RESUMEN
As the COVID-19 pandemic unfolded in the spring of 2020, governments around the world began to implement policies to mitigate and manage the outbreak. Significant research efforts were deployed to track and analyse these policies in real-time to better inform the response. While much of the policy analysis focused narrowly on social distancing measures designed to slow the spread of disease, here, we present a dataset focused on capturing the breadth of policy types implemented by jurisdictions globally across the whole-of-government. COVID Analysis and Mapping of Policies (COVID AMP) includes nearly 50,000 policy measures from 150 countries, 124 intermediate areas, and 235 local areas between January 2020 and June 2022. With up to 40 structured and unstructured characteristics encoded per policy, as well as the original source and policy text, this dataset provides a uniquely broad capture of the governance strategies for pandemic response, serving as a critical data source for future work in legal epidemiology and political science.
Asunto(s)
COVID-19 , Humanos , COVID-19/epidemiología , COVID-19/prevención & control , Pandemias , Distanciamiento Físico , Formulación de Políticas , SARS-CoV-2RESUMEN
Glioblastoma Multiforme (GBM) is the most common form of primary brain tumor with 30-50% of patients presenting with epilepsy. These tumor-associated seizures are often resistant to traditional antiepileptic drug treatment and persist after tumor resection. This suggests that changes in the peritumoral tissue underpin epileptogenesis. It is known that glioma cells extrude pathological concentrations of glutamate which is thought to play a role in tumor progression and the development of epilepsy. Given that pathological concentrations of glutamate have been shown to dephosphorylate and downregulate the potassium chloride cotransporter KCC2, we hypothesized that glioma-induced alterations in KCC2 in the peritumoral region may play a role in tumor-associated epilepsy. Consistent with this hypothesis, we observe a decrease in total KCC2 expression and a dephosphorylation of KCC2 at residue Ser940 in a glioma model which exhibits hyperexcitability and the development of spontaneous seizures. To determine whether the reduction of KCC2 could potentially contribute to tumor-associated epilepsy, we generated mice with a focal knockdown of KCC2 by injecting AAV2-Cre-GFP into the cortex of floxed KCC2 mice. The AAV2-Cre-mediated knockdown of KCC2 was sufficient to induce the development of spontaneous seizures. Further, blocking NKCC1 with bumetanide to offset the loss of KCC2 reduced the seizure susceptibility in glioma-implanted mice. These findings support a mechanism of tumor-associated epilepsy involving downregulation of KCC2 in the peritumoral region leading to compromised GABAergic inhibition and suggest that modulating chloride homeostasis may be useful for seizure control.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Epilepsia/metabolismo , Glioma/metabolismo , Simportadores/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Encéfalo/efectos de los fármacos , Neoplasias Encefálicas/complicaciones , Bumetanida/farmacología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Epilepsia/complicaciones , Glioma/complicaciones , Ácido Glutámico/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Fosforilación , Ratas , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Simportadores/genética , Técnicas de Cultivo de Tejidos , Cotransportadores de K ClRESUMEN
Stress is the most commonly reported precipitating factor for seizures. The proconvulsant actions of stress hormones are thought to mediate the effects of stress on seizure susceptibility. Interestingly, epileptic patients have increased basal levels of stress hormones, including corticotropin-releasing hormone (CRH) and corticosterone, which are further increased following seizures. Given the proconvulsant actions of stress hormones, we proposed that seizure-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis may contribute to future seizure susceptibility. Consistent with this hypothesis, our data demonstrate that pharmacological induction of seizures in mice with kainic acid or pilocarpine increases circulating levels of the stress hormone, corticosterone, and exogenous corticosterone administration is sufficient to increase seizure susceptibility. However, the mechanism(s) whereby seizures activate the HPA axis remain unknown. Here we demonstrate that seizure-induced activation of the HPA axis involves compromised GABAergic control of CRH neurons, which govern HPA axis function. Following seizure activity, there is a collapse of the chloride gradient due to changes in NKCC1 and KCC2 expression, resulting in reduced amplitude of sIPSPs and even depolarizing effects of GABA on CRH neurons. Seizure-induced activation of the HPA axis results in future seizure susceptibility which can be blocked by treatment with an NKCC1 inhibitor, bumetanide, or blocking the CRH signaling with Antalarmin. These data suggest that compromised GABAergic control of CRH neurons following an initial seizure event may cause hyperexcitability of the HPA axis and increase future seizure susceptibility.
Asunto(s)
Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Convulsiones/sangre , Convulsiones/etiología , Animales , Hormona Liberadora de Corticotropina/sangre , Susceptibilidad a Enfermedades , Masculino , Ratones , Ratones Endogámicos C57BL , Convulsiones/fisiopatologíaRESUMEN
The GABA type A receptor (GABA(A)R) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P(o)) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA(A)R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P(o). Specifically, we found that currents were outwardly rectifying at low P(o) and linear at high P(o). We confirmed the correlation between P(o) and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA(A)R. Our results show that the direction of the driving force on the permeant ion, as well as P(o), must be considered together for a complete understanding of drug actions on ligand-gated ion channels.
Asunto(s)
Canales Iónicos Activados por Ligandos/fisiología , Potenciales de la Membrana/fisiología , Receptores de GABA-A/fisiología , Regulación Alostérica , Canales de Cloruro , Agonistas del GABA/farmacología , Humanos , Canales Iónicos Activados por Ligandos/efectos de los fármacos , Piperidinas/farmacología , Probabilidad , Conformación Proteica , Receptores de GABA-A/efectos de los fármacosRESUMEN
The GABA type A receptor (GABA(A)R) is a member of the pentameric ligand gated ion channel (pLGIC) family that mediates ionotropic neurotransmission. Residues in the intracellular loop domain (ILD) have recently been shown to define part of the ion permeation pathway in several closely related members of the pentameric ligand gated ion channel family. In this study, we investigated the role the ILD of the GABA(A)R α1 subunit plays in channel function. Deletion of the α1 ILD resulted in a significant increase in GABA EC(50) and maximal current amplitude, suggesting that the ILD must be intact for proper receptor function. To test this hypothesis, we conducted a mutagenic screen of all amino acids harboring ionizable side chains within this domain to investigate the contribution of individual charged residues to ion permeation. Using macroscopic and single channel voltage-clamp recording techniques, we found that mutations within a subdomain of the α1 ILD near M3 altered GABA apparent affinity; interestingly, α1(K312E) exhibited reduced partial agonist efficacy. We introduced point mutations near M4, including α1(K383E) and α1(K384E), that enhanced receptor desensitization. Mutation of 5 charged residues within a 39-residue span contiguous with M4 reduced relative anion permeability of the channel and may represent a weak intracellular selectivity filter. Within this subdomain, the α1(K378E) mutation induced a significant reduction in single channel conductance, consistent with our hypothesis that the GABA(A)R α1 ILD contributes directly to the permeation pathway.
Asunto(s)
Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Secuencia de Aminoácidos , Animales , Eliminación de Gen , Células HEK293 , Humanos , Ligandos , Datos de Secuencia Molecular , Mutación , Técnicas de Placa-Clamp , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , TorpedoRESUMEN
Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A-D). We show that the potency of a variety of endogenous and synthetic glycine-site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand-binding domains and coexpressed these with NR1 subunits in Xenopus laevis oocytes. Glycine concentration-response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC(50) values similar to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating.
Asunto(s)
Glicina/farmacología , Oocitos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Quimera , Relación Dosis-Respuesta a Droga , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Glicina/antagonistas & inhibidores , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Técnicas de Placa-Clamp , ARN Complementario/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Xenopus laevisRESUMEN
BACKGROUND: The gamma-aminobutyric acid type A receptor (GABAA-R) beta subunits are critical targets for the actions for several intravenous general anesthetics, but the precise nature of the anesthetic binding sites are unknown. In addition, little is known about the role the fourth transmembrane (M4) segment of the receptor plays in receptor function. The aim of this study was to better define the propofol binding site on the GABAA-R by conducting a tryptophan scan in the M4 segment of the beta2 subunit. METHODS: Seven tryptophan mutations were introduced into the C-terminal end of the M4 segment of the GABAA-R beta2 subunit. GABAA-R subunit complementary DNAs were transfected into human embryonic kidney 293 cells grown on glass coverslips. After transfection (36-72 h), coverslips were transferred to a perfusion chamber to assay receptor function. Cells were whole cell patch clamped and exposed to GABA, propofol, etomidate, and pregnenolone. Chemicals were delivered to the cells using two 10-channel infusion pumps and a rapid solution exchanger. RESULTS: All tryptophan mutations were well tolerated, and with one exception, all resulted in minimal changes in receptor activation by GABA. One mutation, beta2(Y444W), selectively suppressed the ability of propofol to enhance receptor function while retaining normal sensitivity to etomidate and pregnenolone. CONCLUSIONS: This is the first report of a mutation that selectively reduces propofol sensitivity without altering the action of etomidate. The reduction in propofol sensitivity is consistent with the loss of a hydrogen bond within the propofol binding site. These results also suggest a possible orientation of the propofol molecule within its binding site.
Asunto(s)
Anestésicos Intravenosos/farmacología , Propofol/farmacología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/genética , Tirosina/genética , Secuencia de Aminoácidos/genética , Anestésicos Generales/farmacología , Células Cultivadas , Etomidato/farmacología , Humanos , Bombas de Infusión , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/métodos , Mutación/genética , Técnicas de Placa-Clamp , Pregnenolona/farmacología , Transfección , Triptófano/genéticaRESUMEN
In mammals, the most poorly understood P-type ATPases are those of the P(5) subfamily. To begin characterization of the mammalian P(5)-ATPases, BLAST searches of DNA sequence databases were performed. Five genes were identified in the mouse, human, dog, and rat genomes, and the coding sequences of the mouse genes, termed Atp13a1-Atp13a5, were determined. The intron/exon organization of Atp13a1 differs entirely from those of Atp13a2-5, which are closely related. Amino acid sequence comparisons between the five mouse and two yeast P(5)-ATPases suggest that Atp13a1 is orthologous to the yeast Cod1 gene and that Atp13a2-5 are orthologous to yeast Yor291w. Northern blot analysis showed that Atp13a1, Atp13a2, and Atp13a3 mRNAs were expressed in all mouse tissues, whereas Atp13a4 and Atp13a5 mRNAs were restricted to brain and stomach. While the substrate specificity of these transporters is unknown, their importance is underscored by the presence of homologs in fish, insects, worms, and other eukaryotes.