Nitric Oxide Regulates Neurogenesis in the Hippocampus following Seizures

Hippocampal neurogenesis is changed by brain injury. When neuroinflammation accompanies injury, activation of resident microglial cells promotes the release of inflammatory cytokines and reactive oxygen/nitrogen species like nitric oxide (NO). In these conditions, NO promotes proliferation of neural stem cells (NSC) in the hippocampus. However, little is known about the role of NO in the survival and differentiation of newborn cells in the injured dentate gyrus. Here we investigated the role of NO following seizures in the regulation of proliferation, migration, differentiation, and survival of NSC in the hippocampus using the kainic acid (KA) induced seizuremouse model. We show that NO increased the proliferation of NSC and the number of neuroblasts following seizures but was detrimental to the survival of newborn neurons. NO was also required for the maintenance of long-term neuroinflammation. Taken together, our data show that NO positively contributes to the initial stages of neurogenesis following seizures but compromises survival of newborn neurons.Foundation for Science and Technology (FCT, Portugal); COMPETE; FEDER [PTDC/SAU-NEU/102612/2008, PTDC/NEU-OSD/0473/2012, PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014]; FCT, Portugal [SFRH/BPD/78901/2011, SFRH/BD/77903/2011

Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide reductase from Marinobacter hydrocarbonoclasticus – An electrochemical study

PTDC/BBB-BQB/0129/2014, FCT/MCTES, UID/Multi/04378/2019, UID/QUI/50006/2019, Centro de Quimica Estrutural multiannual funding 2020-2023, UID/QUI/00100/2019.Reduction of N2O to N2 is catalysed by nitrous oxide reductase in the last step of the denitrification pathway. This multicopper enzyme has an electron transferring centre, CuA, and a tetranuclear copper-sulfide catalytic centre, “CuZ”, which exists as CuZ*(4Cu1S) or CuZ(4Cu2S). The redox behaviour of these metal centres in Marinobacter hydrocarbonoclasticus nitrous oxide reductase was investigated by potentiometry and for the first time by direct electrochemistry. The reduction potential of CuA and CuZ(4Cu2S) was estimated by potentiometry to be +275 ± 5 mV and +65 ± 5 mV vs SHE, respectively, at pH 7.6. A proton-coupled electron transfer mechanism governs CuZ(4Cu2S) reduction potential, due to the protonation/deprotonation of Lys397 with a pKox of 6.0 ± 0.1 and a pKred of 9.2 ± 0.1. The reduction potential of CuA, in enzyme samples with CuZ*(4Cu1S), is controlled by protonation of the coordinating histidine residues in a two-proton coupled electron transfer process. In the cyclic voltammograms, two redox pairs were identified corresponding to CuA and CuZ(4Cu2S), with no additional signals being detected that could be attributed to CuZ*(4Cu1S). However, an enhanced cathodic signal for the activated enzyme was observed under turnover conditions, which is explained by the binding of nitrous oxide to CuZ0(4Cu1S), an intermediate species in the catalytic cycle.authorsversionpublishe

Human gingival fibroblasts response to different endodontic sealers: an in vitro study

Endodontic treatment aims to eliminate infection of the root canals and fill the dental pulp space. The biocompatibility studies of the sealers used in root canals obturation are crucial since they are applied in direct contact with periradicular tissues. Objective: The aim of this study was to evaluate the cytotoxicity of three root canal sealers—AH Plus, Bio MTA+, and Bio C sealer—on immortalized human gingival fibroblasts. Methods: AH Plus, Bio MTA+, and Bio C sealers were evaluated through incubation in real-time and material-conditioned media. Cells were incubated for 24 h and 72 h, at three different concentrations (1, 10, and 100 mg/mL) of each sealer. The cytotoxic activity of the sealers was assessed by Methyl tetrazolium (MTT) and Sulforhodamine B (SRB) assays. Cell morphology and cytogenetic alterations were studied microscopically. Results: MTT and SRB assays revealed similar results within both approaches. Cell culture exposed to sealers through incubation in real-time revealed a cytotoxic effect of AH Plus at 100 mg/mL. Material- conditioned media study revealed a cytotoxic effect of Bio MTA+ and Bio C, increasing with higher compound concentration and reaching 50% with 100 mg/mL. Regarding the cell’s morphology, Bio C sealer revealed a decrease in cell confluence and several morphological changes. AH Plus and Bio MTA+ did not seem to affect the cell confluence however morphology alterations were observed. In the cytogenetic study, a severe decrease of the mitotic index and a large number of chromosomal aberrations were observed. The present study represents an advance in the understanding of the biocompatibility of AH Plus, Bio MTA+, and Bio C sealers. These sealers demonstrated some cytotoxicity, depending on the concentration used. Although more validation studies are still needed, this study brings very relevant results in terms of cytotoxicity, cell morphology, and cytogenetic alterations. Conclusions: These results could help in the selection of the most appropriate compounds to be used in clinical practice as well as to determine the maximum recommended amounts of each sealer. Clinical Relevance: This study highlights the potential cytotoxic effects of three commonly used root canal sealers on human gingival fibroblasts, with varying degrees of impact depending on the concentration used. The results emphasize the importance of careful consideration when selecting and applying these materials in clinical practice.info:eu-repo/semantics/publishedVersio

Spectroscopic Definition of the CuZ° Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into the Catalytic Mechanism

Fundacao para a Ciencia e Tecnologia - PTDC/QUI-BIQ/116481/2010, PTDC/BBB-BQB/0129/2014, SFRH/BD/87898/2012 ; Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO - FCT/MEC - UID/Multi/04378/2013 ; ERDF - POCI-01-0145-FEDER-007728Spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of CuZ°, an intermediate form of the Cu4S active site of nitrous oxide reductase (N2OR) that is observed in single turnover of fully reduced N2OR with N2O. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that CuZ° is a 1-hole (i.e., 3CuICuII) state with spin density delocalized evenly over CuI and CuIV. Resonance Raman spectroscopy shows two Cu-S vibrations at 425 and 413 cm-1, the latter with a -3 cm-1 O18 solvent isotope shift. DFT calculations correlated to these spectral features show that CuZ° has a terminal hydroxide ligand coordinated to CuIV, stabilized by a hydrogen bond to a nearby lysine residue. CuZ° can be reduced via electron transfer from CuA using a physiologically relevant reductant. We obtain a lower limit on the rate of this intramolecular electron transfer (IET) that is >104 faster than the unobserved IET in the resting state, showing that CuZ° is the catalytically relevant oxidized form of N2OR. Terminal hydroxide coordination to CuIV in the CuZ° intermediate yields insight into the nature of N2O binding and reduction, specifying a molecular mechanism in which N2O coordinates in a μ-1,3 fashion to the fully reduced state, with hydrogen bonding from Lys397, and two electrons are transferred from the fully reduced μ4S2- bridged tetranuclear copper cluster to N2O via a single Cu atom to accomplish N-O bond cleavage.authorsversionpublishe

X-chromosome terminal deletion in a female with premature ovarian failure: Haploinsufficiency of X-linked genes as a possible explanation

<p>Abstract</p> <p>Background</p> <p>Premature ovarian failure (POF) has repeatedly been associated to X-chromosome deletions. <it>FMR1 </it>gene premutation allele's carrier women have an increased risk for POF. We intent to determine the cause of POF in a 29 year old female, evaluating both of these situations.</p> <p>Methods</p> <p>Concomitant analysis of <it>FMR1 </it>gene CGG repeat number and karyotype revealed an X-chromosome terminal deletion. Fluorescence <it>in situ </it>further characterized the breakpoint. A methylation assay for <it>FMR1 </it>gene allowed to determine its methylation status, and hence, the methylation status of the normal X-chromosome.</p> <p>Results</p> <p>We report a POF patient with a 46,X,del(X)(q26) karyotype and with skewed X-chromosome inactivation of the structural abnormal X-chromosome.</p> <p>Conclusions</p> <p>Despite the hemizygosity of <it>FMR1 </it>gene, the patient does not present Fragile X syndrome features, since the normal X-chromosome is not subject to methylation. The described deletion supports the hypothesis that haploinsufficiency of X-linked genes can be on the basis of POF, and special attention should be paid to X-linked genes in region Xq28 since they escape inactivation and might have a role in this disorder. A full clinical and cytogenetic characterization of all POF cases is important to highlight a pattern and help to understand which genes are crucial for normal ovarian development.</p

Ion Energy Measurements in Plasma Immersion Ion Implantation

This thesis investigates ion energy distributions (IEDs) during plasma immersion ion implantation (PIII). PIII is a surface modification technique where an object is placed in a plasma and pulse biased with large negative voltages. The energy distribution of implanted ions is important in determining the extent of surface modifications. IED measurements were made during PIII using a pulse biased retarding field energy analyser (RFEA) in a capacitive RF plasma. Experimental results were compared with those obtained from a two dimensional numerical simulation to help explain the origins of features in the IEDs. Time resolved IED measurements were made during PIII of metal and insulator materials and investigated the effects of the use of a metal mesh over the surface and the effects of insulator surface charging. When the pulse was applied to the RFEA, the ion flux rapidly increased above the pulse-off value and then slowly decreased during the pulse. The ion density during the pulse decreased below values measured when no pulse was applied to the RFEA. This indicates that the depletion of ions by the pulsed RFEA is greater than the generation of ions in the plasma. IEDs measured during pulse biasing showed a peak close to the maximum sheath potential energy and a spread of ions with energies between zero and the maximum ion energy. Simulations showed that the peak is produced by ions from the sheath edge directly above the RFEA inlet and that the spread of ions is produced by ions which collide in the sheath and/or arrive at the RFEA with trajectories not perpendicular to the RFEA front surface. The RFEA discriminates ions based only on the component of their velocity perpendicular to the RFEA front surface. To minimise the effects of surface charging during PIII of an insulator, a metal mesh can be placed over the insulator and pulse biased together with the object. Measurements were made with metal mesh cylinders fixed to the metal RFEA front surface. The use of a mesh gave a larger ion flux compared to the use of no mesh. The larger ion flux is attributed to the larger plasma-sheath surface area around the mesh. The measured IEDs showed a low, medium and high energy peak. Simulation results show that the high energy peak is produced by ions from the sheath above the mesh top. The low energy peak is produced by ions trapped by the space charge potential hump which forms inside the mesh. The medium energy peak is produced by ions from the sheath above the mesh corners. Simulations showed that the IED is dependent on measurement position under the mesh. To investigate the effects of insulator surface charging during PIII, IED measurements were made through an orifice cut into a Mylar insulator on the RFEA front surface. With no mesh, during the pulse, an increasing number of lower energy ions were measured. Simulation results show that this is due to the increase in the curvature of the sheath over the orifice region as the insulator potential increases due to surface charging. The surface charging observed at the insulator would reduce the average energy of ions implanted into the insulator during the pulse. Compared to the case with no mesh, the use of a mesh increases the total ion flux and the ion flux during the early stages of the pulse but does not eliminate surface charging. During the pulse, compared to the no mesh case, a larger number of lower energy ions are measured. Simulation results show that this is caused by the potential in the mesh region which affects the trajectories of ions from the sheaths above the mesh top and corners and results in more ions being measured with trajectories less than ninety degrees to the RFEA front surface

High resolution melting : improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort

© 2012 Susana et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology: In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results: HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5’UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions: HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations.info:eu-repo/semantics/publishedVersio

Differential contribution of the guanylyl cyclase-cyclic GMP-protein kinase g pathway to the proliferation of neural stem cells stimulated by nitric oxide

Nitric oxide (NO) is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP) and the cGMP-dependent kinase (PKG) are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ) were used. We observed that long-term exposure to the NO donor (24 h), NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h) increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner. Copyright (C) 2012 S. Karger AG, BaselCalouste Gulbenkian Foundation; L'Oreal; UNESCO; Foundation for Science and Technology (FCT, Portugal) [SFRH/BPD/78901/2011, SFRH/BD/23754/2005, SFRH/BD/38127/2007]; COMPETE; FEDER [PTDC/SAU-NEU/102612/2008]info:eu-repo/semantics/publishedVersio