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Keeping schools open was an active strategy in Sweden to meet the threats of the COVID-19 pandemic. In this article we analyze how a collection of welfare state agents with different tasks, resources and interests in interaction formed an assemblage in their responses to the pandemic and how education thereby became part of a strategy to keep the society going. The inquiries concern what this tells us about education as framed and constrained as a part of society. Our observations are based on statements presented by the government and public agencies, mass media and websites. We identified an assemblage of interwoven agents such as institutions, laws, regulations and recommendations, pandemic manuals, statistics and media. All these were brought together by actions and ideas to handle a pandemic when there were no preventive vaccines. The overarching principle was to educate the population to competent actions in dealing with the pandemic. To keep schools open was part of that principle combined with caretaking ambitions. This assemblage looked like a centralistic machine but it was not; risks were pushed back to local authorities and schools. In conclusion, we note that education is vital in the overarching strategy to deal with the pandemic in Sweden in terms of trust in people and governmentality.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.
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Canal de Potasio ERG1/antagonistas & inhibidores , Medición de Riesgo/métodos , Torsades de Pointes/inducido químicamente , Teorema de Bayes , Simulación por Computador , Humanos , Modelos Biológicos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Seguridad , Torsades de Pointes/fisiopatologíaRESUMEN
Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.
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For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The C: omprehensive I: n Vitro P: roarrhythmia A: ssay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell-derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.
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Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Corazón/efectos de los fármacos , Animales , Humanos , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/diagnóstico , Torsades de Pointes/inducido químicamente , Torsades de Pointes/diagnósticoRESUMEN
The T-type calcium channel inhibitor Mibefradil was reported to protect the heart from atrial remodeling, a key process involved in the development of atrial fibrillation and arrhythmias. Mibefradil is not a selective T-type calcium channel inhibitor and also affects the function of different ion channels. Our aim was to develop a selective T-type calcium channel inhibitor to validate the importance of T-type-related pharmacology in atrial fibrillation. Structural optimisation of a previously disclosed hit series focussed on minimising exposure to the central nervous system and improving pharmacokinetic properties, while maintain adequate potency and selectivity. This resulted in the design of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide, a novel, selective, peripherally restricted chemical probe to verify the role of T-type calcium channel inhibition on atrial fibrillation protection.
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Benzamidas/química , Bloqueadores de los Canales de Calcio/química , Canales de Calcio Tipo T/química , Animales , Benzamidas/síntesis química , Benzamidas/farmacocinética , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/farmacocinética , Canales de Calcio Tipo T/metabolismo , Perros , Evaluación Preclínica de Medicamentos , Semivida , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Relación Estructura-ActividadRESUMEN
Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca(v)3.2) inhibitor with in vivo antihypertensive effect in rats.
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Acetamidas/farmacología , Antihipertensivos/farmacología , Canales de Calcio Tipo T/metabolismo , Descubrimiento de Drogas , Acetamidas/síntesis química , Acetamidas/química , Animales , Antihipertensivos/síntesis química , Antihipertensivos/química , Relación Dosis-Respuesta a Droga , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.
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Cromogranina B/fisiología , Islotes Pancreáticos/metabolismo , Hormonas Pancreáticas/metabolismo , Animales , Cromogranina B/genética , Exocitosis , Insulina/metabolismo , Secreción de Insulina , Ratones , Ratones NoqueadosRESUMEN
Huntington's disease is a severe progressive neurodegenerative disorder caused by a CAG expansion in the IT15 gene, which encodes huntingtin. The disease primarily affects the neostriatum and cerebral cortex and also associates with increased incidence of diabetes. Here, we show that mutant huntingtin disrupts intracellular transport and insulin secretion by direct interference with microtubular beta-tubulin. We demonstrate that mutant huntingtin impairs glucose-stimulated insulin secretion in insulin-producing beta-cells, without altering stored levels of insulin. Using VSVG-YFP, we show that mutant huntingtin retards post-Golgi transport. Moreover, we demonstrate that the speed of insulin vesicle trafficking is reduced. Using immunoprecipitation of mutant and wild-type huntingtin in combination with mass spectrometry, we reveal an enhanced and aberrant interaction between mutant huntingtin and beta-tubulin, implying the underlying mechanism of impaired intracellular transport. Thus, our findings have revealed a novel pathogenetic process by which mutant huntingtin may disrupt hormone exocytosis from beta-cells and possibly impair vesicular transport in any cell that expresses the pathogenic protein.
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Enfermedad de Huntington/metabolismo , Insulina/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Unión Proteica , Transporte de Proteínas , Ratas , Vesículas Transportadoras/genética , Tubulina (Proteína)/genéticaRESUMEN
Biphasic insulin secretion is required for proper insulin action and is observed not only in vivo, but also in isolated pancreatic islets and even single beta-cells. Late events in the granule life cycle are thought to underlie this temporal pattern. In the last few years, we have therefore combined live cell imaging and electrophysiology to study insulin secretion at the level of individual granules, as they approach the plasma membrane, undergo exocytosis and finally release their insulin cargo. In the present paper, we review evidence for two emerging concepts that affect insulin secretion at the level of individual granules: (i) the existence of specialized sites where granules dock in preparation for exocytosis; and (ii) post-exocytotic regulation of cargo release by the fusion pore.
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Gránulos Citoplasmáticos/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Exocitosis , Humanos , Insulina/metabolismo , CinéticaRESUMEN
Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.
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Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calcio/fisiología , Proteína 25 Asociada a Sinaptosomas/metabolismo , Calorimetría , Dicroismo Circular , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Secretagoginas , Transducción de Señal , Espectrofotometría Ultravioleta , Sinaptosomas/metabolismo , Triptófano/análisisRESUMEN
Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by approximately 0.1 and approximately 2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.
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Nucleótidos de Adenina/metabolismo , Islotes Pancreáticos/metabolismo , Vesículas Secretoras/metabolismo , Adenosina Trifosfato/farmacología , Amiloide/metabolismo , Células Cultivadas , Exocitosis/fisiología , Concentración de Iones de Hidrógeno , Insulina/metabolismo , Secreción de Insulina , Polipéptido Amiloide de los Islotes Pancreáticos , Potenciales de la Membrana/fisiología , Péptidos/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Factores de Tiempo , TransfecciónRESUMEN
Postnatal constriction of the full-term ductus arteriosus produces cell death and remodeling of the ductus wall. Using a bioluminescence imaging technique, we found that after birth, the lamb ductus develops ATP, glucose, and glycogen depletion in addition to hypoxia. In vitro studies showed that cell death correlates best with ATP depletion and is most marked when both glucose and oxygen are severely depleted; in addition, the degree of ATP depletion found in vivo is sufficient to account for the extensive degree of cell death that occurs after birth. Under hypoxic conditions, the immature ductus is more capable of preserving its ATP supply than the mature ductus as a result of increased glucose availability, glycogen stores, and glucose utilization. However, the immature ductus is just as susceptible as the mature ductus to ATP depletion when glucose supplies are restricted. The extensive degree of cell death that occurs in the newborn ductus after birth is associated primarily with ATP depletion. The increased glycolytic capacity of the immature ductus may enable it to tolerate episodes of hypoxia and nutrient shortage, making it more resistant to developing postnatal cell death and permanent closure.
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Adenosina Trifosfato/metabolismo , Apoptosis , Conducto Arterial/citología , Conducto Arterial/metabolismo , Animales , Animales Recién Nacidos , Feto/citología , Feto/metabolismo , Glucosa/metabolismo , Glucólisis , Hipoglucemia/metabolismo , Hipoglucemia/patología , Hipoxia/metabolismo , Hipoxia/patología , Técnicas In Vitro , OvinosRESUMEN
Chronic hypoxia is a clinically important condition known to cause vascular abnormalities. To investigate the cellular mechanisms involved, we kept rings of a rat tail artery for 4 days in hypoxic culture (HC) or normoxic culture (NC) (PO(2) = 14 vs. 110 mmHg) and then measured contractility, oxygen consumption (JO(2)), and lactate production (J(lac)) in oxygenated medium. Compared with fresh rings, basal ATP turnover (J(ATP)) was decreased in HC, but not in NC, with a shift from oxidative toward glycolytic metabolism. JO(2) during mitochondrial uncoupling was reduced by HC but not by NC. Glycogen stores were increased 40-fold by HC and fourfold by NC. Maximum tension in response to norepinephrine and the JO(2) versus tension relationship (JO(2) vs. high K(+) elicited force) were unaffected by either HC or NC. Force transients in response to caffeine were increased in HC, whereas intracellular Ca(2+) wave activity during adrenergic stimulation was decreased. Protein synthesis rate was reduced by HC. The results show that long-term hypoxia depresses basal energy turnover, impairs mitochondrial capacity, and alters Ca(2+) homeostasis, but does not affect contractile energetics. These alterations may form a basis for vascular damage by chronic hypoxia.
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Arterias/metabolismo , Hipoxia de la Célula/fisiología , Metabolismo Energético/fisiología , Músculo Liso Vascular/metabolismo , Oxígeno/metabolismo , Animales , Arterias/efectos de los fármacos , Señalización del Calcio/fisiología , Metabolismo Energético/efectos de los fármacos , Femenino , Glucosa/deficiencia , Glucosa/metabolismo , Glucógeno/metabolismo , Técnicas In Vitro , Líquido Intracelular/metabolismo , Ácido Láctico/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/farmacología , Oxígeno/farmacología , Consumo de Oxígeno , Biosíntesis de Proteínas , Ratas , Cola (estructura animal)/irrigación sanguínea , Desacopladores/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacologíaRESUMEN
Inhibition of oxidative metabolism is often found to decrease contractility of systemic vascular smooth muscle, but not to reduce global [Ca(2+)](i). In the present study, we probe the hypothesis that it is associated with an altered pattern of intracellular Ca(2+) oscillations (waves) influencing force development. In the rat tail artery, mitochondrial inhibitors (rotenone, antimycin A, and cyanide) reduced alpha(1)-adrenoceptor-stimulated force by 50% to 80%, but did not reduce global [Ca(2+)](i). Less relaxation (about 30%) was observed after inhibition of myosin phosphatase activity with calyculin A, suggesting that part of the metabolic sensitivity involves the regulation of myosin 20-kDa light chain phosphorylation, although no decrease in phosphorylation was found in freeze-clamped tissue. Confocal imaging revealed that the mitochondrial inhibitors increased the frequency but reduced the amplitude of asynchronous cellular Ca(2+) waves elicited by alpha(1) stimulation. The altered wave pattern, in association with increased basal [Ca(2+)](i), accounted for the unchanged global [Ca(2+)](i). Inhibition of glycolytic ATP production by arsenate caused similar effects on Ca(2+) waves and global [Ca(2+)](i), developing gradually in parallel with decreased contractility. Inhibition of wave activity by the InsP(3) receptor antagonist 2-APB correlated closely with relaxation. Furthermore, abolition of waves with thapsigargin in the presence of verapamil reduced force by about 50%, despite unaltered global [Ca(2+)](i), suggesting that contraction may at least partly depend on Ca(2+) wave activity. This study therefore indicates that mitochondrial inhibition influences Ca(2+) wave activity, possibly due to a close spatial relationship of mitochondria and the sarcoplasmic reticulum and that this contributes to metabolic vascular relaxation.