Building up the inhibitory synapses

2nonenoneCherubini E; Zacchi PCherubini, E; Zacchi,

Asbestos Fibers Enhance the TMEM16A Channel Activity in Xenopus Oocytes

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of 'resting' oocyte [Ca2+]i following asbestos exposure. Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca2+]e, and to specific blockers of TMEM16A Ca2+-activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the 'resting' [Ca2+]i likelihood by increasing the cell membrane permeability to Ca2 in favor of a tonic activation of TMEME16A channels. Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc. Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos-membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes

THE CLINICOPATHOLOGICAL AND PROGNOSTIC SIGNIFICANCES OF C1Q EXPRESSION IN GLIOMAS: A BIOINFORMATICS ANALYSIS

Introduction. The complement system represents an important component of the inflammatory response and acts as a functional bridge between the innate and adaptive immune response. The contribution of the complement component C1q in the pathophysiology of brain cancers has been recently considered in light of its well-known involvement in carcinogenesis. Brain malignancies arise from cells of the CNS and are classified according to the tissue of phylogenetic origin. Gliomas represent the most common and aggressive form of brain tumours in adults. They derive from glial cells that help to support the functions of the other main brain cells type, the neurons (1). These are a heterogeneous group of diseases with multiple subtypes (1, 2). Glioblastoma multiforme (GBM) is the most common and fatal form of a primary brain tumour, accounting for approximately 60% of all glioma cases (3), whereas grade-II and -III gliomas are the second most common type of glioma in adults (~30%) (3). C1q molecule, together with other complement components, can be locally produced within the CNS by microglia and astrocytes, rendering it an attractive player in primary brain tumour development (4). The role of C1q in gliomas microenvironment is still poorly characterized and it is still quite puzzling whether it exerts a beneficial or a harmful activity for cancer progression. In the present study we performed a bioinformatics analysis aimed at investigating if C1q can serve as a potential prognostic marker for gliomas. Methods. The expression levels of C1qA, C1qB and C1qC genes in gliomas were analysed using Oncomine analysis. Available genomics data from The Cancer Genome Atlas project was used for Kaplan–Meier survival analysis to generate survival probability plots, using UALCAN analysis. Results. From the analysis performed on several data- sets using Oncomine, we showed a significantly higher mRNA expression levels for C1qA, C1qB and C1qC chains were detected in gliomas (different histotypes and grades) as compared to normal brain tissue (Fig. 1). We observed a positive correlation between the mRNA expression of C1qA, C1qB and C1qC mRNA poly- peptide chains and the unfavorable prognosis only in gliomas grade-II and -III, where the survival probability is indeed reduced (P <0.05) (Fig. 2). No correlation was observed in glioblastoma multiforme (Fig. 2). By immu- nohistochemical approaches we detected a high depo- sition of C1q in the tumor microenvironment of both in grade-II and -III gliomas and in GBMs examined (Fig. 3a glioma, 3b glioblastoma multiforme; 20x Magnification). Moreover, in double immunocytochemical experiments we demonstrated that CD68 positive infiltrating cells are actively synthesizing C1q in the tumor micro-envi- ronment. CD68 expression is characteristic of tumor- associated macrophages, whose enrichment in glioma has been associated with poor prognosis (5). Conclusion. In our study C1q expression was significantly correlated with poor survival probability in gliomas grade-II and -III while this is not the case for GBM. These data altogether underline how complex, multifaceted and still poorly understood is the role C1q can exert on tumor progression, and how the very same molecule can differentially affect the outcome depending on the biological context it comes to act

Prognostic implications of the complement protein C1q in gliomas

Associazione Italiana per la Ricerca sul Cancro (AIRC); Program Innovative Tools for Cancer Risk Assessment and Diagnosis

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects

While the essential role of the adenosine deaminase ADAR2 in RNA editing is well established, how it is regulated remains largely unknown. Here, the prolyl isomerase Pin1 and the E3 ubiquitin ligase WWP2 are shown to play a role in regulating ADAR2 localisation and stability

Pin1-dependent signaling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction

The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2' s ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABA A receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1\ufffd/\ufffd) associated with an increase in amplitude of spontaneous GABA A -mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction. \ufffd 2014 Macmillan Publishers Limited. All rights reserved

Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance

Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification