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The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid. Proteins 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Aβ stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-a (TNF-α). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-l-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression. [ABSTRACT FROM AUTHOR]

Inward-rectifier channels in cardiac cells ( I) stabilize the resting membrane potential near the K equilibrium potential. Here we investigate the role of I in the regulation of action potentials and link this to the influx of Ca during beating. Inward Ca current alters the open-channel probability of outward I current. Thus Ca ions depolarize cells not only by carrying an inward current but also by blocking an outward current. [ABSTRACT FROM AUTHOR]

UV-photoexcitation of TiO2 in contact with aqueous solutions of azo dyes does not imply only its photocatalytic degradation, but the reaction fate of the dye depends on the experimental conditions. In fact, we demonstrate that the presence of sodium formate is the switch from a degradative pathway of the dye to its transformation into useful products. Laser flash photolysis experiments show that charge separation is extremely long lived in nanostructured TiO2 thin films, making them suitable to drive both oxidation and reduction reactions. ESR spin trapping and photoluminescence experiments demonstrate that formate anions are very efficient in intercepting holes, thereby inhibiting OH radicals formation. Under these conditions, electrons promoted in the conduction band of TiO2 and protons deriving from the oxidation of formate on photogenerated holes lead to the reductive cleavage of N=N bonds with formation and accumulation of reduced intermediates. Negative ion ESI–MS findings provide clear support to point out this new mechanism. This study provides a facile solution for realizing together wastewater purification and photocatalytic conversion of a waste (discharged dye) into useful products (such as sulfanilic acid used again for synthesis of new azo dyes). Moreover, the use of TiO2 deposited on an FTO (Fluorine Tin Oxide) glass circumvents all the difficulties related to the use of slurries. The obtained photocatalyst is easy to handle and to recover and shows an excellent stability allowing complete recyclability. [ABSTRACT FROM AUTHOR]

Cadmium sulfide (CdS)-based photocatalysts are prepared following a hydrothermal procedure (with CdCl 2 and thiourea as precursors). The HydroThermal material annealed (CdS-HTa) is crystalline with a band gap of 2.31 eV. Photoelectrochemical investigation indicates a very reducing photo-potential of -0.9 V, which is very similar to that of commercial CdS. CdS-HTa, albeit having similar reducing properties, is more active than commercial CdS in the reductive dehalogenation of 2,2-dichloropropionic acid (dalapon) to propionic acid. Spectroscopic, electro-, and photoelectrochemical investigation show that photocatalytic properties of CdS are correlated to its electronic structure. The reductive dehalogenation of dalapon has a double significance: on one hand, it represents a demanding reductive process for a photocatalyst, and on the other hand, it has a peculiar interest in water treatment because dalapon can be considered a representative molecule of persistent organic pollutants and is one of the most important disinfection by products, whose removal from the water is the final obstacle to its complete reuse. HPLC-MS investigation points out that complete disappearance of dalapon passes through 2-monochloropropionic acid and leads to propionic acid as the final product. CdS-HTa requires very mild working conditions (room temperature, atmospheric pressure, natural pH), and it is stable and recyclable without significant loss of activity.

Metformin hydrochloride (MH) has recently been repurposed as an anticancer agent, showing antiproliferative activity in vitro and in vivo. In particular, experimental evidence has suggested its potential clinical efficacy in glioblastoma (GBM), a very aggressive tumor frequently characterized by gloomy prognosis. Unfortunately, the published literature concerning experimental applications of MH in glioblastoma animal models report no data on metformin levels reached in the brain, which, considering the high hydrophilicity of the drug, are likely very low. Therefore, new sensitive analytical methods to be applied on biological tissues are necessary to improve our knowledge of MH in vivo biodistribution and biological effects on tumors. In this research work, a GC-MS method for MH quantification in brain tissues is proposed. MH has been derivatized using N-methyl-bis(trifluoroacetamide), as already described in the literature, but the derivatization conditions have been optimized; moreover, deuterated MH has been selected as the best internal standard, after a comparative evaluation including other internal standards employed in published methods. After ascertaining method linearity, its accuracy, precision, specificity, repeatability, LOD and LOQ (0.373 µM and 1.242 µM, respectively, corresponding to 0.887 and 2.958 pmol/mg of wet tissue) have been evaluated on mouse brain tissue samples, obtained through a straightforward preparation procedure involving methanolic extraction from lyophilized brain homogenates and solid phase purification. The method has been validated on brain samples obtained from mice, either healthy or xenografted with GBM cells, receiving metformin dissolved in the drinking water. This analytical method can be usefully applied in preclinical studies aiming at clarifying MH mechanism of action in brain tumors., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michele Mazzanti reports financial support was provided by AIRC Italian Foundation for Cancer Research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As a prime mover in Alzheimer's disease (AD), microglial activation requires membrane translocation, integration, and activation of the metamorphic protein chloride intracellular channel 1 (CLIC1), which is primarily cytoplasmic under physiological conditions. However, the formation and activation mechanisms of functional CLIC1 are unknown. Here, we found that the human antimicrobial peptide (AMP) LL-37 promoted CLIC1 membrane translocation and integration. It also activates CLIC1 to cause microglial hyperactivation, neuroinflammation, and excitotoxicity. In mouse and monkey models, LL-37 caused significant pathological phenotypes linked to AD, including elevated amyloid-β, increased neurofibrillary tangles, enhanced neuronal death and brain atrophy, enlargement of lateral ventricles, and impairment of synaptic plasticity and cognition, while Clic1 knockout and blockade of LL-37-CLIC1 interactions inhibited these phenotypes. Given AD's association with infection and that overloading AMP may exacerbate AD, this study suggests that LL-37, which is up-regulated upon infection, may be a driving force behind AD by acting as an endogenous agonist of CLIC1., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings., Methods: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos)., Results: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity., Conclusions: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach., (© 2022. The Author(s).)

PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABA A receptors (GABA A Rs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABA A Rs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABA A R-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABA A Rs kinetics without altering their conductance. In particular, GABA A Rs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABA A R-mediated tonic transmission and GABA A Rs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.

Background/aim: Chloride intracellular channel 1 (CLIC1) represents a promising target for personalized therapy. Our aim was to assess CLIC1 expression in clear cell renal cell carcinoma (cc RCC) and identify its possible prognostic role., Materials and Methods: Fifty cases of cc RCC were evaluated and selected for immunohistochemistry. CLIC1 expression was correlated with tumor grade, invasion and heterogeneity., Results: A total of 87.5% of the cases were CLIC1 positive, with either a homogeneous (31.42%) or a heterogeneous (68.57%) pattern. Low, mild and strong CLIC1 expressing tumors were defined based on nuclear (N), cytoplasmic (C), membrane (M) or combinations of them (NC, NM, CM, NCM) in terms of CLIC1 distribution. A significant correlation was found between tumor grade and percent of positive tumor cells (p=0.017). For G3 tumors, CLIC1 cytoplasmic expression was strongly correlated with high expression status (p=0.025) and tumor heterogeneity (p=0.004). CLIC1 expression was also correlated with metastasis (p=0.046)., Conclusion: We defined four cc RCC groups depending on G, CLIC1 expression and pattern: i) G3/NM/low CLIC1 + , ii) G2/CM/mild CLIC1 + iii) G1 or G2/NM or CM /high CLIC1 + , and iv) G2/M /high CLIC1 ., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Glioblastoma (GB) is the most lethal, aggressive, and diffuse brain tumor. The main challenge for successful treatment is targeting the cancer stem cell (CSC) subpopulation responsible for tumor origin, progression, and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability. In vitro , CLIC1 inhibition leads to a significant arrest of GB CSCs in G 1 phase of the cell cycle. Furthermore, CLIC1 knockdown impairs tumor growth in vivo Here, we demonstrate that CLIC1 membrane localization and function is specific for GB CSCs. Mesenchymal stem cells (MSC) do not show CLIC1-associated chloride permeability, and inhibition of CLIC1 protein function has no influence on MSC cell-cycle progression. Investigation of the basic functions of GB CSCs reveals a constitutive state of oxidative stress and cytoplasmic alkalinization compared with MSCs. Both intracellular oxidation and cytoplasmic pH changes have been reported to affect CLIC1 membrane functional expression. We now report that in CSCs these three elements are temporally linked during CSC G 1 -S transition. Impeding CLIC1-mediated chloride current prevents both intracellular ROS accumulation and pH changes. CLIC1 membrane functional impairment results in GB CSCs resetting from an allostatic tumorigenic condition to a homeostatic steady state. In contrast, inhibiting NADPH oxidase and NHE1 proton pump results in cell death of both GB CSCs and MSCs. Our results show that CLIC1 membrane protein is crucial and specific for GB CSC proliferation, and is a promising pharmacologic target for successful brain tumor therapies. Mol Cancer Ther; 17(11); 2451-61. ©2018 AACR ., (©2018 American Association for Cancer Research.)