Heterozygous Reelin Mutations Cause Autosomal-Dominant Lateral Temporal Epilepsy

Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic epilepsy syndrome clinically characterized by focal seizures with prominent auditory symptoms. ADLTE is genetically heterogeneous, and mutations in LGI1 account for fewer than 50% of affected families. Here, we report the identification of causal mutations in reelin (RELN) in seven ADLTE-affected families without LGI1 mutations. We initially investigated 13 ADLTE-affected families by performing SNP-array linkage analysis and whole-exome sequencing and identified three heterozygous missense mutations co-segregating with the syndrome. Subsequent analysis of 15 small ADLTE-affected families revealed four additional missense mutations. 3D modeling predicted that all mutations have structural effects on protein-domain folding. Overall, RELN mutations occurred in 7/40 (17.5%) ADLTE-affected families. RELN encodes a secreted protein, Reelin, which has important functions in both the developing and adult brain and is also found in the blood serum. We show that ADLTE-related mutations significantly decrease serum levels of Reelin, suggesting an inhibitory effect of mutations on protein secretion. We also show that Reelin and LGI1 co-localize in a subset of rat brain neurons, supporting an involvement of both proteins in a common molecular pathway underlying ADLTE. Homozygous RELN mutations are known to cause lissencephaly with cerebellar hypoplasia. Our findings extend the spectrum of neurological disorders associated with RELN mutations and establish a link between RELN and LGI1, which play key regulatory roles in both the developing and adult brain.     

Titin Domains Progressively Unfolded by Force Are Homogenously Distributed along the Molecule

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule''s length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.     

Environment, inflammation, and cancer

Rudolf Virchow postulated that a critical feature of tumors was the presence of leukocytes, providing the first indication that inflammation may play a role in tumorigenesis. We now have a wealth of experimental and clinical data demonstrating a clear relationship between inflammatory responses and the roles they play at different stages of tumor development. The details of the dynamic relationship between tumor cells and specific subtypes of immune cells and mesenchymal cells are being revealed as critical to cancer progression which has led to the development of potential new targets for cancer treatment. This review describes some of the key molecular and cellular events demonstrating the critical role of inflammation on promoting tumorigenesis with attention on novel therapeutics and their potential clinical success.     

Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, ??-Tubulin, ??-Catenin, ??-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, ??1-Antitrypsin (AAT) and ??-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.     

Intracellular signaling cascades triggered by the NK1 fragment of hepatocyte growth factor in human prostate epithelial cell line PNT1A

Hepatocyte Growth Factor (HGF)/c-MET signaling has an emerging role in promoting cell proliferation, survival, migration, wound repair and branching in a variety of cell types. HGF plays a crucial role as a mediator of stromal–epithelial interactions in the normal prostate but the precise biological function of HGF/c-Met interaction in the normal prostate and in prostate cancer is not clear. HGF has two naturally occurring splice variants and NK1, the smallest of these HGF variants, consists of the HGF amino terminus through the first kringle domain. We evaluated the intracellular signaling cascades and the morphological changes triggered by NK1 in human prostate epithelial cell line PNT1A which shows molecular and biochemical properties close to the normal prostate epithelium. We demonstrated that these cells express a functional c-MET, and cell exposure to NK1 induces the phosphorylation of tyrosines 1313/1349/1356 residues of c-MET which provide docking sites for signaling molecules. We observed an increased phosphorylation of ERK1/2, Akt, c-Src, p125FAK, SMAD2/3, and STAT3, down-regulation of the expression of epithelial cell–cell adhesion marker E-cadherin, and enhanced expression levels of mesenchymal markers vimentin, fibronectin, vinculin, α-actinin, and α-smooth muscle actin. This results in cell proliferation, in the appearance of a mesenchymal phenotype, in morphological changes resembling cell scattering and in wound healing. Our findings highlight the function of NK1 in non-tumorigenic human prostatic epithelial cells and provide a picture of the signaling pathways triggered by NK1 in a unique cell line.     

Spatio‐temporal distribution of Ceratitis capitata population in a heterogeneous landscape in Central Italy

The spatio‐temporal dynamics of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was investigated to evaluate the effect of the landscape elements and host plants on pest distribution, in an agricultural landscape of 500 ha located in Central Italy. Two farms (farm 1 and farm 2) are located in the experimental area, composing mixed fruit orchards and surrounded by hedgerows, small woodlots, private gardens and cereal fields. Ceratitis capitata population fluctuation was monitored, from 2006 to 2008, using traps baited with trimedlure. Geostatistical methods such as Inverse distance squared weighted were used to obtain distributional maps of adults, mainly males. Results showed that the adult Mediterranean fruit flies were primarily distributed inside farm 1, with the maximum density found in the months of September and October. Away from the principal host plants, particularly in cereal fields, the number of trapped individuals was always low or zero. In both farms, flies were caught sequentially in traps located on host plants (i.e. peach, apple, pear, oriental persimmon and prickly pear) at varying times of maturation, especially when fruits remained on the trees. Distributional maps provided evidence that allowed to identify habitats in which the fly developed early in the season (mixed peach orchards) and afterwards during the periodic flights.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.