pulex throughout Africa. The presence of this ND5 haplotype in Sardinia shows that this invasive clone also poses an invasive threat to native populations in Europe.     

Transglutaminase 2 as a biomarker of osteoarthritis: an update

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca²⁺-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.     

TpF1 from Treponema pallidum activates inflammasome and promotes the development of regulatory T cells

Human syphilis is a multistage disease, with diverse and wide-ranging manifestations caused by Treponema pallidum. Despite the fact that a cell-mediated immune response takes part in the course of syphilis, T. pallidum often manages to evade host immunity and, in untreated individuals, may trigger chronic infection. With this study, we demonstrate for the first time, to our knowledge, that Treponema pallidum induces a regulatory T (Treg) response in patients with secondary syphilis and we found that the miniferritin TpF1, produced by the bacterium, is able to expand this response and promote the production of TGF-β. Accordingly, TpF1 stimulates monocytes to release IL-10 and TGF-β, the key cytokines in driving Treg cell differentiation. Interestingly, we also found that TpF1 stimulates monocytes to synthesize and release several proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, the latter following the activation of the multiprotein complex inflammasome. Collectively, these data strongly support a central role for TpF1 both in the inflammation process, which occurs in particular during the early stage of syphilis, and in the long-term persistence of the spirochete within the host by promoting Treg response and TGF-β production.     

Neuroglobin and cytoglobin as potential enzyme or substrate

The possible enzymatic activities of neuro- and cytoglobin as well as their potential function as substrates in enzymatic reactions were studied. Neuro- and cytoglobin are found to show no appreciable superoxide dismutase, catalase, and peroxidase activities. However, the internal disulfide bond (CD7-D5) of human neuroglobin can be reduced by thioredoxin reductase. Furthermore, our in vivo and in vitro studies show that Escherichia coli cells contain an enzymatic reducing system that keeps the heme iron atom of neuroglobin in the Fe(2+) form in the presence of dioxygen despite the high autoxidation rate of the molecule. This reducing system needs a low-molecular-weight compound as co-factor. In vitro tests show that both NADH and NADPH can play this role. Furthermore, the reducing system is not specific for neuroglobin but allows the reduction of the ferric forms of other globins such as cytoglobin and myoglobin. A similar reducing system is present in eukaryotic tissue protein extracts.     

Helicobacter pylori and gastric autoimmunity

Host specific T-cell response is critical for the outcome of Helicobacter pylori infection. In genetically susceptible individuals, H. pylori can activate gastric CD4+ Th1 cells that recognize cross-reactive epitopes shared by H. pylori proteins and self H+, K+-ATPase, leading to gastric autoimmunity via molecular mimicry.     

Mucin depleted foci, colonic preneoplastic lesions lacking Muc2, show up-regulation of Tlr2 but not bacterial infiltration

Mucin depleted foci (MDF) are precancerous lesions of the colon in carcinogen-treated rodents and humans at high risk. Since MDF show signs of inflammation we hypothesized that the defective mucous production would expose them to the risk of being penetrated by intestinal bacteria, which can be sensed by Toll-like receptors (Tlrs) and activate inflammatory pathways. To verify this hypothesis we tested the expression of 84 genes coding for Tlrs and associated pathways using RT-qPCR in MDF (n = 7) from 1,2-dimethylhydrazine (DMH)-treated rats. Among the 84 tested genes, 26 were differentially expressed in MDF with 5 genes significantly up-regulated and 21 down-regulated when compared to the normal mucosa. Tlr2, as well as other downstream genes (Map4k4, Hspd1, Irak1, Ube2n), was significantly up-regulated. Among the genes regulating the NFkB pathway, only Map4k4 was significantly up-regulated, while 19 genes were not varied and 6 were down-regulated. Tlr2 protein was weakly expressed both in normal mucosa and MDF. To determine whether inflammation observed in MDF could be caused by bacteria contacting or infiltrating crypts, we performed fluorescence in situ hybridization (FISH) experiments with a rRNA universal bacterial probe. None of the 21 MDF tested, showed bacteria inside the crypts, while among the colonic tumors (n = 15), only one had very few bacteria on the surface and on the surrounding normal mucosa. In conclusion, the up-regulation of Tlr2 in MDF, suggests a link between this receptor and carcinogenesis, possibly related to a defective barrier function of these lesions. The data of FISH experiments do not support the hypothesis that inflammation in MDF and tumors is stimulated by bacterial infiltration.     

A human islet cell culture system for high-throughput screening

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.     

MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-β-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3'UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.     

2D-DIGE analysis of ovarian cancer cell responses to cytotoxic gold compounds

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.     

Identification of a posttranslational mechanism for the regulation of hERG1 K+ channel expression and hERG1 current density in tumor cells

A common feature of tumor cells is the aberrant expression of ion channels on their plasma membrane. The molecular mechanisms regulating ion channel expression in cancer cells are still poorly known. K(+) channels that belong to the human ether-a-go-go-related gene 1 (herg1) family are frequently misexpressed in cancer cells compared to their healthy counterparts. We describe here a posttranslational mechanism for the regulation of hERG1 channel surface expression in cancer cells. This mechanism is based on the activity of hERG1 isoforms containing the USO exon. These isoforms (i) are frequently overexpressed in human cancers, (ii) are retained in the endoplasmic reticulum, and (iii) form heterotetramers with different proteins of the hERG family. (iv) The USO-containing heterotetramers are retained intracellularly and undergo ubiquitin-dependent degradation. This process results in decreased hERG1 current (I(hERG1)) density. We detailed such a mechanism in heterologous systems and confirmed its functioning in tumor cells that endogenously express hERG1 proteins. The silencing of USO-containing hERG1 isoforms induces a higher I(hERG1) density in tumors, an effect that apparently regulates neurite outgrowth in neuroblastoma cells and apoptosis in leukemia cells.