Molecular cloning and permanent expression in a neuroblastoma cell line of a fast inactivating potassium channel from bovine adrenal medulla

AbstractUsing a cDNA library from bovine adrenal medulla, we have isolated cDNAs coding for a potassium channel. Then cDNAs encode a 660-amino acid protein that has a molecular weight of 73.288 kDa and no amino-terminal signal peptide. We have called it BAK4. Analysis of its sequence reveals close similarity (94% homology) with a recently described potassium channel from rat brain (RCK4) and heart (RHK1). Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK4 DNA. Expression of the DNA was under the control of a heat-shock promoter. Several clones, that were isolated by neomycin resistance selection, had integrated the plasmid DNA in a stable form. Upon heat induction, these cells produced BAK4 RNA and a potassium outward current, not present in control non-transfected cells. The current, which was transient and decayed markedly during the duration of 200 ms-pulses, can be described as ??? potassium current. The expression of these types of channels in brain (RCK4,RHK1), heart (RHK1) and adrenal medulla (BAK4) suggest their possible implication in important functions for the cell

Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy

We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding eventsThis work was funded by the European Research Council ERC-AdG-340177 (3DNanoMech) grant to RG and by the Spanish Ministry of Economy (MINECO) through grants CSD2010-00024, MAT2013-44858-R to RG and BFU2011-30161- C02-01 and BFU2014-59009-P to VC.Peer reviewe

Naloxone inhibits nicotine-induced receptor current and catecholamine secretion in bovine chromaffin cells

Nicotine-induced catecholamine (CA) secretion and inward ionic currents were inhibited by the opioid antagonist naloxone in cultured bovine chromaffin cells. Naloxone inhibited nicotine-induced CA secretion, as detected by an on-line real-time electrochemical technique, in a dose-dependent manner (IC50=29 [mu]M). In voltage-clamped chromaffin cells, nicotine (10 [mu]M) evoked an average peak inward current of -146 pA that was inhibited by low concentrations of naloxone (42% at 0.1 [mu]M). The antagonist also inhibited total charge influx associated with nicotinic receptor activation (53% at 0.1 [mu]M). This provides strong evidence that naloxone modulation of nicotine-induced CA secretion does not involve opioid receptors but results from the direct interaction with the nicotinic receptor itself, which might also be the case for other related opioid compounds.http://www.sciencedirect.com/science/article/B6SYR-43MC9YT-C/1/1994796a9d2862757ab52186bcacc5f

Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis

© 2020 by the authors.Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a β-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.This research was funded, in part, by contract numbers PID2019-105858RB-I00 (MCI, AEI, FEDER, UE) and RTI2018-097609-B-C21 (MICIU, AEI, FEDER, UE) to C.O.M. and J.M.G.F. and grants from the Spanish Ministerio de Economía y Competitividad (project SAF2017-89288-R from MINECO/AEI/FEDER/UE), from Junta de Comunidades de Castilla-La Mancha (project SBPLY/19/180501/000067), and ISCIII (AC19/00075) and ERANET Euronanomed Program (project NANO4GLIO) to V.C. It also benefited from the framework of COST Action Nano2Clinic (CA17140), supported by COST (European Cooperation in Science and Technology).Publisher’s versio

Prevalence of chronic head, neck and low back pain and associated factors in women residing in the Autonomous Region of Madrid (Spain)

AbstractObjectiveTo compare the prevalence of chronic headache (CH), chronic neck pain (CNP) and chronic low back pain (CLBP) in the autonomous region of Madrid by analyzing gender differences and to determine the factors associated with each pain location in women in 2007.MethodsWe analyzed data obtained from adults aged 16 years or older (n = 12,190) who participated in the 2007 Madrid Regional Health Survey. This survey includes data from personal interviews conducted in a representative population residing in family dwellings in Madrid. The presence CH, CNP, and CLBP was analyzed. Sociodemographic features, self-perceived health status, lifestyle habits, psychological distress, drug consumption, use of healthcare services, the search for alternative solutions, and comorbid diseases were analyzed by using logistic regression models.ResultsThe prevalence of CH, CNP and CLBP was significantly higher (P<0.001) in women (7.3%, 8.4%, 14.1%, respectively) than in men (2.2%, 3.2%, 7.8%, respectively). In women, CH, CNP and CBLP were significantly associated with having ≥3 chronic diseases (OR 7.1, 8.5, 5.8, respectively), and with the use of analgesics and drugs for inflammation (OR: 3.5, 1.95, 2.5, respectively). In the bivariate analysis, the factors associated with pain in distinct body locations differed between men and women.ConclusionsThis study found that CH, CNP and CLBP are a major public health problem in women in central Spain. Women have a higher overall prevalence of chronic pain than men. Chronic pain was associated with a higher use of analgesics and healthcare services

Carbon nanohorns functionalized with polyamidoamine dendrimers as efficient biocarrier materials for gene therapy

[EN] Carbon nanohorns are suitable platforms for use in biological applications. Their high surface areas allow the incorporation of molecular entities, such as polyamidoamine dendrimers. In this work, we report the synthesis, structural characterization and biological data of new hybrid systems of carbon nanohorns that hold polyamidoamine dendrimers. One of these derivatives has been employed as an agent for gene delivery. The system is able to release interfering genetic material diminishing the levels of a house-keeping protein and a protein directly involved in prostate cancer development. Importantly, this hybrid material is also far less toxic than the corresponding free dendrimer. These results allow us to conclude that these nanomaterials can be exploited as useful non-viral agents for gene therapy.M.A.H., N.R., M.L. and E.V. are grateful to DGICYT of Spain for funding through the Project CTQ2007-60037/BQU and to Consejeria de Educacion y Ciencia (JCCM) for funding projects PBI-06-0020 and PCI08-0040. J.G. also acknowledges the Ministerio de Ciencia e Innovacion (MICINN) (Spain) (BFU2011-30161-C02-02), MICINN (Spain)-Fondo Europeo de Desarrollo Regional (FEDER, European Union) (Project CTQ2006-08871) and JCCM (Project PCI08-0033). This work has been supported, in part, by Grants PI081434 from Fondo de Investigaciones Sanitarias, BFU2011-30161-C02-01 from MICINN and PII1109-0163-4002 and POII10-0274-3182 from Consejeria de Educacion, JCCM to V.C. J.G., F.C.P.-M and B.C. are recipients of Torres-Quevedo research contracts funded by MICINN (Spain) and Nano-Drugs S.L. Authors are very grateful to Dr. V. Sue Myers at UT-Austin and Claudio Gamboz of Settore Microscopia Elettronica at University of Trieste for their help with the TEM measurements. We also thank Ana Belen Garcia for her expert technical assistance. Authors are also very grateful to Dr. Sonia Merino and Dr. Prado Sanchez-Verdti for fruitful discussions.Guerra, J.; Herrero, MA.; Carrión, B.; Pérez-Martínez, FC.; Lucío, MI.; Rubio, N.; Meneghetti, M.... (2012). Carbon nanohorns functionalized with polyamidoamine dendrimers as efficient biocarrier materials for gene therapy. Carbon. 50(8):2832-2844. https://doi.org/10.1016/j.carbon.2012.02.0502832284450

Docetaxel-Loaded Nanoparticles Assembled from β-Cyclodextrin/Calixarene Giant Surfactants: Physicochemical Properties and Cytotoxic Effect in Prostate Cancer and Glioblastoma Cells

Giant amphiphiles encompassing a hydrophilic β-cyclodextrin (βCD) component and a hydrophobic calix[4]arene (CA4) module undergo self-assembly in aqueous media to afford core-shell nanospheres or nanocapsules, depending on the nanoprecipitation protocol, with high docetaxel (DTX) loading capacity. The blank and loaded nanoparticles have been fully characterized by dynamic light scattering (DLS), ζ-potential measurements and cryo-transmission electron microscopy (cryo-TEM). The data are compatible with the distribution of the drug between the nanoparticle core and the shell, where it is probably anchored by inclusion of the DTX aromatic moieties in βCD cavities. Indeed, the release kinetics profiles evidenced an initial fast release of the drug, which likely accounts for the fraction hosted on the surface, followed by a slow and sustained release rate, corresponding to diffusion of DTX in the core, which can be finely tuned by modification of the giant amphiphile chemical structure. The ability of the docetaxel-loaded nanoparticles to induce cellular death in different prostate (human LnCap and PC3) and glioblastoma (human U87 and rat C6) cells was also explored. Giant amphiphile-based DTX formulations surpassing or matching the antitumoral activity of the free DTX formulation were identified in all cases with no need to employ any organic co-solvent, thus overcoming the DTX water solubility problems. Moreover, the presence of the βCD shell at the surface of the assemblies is intended to impart stealth properties against serum proteins while permitting nanoparticle surface decoration by supramolecular approaches, paving the way for a new generation of molecularly well-defined antitumoral drug delivery systems with improved specificity and efficiency. Altogether, the results provide a proof of concept of the suitability of the approach based on βCD-CA4 giant amphiphiles to access DTX carriers with tunable properties

The styryl benzoic acid derivative DC10 potentiates radiotherapy by targeting the xCT-glutathione axis

Metastatic tumors with moderate radiosensitivity account for most cancer-related deaths, highlighting the limitations of current radiotherapy regimens. The xCT-inhibitor sulfasalazine (SAS) sensitizes cancer cells to radiotherapy by blocking cystine uptake via the xCT membrane antiporter, and thereby glutathione (GSH) synthesis protecting against radiation-induced oxidative stress. The expression of xCT in multiple tumor types implies it as a target generic to cancer rather than confined to few subtypes. However, SAS has limited clinical potential as a radiosensitizer due to side effects and low bioavailability. Using SAS as a starting point, we previously developed synthetic xCT-inhibitors through scaffold hopping and structure optimization aided by structure-activity relationship analysis (SAR). Notably, the compound DC10 exhibited inhibition of GSH synthesis. In this study, we validated DC10 as a radiosensitizer in the xCT-expressing cancer cell lines A172, A375 and MCF7, and mice harboring melanoma xenografts. After DC10 treatment, we measured 14C-cystine uptake in the cancer cells using liquid scintillation counting, and intracellular GSH levels and reactive oxygen species (ROS) using luminescence assays. We performed immunoblotting of H2AX and ATM to assess DNA damage after treatment with DC10 and radiotherapy. We then assessed the effect of adding DC10 to radiation upon cancer cell colony formation. Blood samples from mice treated with DC10 underwent biochemical analysis to assess toxicity. Finally, mice with A375 melanomas in the flank, received DC10 and radiotherapy in combination, as monotherapies or no treatment. Notably, DC10 reduced cystine uptake and GSH synthesis and increased ROS levels in a dose-dependent manner. Furthermore, DC10 interacted synergistically with radiation to increase DNA damage and reduce tumor cell colony formation. Mice receiving DC10 were clinically unaffected, whereas blood samples analysis to assess bone marrow suppression, liver or kidney toxicity revealed no significant differences between treated mice and untreated controls. Importantly, DC10 potentiated the anti-tumor efficacy of radiation in mice with melanoma xenografts. We conclude that DC10 is well tolerated and acts as a radiosensitizer by inhibiting cystine uptake, leading to GSH depletion and increased oxidative stress. Our findings demonstrate the feasibility of using synthetic xCT-inhibitors to overcome radioresistance.publishedVersio

The presence of headache at onset in SARS-CoV-2 infection is associated with long-term post-COVID headache and fatigue:A case-control study

OBJECTIVE: To investigate the association of headache during the acute phase of SARS-CoV-2 infection with long-term post-COVID headache and other post-COVID symptoms in hospitalised survivors. METHODS: A case-control study including patients hospitalised during the first wave of the pandemic in Spain was conducted. Patients reporting headache as a symptom during the acute phase and age- and sex-matched patients without headache during the acute phase participated. Hospitalisation and clinical data were collected from medical records. Patients were scheduled for a telephone interview 7 months after hospital discharge. Participants were asked about a list of post-COVID symptoms and were also invited to report any additional symptom they might have. Anxiety/depressive symptoms and sleep quality were assessed with the Hospital Anxiety and Depression Scale and the Pittsburgh Sleep Quality Index. RESULTS: Overall, 205 patients reporting headache and 410 patients without headache at hospitalisation were assessed 7.3 months (Standard Deviation 0.6) after hospital discharge. Patients with headache at onset presented a higher number of post-COVID symptoms (Incident Rate Ratio: 1.16, 95% CI: 1.03–1.30). Headache at onset was associated with a previous history of migraine (Odd Ratio: 2.90, 95% Confidence Interval: 1.41–5.98) and with the development of persistent tension-type like headache as a new post-COVID symptom (Odd Ratio: 2.65, 95% CI: 1.66–4.24). Fatigue as a long-term symptom was also more prevalent in patients with headache at onset (Odd Ratio: 1.55, 95% CI: 1.07–2.24). No between-group differences in the prevalence of anxiety/depressive symptoms or sleep quality were seen. CONCLUSION: Headache in the acute phase of SARS-CoV-2 infection was associated with higher prevalence of headache and fatigue as long-term post-COVID symptoms. Monitoring headache during the acute phase could help to identify patients at risk of developing long-term post-COVID symptoms, including post-COVID headache