RESUMO
Systemic risks are characterized by high complexity, multiple uncertainties, major ambiguities, and transgressive effects on other systems outside of the system of origin. Due to these characteristics, systemic risks are overextending established risk management and create new, unsolved challenges for policymaking in risk assessment and risk governance. Their negative effects are often pervasive, impacting fields beyond the obvious primary areas of harm. This article addresses these challenges of systemic risks from different disciplinary and sectorial perspectives. It highlights the special contributions of these perspectives and approaches and provides a synthesis for an interdisciplinary understanding of systemic risks and effective governance. The main argument is that understanding systemic risks and providing good governance advice relies on an approach that integrates novel modeling tools from complexity sciences with empirical data from observations, experiments, or simulations and evidence-based insights about social and cultural response patterns revealed by quantitative (e.g., surveys) or qualitative (e.g., participatory appraisals) investigations. Systemic risks cannot be easily characterized by single numerical estimations but can be assessed by using multiple indicators and including several dynamic gradients that can be aggregated into diverse but coherent scenarios. Lastly, governance of systemic risks requires interdisciplinary and cross-sectoral cooperation, a close monitoring system, and the engagement of scientists, regulators, and stakeholders to be effective as well as socially acceptable.
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Gestão de RiscosRESUMO
Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.
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Proteínas de Bactérias/metabolismo , Citrobacter rodentium/crescimento & desenvolvimento , Citrobacter rodentium/genética , Infecções por Enterobacteriaceae/microbiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Regulon , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Camundongos , Proteoma/análiseRESUMO
Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation-mimicking SEPT9 mutant rescued these effects. Collectively, this study provides the first global analysis of phosphorylation-mediated processes during infection with an extracellular, diarrheagenic bacterial pathogen.
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Escherichia coli Enteropatogênica/patogenicidade , Interações Hospedeiro-Patógeno , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Transdução de Sinais , Sequência de Aminoácidos , Sistemas de Secreção Bacterianos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Células HeLa , Humanos , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Septinas/metabolismo , Shigella/metabolismo , VirulênciaRESUMO
Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Interações Hospedeiro-Patógeno , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Humanos , Imunoprecipitação , Espectrometria de Massas , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Sistemas de Secreção Tipo III/químicaRESUMO
The mammalian AMP-activated protein kinase (AMPK) is an obligatory αßγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the ß-subunit (AMPKß) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or ß1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKß is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.
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Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio/metabolismo , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Ativação Enzimática , Células HEK293 , Células Hep G2 , Humanos , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Transporte Proteico , TreoninaRESUMO
Although Escherichia coli can be an innocuous resident of the gastrointestinal tract, it also has the pathogenic capacity to cause significant diarrheal and extraintestinal diseases. Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide. Consequently, pathogenic E. coli is widely studied in humans, animals, food, and the environment. While there are many common features that these pathotypes employ to colonize the intestinal mucosa and cause disease, the course, onset, and complications vary significantly. Outbreaks are common in developed and developing countries, and they sometimes have fatal consequences. Many of these pathotypes are a major public health concern as they have low infectious doses and are transmitted through ubiquitous mediums, including food and water. The seriousness of pathogenic E. coli is exemplified by dedicated national and international surveillance programs that monitor and track outbreaks; unfortunately, this surveillance is often lacking in developing countries. While not all pathotypes carry the same public health profile, they all carry an enormous potential to cause disease and continue to present challenges to human health. This comprehensive review highlights recent advances in our understanding of the intestinal pathotypes of E. coli.
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Diarreia/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/patogenicidade , Adulto , Antibacterianos/uso terapêutico , Pré-Escolar , Diarreia/diagnóstico , Diarreia/microbiologia , Diarreia/fisiopatologia , Surtos de Doenças , Reservatórios de Doenças/microbiologia , Transmissão de Doença Infecciosa , Farmacorresistência Bacteriana/efeitos dos fármacos , Monitoramento Epidemiológico , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/transmissão , Vacinas contra Escherichia coli , Evolução Molecular , Humanos , Lactente , FilogeniaRESUMO
The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by beta-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKalpha1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKalpha1 by LKB1 in response to lipolytic signals. Activation of AMPKalpha1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKalpha1 in vitro. Functional analysis of an AMPKalpha1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA-activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.
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Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Lipólise , Proteínas Quinases Ativadas por AMP/genética , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Ácidos Graxos/metabolismo , Glicerol/metabolismo , Isoproterenol/farmacologia , Camundongos , Fosforilação , Mutação Puntual , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Type III secretion systems (T3SSs) are central virulence mechanisms used by a variety of Gram-negative bacteria to inject effector proteins into host cells. The needle polymer is an essential part of the T3SS that provides the effector proteins a continuous channel into the host cytoplasm. It has been shown for a few T3SSs that two chaperones stabilize the needle protein within the bacterial cytosol to prevent its premature polymerization. In this study, we characterized the chaperones of the enteropathogenic Escherichia coli (EPEC) needle protein EscF. We found that Orf2 and Orf29, two poorly characterized proteins encoded within the EPEC locus of enterocyte effacement (LEE), function as the needle protein cochaperones. Our finding demonstrated that both Orf2 and Orf29 are essential for type III secretion (T3S). In addition, we found that Orf2 and Orf29 associate with the bacterial membrane and form a complex with EscF. Orf2 and Orf29 were also shown to disrupt the polymerization of EscF in vitro. Prediction of the tertiary structures of Orf2 and Orf29 showed high structural homology to chaperones of other T3SS needle proteins. Overall, our data suggest that Orf2 and Orf29 function as the chaperones of the needle protein, and therefore, they have been renamed EscE and EscG.
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Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas do Citoesqueleto/metabolismo , Enterócitos/metabolismo , Escherichia coli Enteropatogênica/química , Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação , Fosfoproteínas/genética , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
Interdisciplinary scientific knowledge is necessary but not sufficient when it comes to addressing sustainable transformations, as science increasingly has to deal with normative and value-related issues. A systems perspective on coupled human-environmental systems (HES) helps to address the inherent complexities. Additionally, a thorough interaction between science and society (i.e., transdisciplinarity = TD) is necessary, as sustainable transitions are sometimes contested and can cause conflicts. In order to navigate complexities regarding the delicate interaction of scientific research with societal decisions these processes must proceed in a structured and functional way. We thus propose HES-based TD processes to provide a basis for reorganizing science in coming decades.
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Ciência , SociedadesRESUMO
The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.
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Sistemas de Secreção Bacterianos , Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Sequência de Aminoácidos , Escherichia coli Enteropatogênica/química , Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Conventional energy strategy defines an energy system vision (the goal), energy scenarios with technical choices and an implementation mechanism (such as economic incentives). Due to the lead of a generic vision, when applied in a specific regional context, such a strategy can deviate from the optimal one with, for instance, the lowest environmental impacts. This paper proposes an approach for developing energy strategies by simultaneously, rather than sequentially, combining multiple energy system visions and technically feasible, cost-effective energy scenarios that meet environmental constraints at a given place. The approach is illustrated by developing a residential heat supply strategy for a Swiss region. In the analyzed case, urban municipalities should focus on reducing heat demand, and rural municipalities should focus on harvesting local energy sources, primarily wood. Solar thermal units are cost-competitive in all municipalities, and their deployment should be fostered by information campaigns. Heat pumps and building refurbishment are not competitive; thus, economic incentives are essential, especially for urban municipalities. In rural municipalities, wood is cost-competitive, and community-based initiatives are likely to be most successful. Thus, the paper shows that energy strategies should be spatially differentiated. The suggested approach can be transferred to other regions and spatial scales.
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Fontes Geradoras de Energia/economia , Formulação de Políticas , Cidades/economia , Temperatura Alta , Humanos , População Rural , SuíçaRESUMO
The disposal of nuclear waste involves extensive time scales. Technical experts consider up to 1 million years for the disposal of spent fuel and high-level waste in their safety assessment. Yet nuclear waste is not only a technical but also a so-called sociotechnical problem and, therefore, requires interdisciplinary collaboration between technical, natural, social sciences, and the humanities in its management. Given that these disciplines differ in their language, epistemics, and interests, such collaboration might be problematic. Based on evidence from cognitive psychology, we suggest that, in particular, a concept like time is presumably critical and can be understood differently. This study explores how different scientific disciplines understand extensive time scales in general and then focuses on nuclear waste. Eighteen qualitative exploratory interviews were conducted with experts for time-related phenomena of different disciplines, among them experts working in nuclear waste management. Analyses revealed two distinct conceptions of time corresponding to idiographic and nomothetic research approaches: scientists from the humanities and social sciences tend to have a more open, undetermined conception of time, whereas natural scientists tend to focus on a more determined conception that includes some undetermined aspects. Our analyses lead to reflections on potential difficulties for interdisciplinary teams in nuclear waste management. We focus on the understanding of the safety assessment, on potential implications for communication between experts from different disciplines (e.g., between experts from the humanities and engineering for risk assessment and risk communication), and we reflect on the roles of different disciplines in nuclear waste management.
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Resíduos Radioativos/efeitos adversos , Gestão de Riscos/métodos , Gerenciamento de Resíduos/métodos , Comportamento Cooperativo , Humanos , Medição de Risco , Fatores de TempoRESUMO
This work reports an unexpected oxidation behavior of Cu nanoparticles embedded in porous Al(2)O(3) confinements that are produced by annealing alucone (an organic-inorganic hybrid material) deposited by molecular layer deposition. An oxidation of such encapsulated Cu nanoparticles by annealing in air produces Cu oxide nanoparticles attached to the outer surface of the hollow Al(2)O(3) nanostructures, which is in strong contrast to bare or compact, nonporous Al(2)O(3)-coated Cu nanoparticles, which result in hollow oxide nanospheres or do not undergo oxidation, respectively. The conversion from encapsulated Cu to supported oxide nanoparticles is explained by a concerted pore-assisted diffusion and oxidation mechanism. The micropores in the films, having diameters of several angstroms, permit a selective out-diffusion of Cu atoms and prevent the inward diffusion of oxygen. The subsequent oxidation occurs at the pore entrances, which work as multiple nucleation sites for the formation of oxide nanoparticles with a small size and good dispersion.
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Óxido de Alumínio/química , Cobre/química , Membranas Artificiais , Nanopartículas Metálicas/química , Oxirredução , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
Transforming growth factor-beta-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain alphaG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic alphaG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of alphaG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.
Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Imunoprecipitação , MAP Quinase Quinase Quinases/genética , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação/genética , Fosforilação/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologiaRESUMO
A general approach for the fabrication of nanoporous Pt-based multimetallic alloy nanowires is reported, which involves electrodeposition of corresponding precursor alloys into porous anodic alumina templates, followed by a mild dealloying process. Nanoporous ternary PtCoNi and PtCoAu as well as quaternary PtRuCoNi nanowires were successfully fabricated, and their microstructure and composition were examined by transmission electron microscopy. Electrochemical tests showed that these porous nanowires exhibit higher electrochemically active surface area and much improved durability compared to commercially available Pt black, and may find potential applications in electrocatalysis and electrochemical sensing.
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UNLABELLED: world has not faced a severe pandemic for decades, except the rather mild H1N1 one in 2009, pandemic influenza models are inherently hypothetical and validation is, thus, difficult. We aim at reconstructing a recent seasonal influenza epidemic that occurred in Switzerland and deem this to be a promising validation strategy for models of influenza spread. METHODS: We present a spatially explicit, individual-based simulation model of influenza spread. The simulation model bases upon (i) simulated human travel data, (ii) data on human contact patterns and (iii) empirical knowledge on the epidemiology of influenza. For model validation we compare the simulation outcomes with empirical knowledge regarding (i) the shape of the epidemic curve, overall infection rate and reproduction number, (ii) age-dependent infection rates and time of infection, (iii) spatial patterns. RESULTS: The simulation model is capable of reproducing the shape of the 2003/2004 H3N2 epidemic curve of Switzerland and generates an overall infection rate (14.9 percent) and reproduction numbers (between 1.2 and 1.3), which are realistic for seasonal influenza epidemics. Age and spatial patterns observed in empirical data are also reflected by the model: Highest infection rates are in children between 5 and 14 and the disease spreads along the main transport axes from west to east. CONCLUSIONS: We show that finding evidence for the validity of simulation models of influenza spread by challenging them with seasonal influenza outbreak data is possible and promising. Simulation models for pandemic spread gain more credibility if they are able to reproduce seasonal influenza outbreaks. For more robust modelling of seasonal influenza, serological data complementing sentinel information would be beneficial.
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Influenza Humana/epidemiologia , Influenza Humana/transmissão , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Simulação por Computador , Epidemias , Feminino , Humanos , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Reprodutibilidade dos Testes , Suíça/epidemiologia , Adulto JovemRESUMO
There is increasing demand for science to contribute to solving societal problems (solutionism). Thereby, scientists may become normative activists for solving certain problems (advocacy). When doing this, they may insufficiently differentiate between scientific and political modes of reasoning and validation (de-differentiationism), which is sometimes linked to questionable forms of utilizing the force of facts (German: Faktengewalt). Scientific findings are simplified and communicated in such a way that they acquire a status as unfalsifiable and absolutely true (truth to power). This becomes critical if the consistency and validation of the findings are questionable and scientific models underlying science activists' actions are doubtful, oversimplified, or incorrect. Herein, we exemplarily elaborate how the integrity of science is endangered by normative solutionist and sociopolitically driven transition management and present mineral scarcity claims that ignore that reserves or resources are dynamic geotechnological-socioeconomic entities. We present the main mineral scarcity models and their fallacious assumptions. We then discuss the phosphorus scarcity fallacy, which is of particular interest as phosphorus is non-substitutable and half of all current food production depends on fertilizers (and thus phosphorus). We show that phosphorus scarcity claims are based on integrating basic geoeconomic knowledge and discuss cognitive and epistemological barriers and motivational and sociopolitical drivers promoting the scarcity fallacy, which affects high-level public media. This may induce unsustainable environmental action. Scientists as honest knowledge brokers should communicate the strengths but also the constraints and limits of scientific modeling and of applying it in reality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11625-021-01006-w.
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AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain alphaG-helix. Mutation of the corresponding AMPK alpha-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the alphaG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (alpha1(-/-), alpha2(-/-)) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the alphaG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Calpha was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic alphaG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic alphaG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.
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Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Multimerização Proteica , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , TreoninaRESUMO
The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na(+)-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten V(max) parameter. [(14)C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Creatina/metabolismo , Células Epiteliais/enzimologia , Túbulos Renais Proximais/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Biotinilação , Western Blotting , Linhagem Celular Transformada , Polaridade Celular , Metabolismo Energético , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Células Epiteliais/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Cinética , Masculino , Potenciais da Membrana , Proteínas de Membrana Transportadoras/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Oócitos , Técnicas de Patch-Clamp , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ribonucleotídeos/farmacologia , Sirolimo/farmacologia , Sódio/metabolismo , Serina-Treonina Quinases TOR , Xenopus laevisRESUMO
AMPK is a metabolic stress-sensing kinase with important functions for red blood cell (RBC) survival. By using a proteomic approach, we identified putative AMPK targets in hemoglobin-depleted lysates of RBC, including metabolic enzymes, cytoskeletal proteins and enzymes involved in the oxidative stress response. These data tie in with the phenotypic observations of AMPKalpha1-deficient RBC and provide reference for future studies.