RESUMO
The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 106 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed.
Assuntos
COVID-19 , SARS-CoV-2 , Chlorocebus aethiops , Animais , Humanos , Pandemias , Células Vero , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Imunológicos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests. METHODS: We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level. RESULTS: 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each. CONCLUSION: The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.
Assuntos
COVID-19 , Mpox , Orthopoxvirus , Humanos , Monkeypox virus/genética , SARS-CoV-2/genéticaRESUMO
Uncoupling protein 1 (Ucp1) provides nonshivering thermogenesis (NST) fueled by the dissipation of energy from macronutrients in brown and brite adipocytes. The availability of thermogenic fuels is facilitated by the uptake of extracellular glucose. This conjunction renders thermogenic adipocytes in brown and white adipose tissue (WAT) a potential target against obesity and glucose intolerance. We employed wild-type (WT) and Ucp1-ablated mice to elucidate this relationship. In three experiments of similar setup, Ucp1-ablated mice fed a high-fat diet (HFD) had either reduced or similar body mass gain, food intake, and metabolic efficiency compared with WT mice, challenging the hypothesized role of this protein in the development of diet-induced obesity. Despite the absence of increased body mass, oral glucose tolerance was robustly impaired in Ucp1-ablated mice in response to HFD. Postprandial glucose uptake was attenuated in brown adipose tissue but enhanced in subcutaneous WAT of Ucp1-ablated mice. These differences were explainable by expression of the insulin-responsive member 4 of the facilitated glucose transporter family and fully in line with the capacity for NST in these very tissues. Thus, the postprandial glucose uptake of adipose tissues serves as a surrogate measure for Ucp1-dependent and independent capacity for NST. Collectively, our findings corroborate Ucp1 as a modulator of adipose tissue glucose uptake and systemic glucose homeostasis but challenge its hypothesized causal effect on the development of obesity.
Assuntos
Glucose/metabolismo , Homeostase/fisiologia , Termogênese/fisiologia , Proteína Desacopladora 1/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Dieta Hiperlipídica , Intolerância à Glucose/metabolismo , Teste de Tolerância a Glucose , Hiperglicemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Transcriptoma/genética , Proteína Desacopladora 1/genéticaRESUMO
In rare cases, monogenetic obesity is caused by nonsense mutations in genes regulating energy balance. A key factor herein is the leptin receptor. Here, we focus on leptin receptor nonsense variants causing obesity, namely the human W31X, murine Y333X and rat Y763X mutations, and explored their susceptibilities to aminoglycoside and PTC124 mediated translational read-through in vitro. In a luciferase based assay, all mutations - when analysed within the mouse receptor - were prone to aminoglycoside mediated nonsense suppression with the highest susceptibility for W31X, followed by Y763X and Y333X. For the latter, the corresponding rodent models appear valuable for in vivo experiments. When W31X was studied in the human receptor, its superior read-through susceptibility - initially observed in the mouse receptor - was eliminated, likely due to the different nucleotide context surrounding the mutation in the two orthologues. The impact of the surrounding context on the read-through opens the possibility to discover novel sequence elements influencing nonsense suppression. As an alternative to toxic aminoglycosides, PTC124 was indicated as a superior nonsense suppressor but inconsistent data concerning its read-through activity are reported. PTC124 failed to rescue W31X as well as different nonsense mutated luciferase reporters, thus, challenging its ability to induce translational read-through.
Assuntos
Aminoglicosídeos/farmacologia , Códon sem Sentido , Luciferases/genética , Oxidiazóis/farmacologia , Receptores para Leptina/genética , Animais , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Obesidade/genética , RatosRESUMO
AIMS/HYPOTHESIS: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. METHODS: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. RESULTS: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. CONCLUSIONS/INTERPRETATION: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.
Assuntos
Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Resistência à Insulina/fisiologia , Leptina/uso terapêutico , Animais , Ingestão de Energia/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fígado Gorduroso/sangue , Feminino , Insulina/metabolismo , Leptina/química , Ácidos Linoleicos Conjugados/toxicidade , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipodistrofia/induzido quimicamente , Lipodistrofia/tratamento farmacológico , Lipodistrofia/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Actin depolymerizing proteins of the actin depolymerizing factor (ADF)/cofilin family are essential for actin dynamics, which is critical for synaptic function. Two ADF/cofilin family members, ADF and n-cofilin, are highly abundant in the brain, where they are present in excitatory synapses. Previous studies demonstrated the relevance of n-cofilin for postsynaptic plasticity, associative learning, and anxiety. These studies also suggested overlapping functions for ADF and n-cofilin. METHODS: We performed pharmacobehavioral, electrophysiologic, and electron microscopic studies on ADF and n-cofilin single mutants and double mutants (named ACC mice) to characterize the importance of ADF/cofilin activity for synapse physiology and mouse behavior. RESULTS: The ACC mice, but not single mutants, exhibited hyperlocomotion, impulsivity, and impaired working memory. Hyperlocomotion and impulsive behavior were reversed by methylphenidate, a psychostimulant commonly used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Also, ACC mice displayed a disturbed morphology of striatal excitatory synapses, accompanied by strongly increased glutamate release. Blockade of dopamine or glutamate transmission resulted in normal locomotion. CONCLUSIONS: Our study reveals that ADHD can result from a disturbed balance between excitation and inhibition in striatal circuits, providing novel insights into the mechanisms underlying this neurobehavioral disorder. Our results link actin dynamics to ADHD, suggesting that mutations in actin regulatory proteins may contribute to the etiology of ADHD in humans.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Cofilina 1/fisiologia , Corpo Estriado/ultraestrutura , Destrina/fisiologia , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Estimulantes do Sistema Nervoso Central/farmacologia , Cofilina 1/genética , Cofilina 1/metabolismo , Destrina/genética , Modelos Animais de Doenças , Antagonistas de Dopamina , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Potenciais Pós-Sinápticos Excitadores , Glutamatos/metabolismo , Comportamento Impulsivo/efeitos dos fármacos , Comportamento Impulsivo/fisiologia , Masculino , Memória de Curto Prazo/fisiologia , Metilfenidato/farmacologia , Camundongos , Camundongos Knockout , Atividade Motora/genética , Comportamento de Nidação , Neurônios/metabolismo , Neurônios/ultraestrutura , Fenótipo , Receptores Dopaminérgicos/fisiologia , Substância Negra/metabolismo , Sinapses/ultraestruturaRESUMO
Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release.
Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Exocitose/fisiologia , Transporte Proteico/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Cofilina 1/genética , Destrina/genética , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/genética , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Fosforilação , Cloreto de Potássio/farmacologia , Prosencéfalo/citologia , Transporte Proteico/genética , Proteínas SNARE/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/ultraestruturaRESUMO
Uncoupling protein (UCP) 3 is a mitochondrial inner membrane protein implicated in lipid handling and metabolism of reactive oxygen species. Its transcription is mainly regulated by peroxisome proliferator-activated receptors (PPAR), a family of nuclear hormone receptors. Employing bandshift assays, RNA interference and reporter gene assays we examine an intronic region in the UCP3 gene harboring a cis-element essential for expression in brown adipocytes. We demonstrate binding of SP1 and SP3 to this element which is adjacent to a direct repeat 1 element mediating activation of UCP3 expression by PPARγ agonists. Transactivation mediated by these elements is interdependent and indispensable for UCP3 expression. Systematic deletion uncovered a third binding element, a putative NF1 site, in close proximity to the SP1/3 and PPARγ binding elements. Data mining demonstrated binding of MyoD and Myogenin to this third element in C2C12 cells, and, furthermore, revealed recruitment of p300. Taken together, this intronic region is the main enhancer driving UCP3 expression with SP1/3 and PPARγ as the core factors required for expression.
Assuntos
Adipócitos Marrons/metabolismo , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , DNA/genética , DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Íntrons , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Proteína MyoD/metabolismo , Miogenina/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Phodopus , Ligação Proteica , Ratos , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/antagonistas & inibidores , Fator de Transcrição Sp3/genética , Proteína Desacopladora 3 , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity.
Assuntos
Profilinas/deficiência , Sinapses/fisiologia , Animais , Região CA1 Hipocampal/fisiologia , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Deleção de Genes , Camundongos , Camundongos Transgênicos , Plasticidade Neuronal/fisiologia , Especificidade de Órgãos , Profilinas/metabolismo , Prosencéfalo/metabolismo , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.