RESUMO
ADAMTS13 regulates the hemostatic activity of von Willebrand factor (VWF). Determined by static assays, proteolytic activity <10IU/dL in patient plasma, in absence of ADAMTS13 autoantibodies, indicates Upshaw-Schulman syndrome (USS); the congenital form of Thrombotic Thrombocytopenic Purpura (TTP). We have recently functionally characterized sixteen USS-associated ADAMTS13 missense variants under static conditions. Here, we used two assays under shear flow conditions to analyze the activity of those seven mutants with sufficiently high residual secretion plus two newly identified variants. One assay determines cleavage of VWF strings bound to the surface of endothelial cells. The other, light transmission aggregometry-based assay, mimics degradation of VWF-platelet complexes, which are likely to be present in the circulation during TTP bouts. We found that 100 ng/ml of all variants were able to cleave about 80-90% of VWF strings even though 5 out of 9 exhibited activity ≤1% in the state-of-the-art static assay at the same concentration. These data indicate underestimation of ADAMTS13 activity by the used static assay. In simulated circulation, two variants, with missense mutations in the vicinity of the catalytic domain, exhibited only minor residual activity while all other variants were able to effectively break down VWF-platelet complexes. In both assays, significant proteolytic activity could be observed down to 100 ng/ml ADAMTS13. It is thus intriguing to postulate that most variants would have ample activity if secretion of 10% of normal plasma levels could be achieved.
Assuntos
Proteína ADAMTS13/genética , Variação Genética , Mutação de Sentido Incorreto , Púrpura Trombocitopênica Trombótica/congênito , Púrpura Trombocitopênica Trombótica/genética , Plaquetas/metabolismo , Domínio Catalítico , Códon sem Sentido , Células Endoteliais/metabolismo , Células HEK293 , Hemostasia , Humanos , Agregação Plaquetária , Proteínas Recombinantes/genética , Resistência ao Cisalhamento , Fatores de Tempo , Fator de von WillebrandRESUMO
Most river systems are impacted by sewage effluent. It remains unclear if there is a lower threshold to the concentration of sewage effluent that can significantly change the structure of the microbial community and its mobile genetic elements in a natural river biofilm. We used novel in situ mesocosms to conduct replicated experiments to study how the addition of low-level concentrations of sewage effluent (nominally 2.5 ppm) affects river biofilms in two contrasting Chalk river systems, the Rivers Kennet and Lambourn (high/low sewage impact, respectively). 16S sequencing and qPCR showed that community composition was not significantly changed by the sewage effluent addition, but class 1 integron prevalence (Lambourn control 0.07% (SE ± 0.01), Lambourn sewage effluent 0.11% (SE ± 0.006), Kennet control 0.56% (SE ± 0.01), Kennet sewage effluent 1.28% (SE ± 0.16)) was significantly greater in the communities exposed to sewage effluent than in the control flumes (ANOVA, F = 5.11, p = 0.045) in both rivers. Furthermore, the difference in integron prevalence between the Kennet control (no sewage effluent addition) and Kennet sewage-treated samples was proportionally greater than the difference in prevalence between the Lambourn control and sewage-treated samples (ANOVA (interaction between treatment and river), F = 6.42, p = 0.028). Mechanisms that lead to such differences could include macronutrient/biofilm or phage/bacteria interactions. Our findings highlight the role that low-level exposure to complex polluting mixtures such as sewage effluent can play in the spread of antibiotic resistance genes. The results also highlight that certain conditions, such as macronutrient load, might accelerate spread of antibiotic resistance genes.
Assuntos
Biofilmes , Esgotos/química , Integrons , Fósforo , Prevalência , Rios/químicaRESUMO
This protocol describes how to sample and preserve microbial water column samples from rivers that can be used for 16S or 18S metabarcoding studies or shotgun sequencing. It further describes how to extract the DNA for sequencing and how to prepare raw Illumina MiSeq amplicon data and analyze it in the R environment.
Assuntos
Rios/microbiologia , Microbiologia da Água , Biodiversidade , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodosRESUMO
Elevated nutrient levels can lead to excessive biofilm growth, but reducing nutrient pollution is often challenging. There is therefore interest in developing control measures for biofilm growth in nutrient-rich rivers that could act as complement to direct reductions in nutrient load. Shading of rivers is one option that can mitigate blooms, but few studies have experimentally examined the differences in biofilm communities grown under shaded and unshaded conditions. We investigated the assembly and diversity of biofilm communities using in situ mesocosms within the River Thames (UK). Biofilm composition was surveyed by 454 sequencing of 16S amplicons (â¼400 bp length covering regions V6/V7). The results confirm the importance of sunlight for biofilm community assembly; a resource that was utilized by a relatively small number of dominant taxa, leading to significantly less diversity than in shaded communities. These differences between unshaded and shaded treatments were either because of differences in resource utilization or loss of diatom-structures as habitats for bacteria. We observed more co-occurrence patterns and network interactions in the shaded communities. This lends further support to the proposal that increased river shading can help mitigate the effects from macronutrient pollution in rivers.
Assuntos
Bactérias/classificação , Biofilmes/crescimento & desenvolvimento , Eutrofização/fisiologia , Rios/microbiologia , Microbiologia da Água , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Sequência de Bases , Biofilmes/classificação , Diatomáceas/genética , Diatomáceas/microbiologia , Ecossistema , Inglaterra , Tipagem Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de RNA , Luz SolarRESUMO
Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world's oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.
Assuntos
Biologia Marinha , Biodiversidade , Sistemas de Gerenciamento de Base de Dados , Metagenômica , Oceanos e MaresRESUMO
Dermatophytes initiate dermatophytosis, but susceptibility to infection is dictated by host genetic factors, although the role of some of these-such as human beta-defensin 2 (hBD-2) genomic (DEFB4) copy number (CN) variation and its induction by IL-22-remains unclear. This was investigated in this cross-sectional study in 442 unrelated Caucasian subjects, including 195 healthy controls and 247 dermatophytosis patients who were divided into five subgroups according to clinical presentation. DNA samples were evaluated for DEFB4 CN variation by relative quantification using the comparative CT method, and serum hBD-2 and IL-22 levels were determined by ELISA. DEFB4 CN in patients was significantly lower and, except in the tinea cruris subgroup, serum hBD-2 levels were higher than in controls. The positive correlation between hBD-2 levels and DEFB4 CN observed in controls was not detected in patients, who also had higher serum IL-22 levels that were positively correlated with hBD-2 levels. Moreover, unlike in control subjects, the serum IL-22 level was negatively correlated with DEFB4 CN in patients. Taken together, these findings suggest an association between decreased DEFB4 CN, elevated serum hBD-2 and IL-22 levels, and dermatophytosis, underscoring a gene/cytokine interaction in the occurrence of this infection.
Assuntos
Dosagem de Genes/genética , Interleucinas/sangue , Tinha/sangue , Tinha/genética , beta-Defensinas/sangue , beta-Defensinas/genética , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos Transversais , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Tinha/microbiologia , Trichophyton , Adulto Jovem , Interleucina 22RESUMO
Leukocytes are a heterogeneous group of cells that display differences in anatomic localization, cell surface phenotype, and function. The different subtypes include e.g., granulocytes, monocytes, dendritic cells, T cells, B cells and NK cells. These different cell types represent the cellular component of innate and adaptive immunity. Using certain toxins such as pertussis toxin, cholera toxin or clostridium difficile toxin, the regulatory functions of Gα(i), Gαs and small GTPases of the Rho family in leukocytes have been reported. A summary of these reports is discussed in this review.
Assuntos
Toxinas Bacterianas/farmacologia , Leucócitos/efeitos dos fármacos , Animais , Proteínas de Bactérias/farmacologia , Toxina da Cólera/farmacologia , Enterotoxinas/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Humanos , Leucócitos/fisiologia , Toxina Pertussis/farmacologia , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Lysophosphatidic acid (LPA) is an activator and chemoattractant of NK cells, which are critical members of the immunological tumor surveillance machinery. Here, we analyzed the influence of LPA on the interaction of human NK cells with tumor cells such as the Burkitt lymphoma cell line Raji and the human melanoma cell line A2058. Thereby we found that LPA inhibits the release of perforin and cytotoxic activity of NK cells. Analysis of signal transduction showed that LPA induces common signaling pathways of chemotaxins such as G(i) protein-dependent actin re-organization, activation of the mitogen-activated protein kinase p38 as well as phosphatidylinositol-3-kinase-dependent signal molecules [protein kinase B/Akt and glycogen synthase kinase-3beta (GSK-3beta)]. In contrast to most chemotaxins, LPA is also able to activate G(s)-dependent signaling molecules. This signaling cascade involves the LPA receptor type-2, increase cAMP levels and protein kinase A (PKA) activation, which in turn are responsible for the modulatory effect of LPA on NK cell-mediated cytotoxicity. Moreover, blocking the regulatory subunits of PKA I abrogates the inhibitory effect of LPA, whereas the catalytic subunits are not involved. Based on our data, one can assume that LPA contributes to the tumor escape from the immunological surveillance machinery.
Assuntos
Linfoma de Burkitt/imunologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células Matadoras Naturais/metabolismo , Lisofosfolipídeos/metabolismo , Melanoma/imunologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citotoxicidade Imunológica , Regulação para Baixo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/imunologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/imunologia , Humanos , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Lisofosfolipídeos/imunologia , Melanoma/metabolismo , Melanoma/patologia , Perforina/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Ácidos Lisofosfatídicos/imunologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/imunologia , Evasão Tumoral , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a bioactive phospholipid that transmits signals through G-protein-coupled receptors to control cellular differentiation, survival, and several functions of immune cells. S1P is a chemoattractant for NK cells, which are critical members of the immunological tumor surveillance machinery. In this study we analyzed the influence of S1P on the interaction of NK cells with tumor cells such as the human melanoma cell line Hs294T and the Burkitt's lymphoma cell line Raji. We found that S1P inhibited the cytotoxic activity of NK cells. Analysis of signal transduction pathways revealed that S1P induced common signalling pathways of chemotaxins such as Gi protein-dependent actin reorganization and activation of the phosphatidylinositol 3-kinase (PI3K) dependent signal molecules, protein kinase B (PKB/Akt) and glycogen synthase kinase-3beta (GSK-3beta). In contrast to most chemotaxins, S1P is also able to activate Gs-dependent signalling molecules. This signalling cascade involves increase of cAMP levels and protein kinase A (PKA) activation. Additionally, blocking the regulatory subunits of PKA I abrogated the inhibitory effect of S1P, whereas the catalytic subunits were not involved. Our data indicate that S1P may contributes to the tumor escape from NK cell-dependent immunological surveillance machinery.
Assuntos
Linfoma de Burkitt/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Matadoras Naturais/metabolismo , Lisofosfolipídeos/farmacologia , Melanoma/patologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Linfoma de Burkitt/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Immunoblotting , Interleucina-2/farmacologia , Melanoma/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingosina/farmacologiaRESUMO
Neutrophils release reactive oxygen species (ROS) as part of the innate inflammatory immune response. Phosphoinositide 3-kinase gamma (PI3Kgamma), which is induced by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), has been identified as an essential intracellular mediator of ROS production. However, the complex signalling reactions that link PI3Kgamma with ROS synthesis by NADPH oxidase have not yet been described in detail. We found that activation of neutrophils by fMLP triggers the association of PI3Kgamma with protein kinase Calpha (PKCalpha). Specific inhibition of PI3Kgamma suppresses fMLP-mediated activation of PKCalpha activity and ROS production, suggesting that the protein kinase activity of PI3Kgamma is involved. Our data suggest that the direct interaction of PI3Kgamma with PKCalpha forms a discrete regulatory module of fMLP-dependent ROS production in neutrophils.
Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/citologia , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-alfa/metabolismo , Explosão Respiratória , Classe Ib de Fosfatidilinositol 3-Quinase , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/efeitos dos fármacosRESUMO
A river water microbial community was studied in response to perturbation with the monoterpene enantiomers R- and S-carvone. The microbial community structure and function was also evaluated after enantiomers exposure was switched. Microbial communities were evaluated by length heterogeneity PCR. The addition of R- and S-carvone enriched for a range of functionally different communities: enantiomer-selective, racemic and ones that contain both. After 5 days incubation, the R- and S-carvone treatments developed a range of dominant microbial communities, which were increasingly dissimilar from the ones in which no carvone degradation had taken place (R-values: R-carvone 0.743, S-carvone 0.5007). There was an increase in the evenness of the microbial community structure upon carvone depletion. After the cross-over, the rate of carvone utilization was significantly faster than after the first carvone addition (P=0.008) as demonstrated by concomitant carvone and oxygen depletion. The main R-degrading community (450-456 bp) appeared enantioselective and largely unable to degrade S-carvone, whereas the S-carvone-degrading community (502-508 bp) appeared to have racemic catabolic capacity. In conclusion, chemical perturbations, such as enantiomers, might generate a significant shift in the river microbial ecology that can have implications for the function of a river in both a spatial and temporal context.
Assuntos
Bactérias , Ecossistema , Eucariotos , Monoterpenos , Rios/microbiologia , Rios/parasitologia , Animais , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Biodegradação Ambiental , Monoterpenos Cicloexânicos , Eucariotos/crescimento & desenvolvimento , Eucariotos/fisiologia , Monoterpenos/metabolismo , Monoterpenos/farmacologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Rios/químicaRESUMO
Little is known about psychological and social factors influencing the frequency of migraine attacks. The aim of the present study was to study whether anger, anger-expression, anxiety, depression, somatization and childhood adversities influence the frequency of migraine attacks. 53 female patients suffering from migraine with a mean monthly headache frequency of 6.85 took part. Groups of patients with 6 or less monthly attacks (N = 27) vs. patients with 7 or more days (N = 26) were formed. Logistic regression analysis revealed, that only the variable "anger-in" resulted in an improvement of prediction of group-membership (rate of correct classified cases: 69.8 %). Tendency to repress anger increases the probability to be a member of the group with frequent attacks. This result points out the relevance of anger-expression and -perception for the psychotherapy of migraine.
Assuntos
Transtornos de Enxaqueca/psicologia , Adulto , Idoso , Ira , Ansiedade/complicações , Ansiedade/psicologia , Depressão/complicações , Depressão/psicologia , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/etiologia , Risco , Transtornos Somatoformes/complicaçõesRESUMO
The inhomogeneous distribution of radon between water and non-aqueous phase liquid (NAPL) allows for utilizing naturally occurring radon as aqueous tracer for the assessment of residual NAPL contamination of aquifers ("NAPL source zones"). For the qualitative assessment (i.e. the localization) of NAPL source zones depleted radon concentrations that occur locally in the groundwater can be used as indicator. However, quantitative estimation of the NAPL saturation of the aquifer pore space requires the knowledge of the radon partition coefficient specific for the NAPL present. While radon partition coefficients are known for a wide range of pure substances, few reliable data is available on radon partitioning into complex NAPL mixtures. At the same time, widely used NAPL mixtures, such as diesel fuel, gasoline, and kerosene, have to be named as main contaminants at many NAPL contaminated sites. The paper presents radon partition coefficients for the three NAPL mixtures mentioned, achieved by application of an analytical method based on radon partitioning between air, water, and NAPL in a closed system, which is described in detail. Based on the experimental results a respective potential theoretical approach employing the regular-solution theory of Hildebrand and Scatchard is discussed and evaluated critically. Finally, the general practical applicability of naturally occurring radon as an indicator for the quantitative evaluation of NAPL source zones is assessed through laboratory experiments carried out in NAPL-contaminated sand columns. The distinct negative correlation between radon concentration and NAPL saturation of the pore space suggests the general applicability of radon for quantitative estimation of NAPL contamination of aquifers.
Assuntos
Monitoramento Ambiental/métodos , Hidrocarbonetos/análise , Radônio/química , Poluentes Químicos da Água/análise , Poluentes Radioativos da Água/química , Hidrocarbonetos/química , Petróleo , Água/química , Poluentes Químicos da Água/química , Abastecimento de Água/análiseRESUMO
We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, is attacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerase chain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome. These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northern analysis on glass slide microarrays, and hybridized with Cy3- and Cy5-labeled probes derived from plants after 2, 6, 12, and 24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of the cloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcription-polymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins (more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defenses used against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, and oxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses. These defense responses require resources that could be made available by decreases in four photosynthetic-related transcripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associated with carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associated transcripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators, chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration that underscores the complexity of response to herbivore attack.