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SUMMARY: Mutation-Simulator allows the introduction of various types of sequence alterations in reference sequences, with reasonable compute-time even for large eukaryotic genomes. Its intuitive system for fine-grained control over mutation rates along the sequence enables the mimicking of natural mutation patterns. Using standard file formats for input and output data, it can easily be integrated into any development and benchmarking workflow for high-throughput sequencing applications. AVAILABILITY AND IMPLEMENTATION: Mutation-Simulator is written in Python 3 and the source code, documentation, help and use cases are available on the Github page at https://github.com/mkpython3/Mutation-Simulator. It is free for use under the GPL 3 license.
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Genoma , Software , Simulação por Computador , Sequenciamento de Nucleotídeos em Larga Escala , MutaçãoRESUMO
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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The success of reef-building corals for >200 million years has been dependent on the mutualistic interaction between the coral host and its photosynthetic endosymbiont dinoflagellates (family Symbiodiniaceae) that supply the coral host with nutrients and energy for growth and calcification. While multiple light scattering in coral tissue and skeleton significantly enhance the light microenvironment for Symbiodiniaceae, the mechanisms of light propagation in tissue and skeleton remain largely unknown due to a lack of technologies to measure the intrinsic optical properties of both compartments in live corals. Here we introduce ISOCT (inverse spectroscopic optical coherence tomography), a non-invasive approach to measure optical properties and three-dimensional morphology of living corals at micron- and nano-length scales, respectively, which are involved in the control of light propagation. ISOCT enables measurements of optical properties in the visible range and thus allows for characterization of the density of light harvesting pigments in coral. We used ISOCT to characterize the optical scattering coefficient (µs) of the coral skeleton and chlorophyll a concentration of live coral tissue. ISOCT further characterized the overall micro- and nano-morphology of live tissue by measuring differences in the sub-micron spatial mass density distribution (D) that vary throughout the tissue and skeleton and give rise to light scattering, and this enabled estimates of the spatial directionality of light scattering, i.e., the anisotropy coefficient, g. Thus, ISOCT enables imaging of coral nanoscale structures and allows for quantifying light scattering and pigment absorption in live corals. ISOCT could thus be developed into an important tool for rapid, non-invasive monitoring of coral health, growth and photophysiology with unprecedented spatial resolution.
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Antozoários/fisiologia , Monitoramento Ambiental/métodos , Tomografia de Coerência Óptica/métodos , Absorção de Radiação , Animais , Antozoários/química , Recifes de Corais , Difusão Dinâmica da Luz/métodosRESUMO
Irradiance and temperature variations during tidal cycles modulate microphytobenthic primary production potentially by changing the radiative energy balance of photosynthetic mats between immersion and emersion and thus sediment daily net metabolism. To test the effect of tidal stages on the radiative energy budget, we used microsensor measurements of oxygen, temperature, and scalar irradiance to estimate the radiative energy budget in a coastal photosynthetic microbial mat during immersion (constant water column of 2 cm) and emersion under increasing irradiance. Total absorbed light energy was higher in immersion than emersion, due to a lower reflectance of the microbial mat, while most (> 97%) of the absorbed light energy was dissipated as heat irrespective of tidal conditions. During immersion, the upward heat flux was higher than the downward one, whereas the opposite occurred during emersion. At highest photon irradiance (800 µmol photon m-2 s-1), the sediment temperature increased ~ 2.5 °C after changing the conditions from immersion to emersion. The radiative energy balance showed that less than 1% of the incident light energy (PAR, 400-700 nm) was conserved by photosynthesis under both tidal conditions. At low to moderate incident irradiances, the light use efficiency was similar during the tidal stages. In contrast, we found an ~ 30% reduction in the light use efficiency during emersion as compared to immersion under the highest irradiance likely due to the rapid warming of the sediment during emersion and increased non-photochemical quenching. These changes in the photosynthetic efficiency and radiative energy budget could affect both primary producers and temperature-dependent bacterial activity and consequently daily net metabolism rates having important ecological consequences.
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Fenômenos Fisiológicos Bacterianos , Sedimentos Geológicos/microbiologia , Água do Mar/microbiologia , Luz Solar , Ondas de Maré , DinamarcaRESUMO
Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O(2)) due to production of superoxide (O(2)(-)). In this study, we show that the PMNs also consume O(2) for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N(G) -monomethyl-L-arginine (L-NMMA) resulted in reduced O(2) consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO(3)(-) (P < 0·04, r = 0·66, n = 10) and NO(2)(-) (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O(2) for production of reactive oxygen species, the PMNs in CF sputum also consume O(2) for production of NO.
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Fibrose Cística/metabolismo , Pulmão/metabolismo , Neutrófilos/imunologia , Óxido Nítrico/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/patologia , Escarro/metabolismo , Adulto , Células Cultivadas , Doença Crônica , Fibrose Cística/complicações , Fibrose Cística/imunologia , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/microbiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Consumo de Oxigênio , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/imunologia , Adulto Jovem , ômega-N-Metilarginina/farmacologiaRESUMO
Long-term measurements of molecular oxygen (O) dynamics in wetlands are highly relevant for understanding the effects of water level changes on net greenhouse gas budgets in these ecosystems. However, such measurements have been limited due to a lack of suitable measuring equipment. We constructed an O optode sensor array for long-term in situ measurements in soil and sediment. The new device consists of a 1.3-m-long, cylindrical, spear-shaped rod equipped with 10 sensor spots along the shaft. Each spot contains a thermocouple fixed with a robust fiberoptic O optode made by immobilizing a layer of Pt(II) meso-tetra(pentafluorophenyl)porphine in polystyrene at the end of a 2-mm polymethyl methacrylate plastic fiber. Temperature and O optode readings are collected continuously by a data logger and a multichannel fiberoptic O meter. The construction and measuring characteristics of the sensor array system are presented along with a novel approach for temperature compensation of O optodes. During in situ application over several months in a peat bog, we used the new device to document pronounced variations in O distribution after marked shifts in water level. The measurements showed anoxic conditions below the water level but also diel variations in O concentrations in the upper layer presumably due to rhizospheric oxidation by the main vegetation The new field instrument thus enables new and more detailed insights to the in situ O dynamics in wetlands.
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Oxigênio , Solo , Ecossistema , Água , Áreas AlagadasRESUMO
A ratiometric luminescence intensity imaging approach is presented, which enables spatial O2 measurements in biofilm reactors with transparent planar O2 optodes. Optodes consist of an O2 sensitive luminescent dye immobilized in a 1-10 µm thick polymeric layer on a transparent carrier, e.g. a glass window. The method is based on sequential imaging of the O2 dependent luminescence intensity, which are subsequently normalized with luminescent intensity images recorded under anoxic conditions. We present 2-dimensional O2 distribution images at the base of a tap water biofilm measured with the new ratiometric method and compare the results with O2 distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O2 distributions with a high signal-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O2 concentration is depleted during incubation.
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Biofilmes , Oxigênio/análise , Abastecimento de Água , Microbiologia da ÁguaRESUMO
New transparent optodes for life-time based microscopic imaging of O2 were developed by spin-coating a µm-thin layer of a highly luminescent cyclometalated iridium(III) coumarin complex in polystyrene onto glass cover slips. Compared to similar thin-film O2 optodes based on a ruthenium(II) polypyridyl complex or a platinum(II) porphyrin, the new planar sensors have i) higher brightness allowing for much shorter exposure times and thus higher time resolution, ii) more homogeneous and smaller pixel to pixel variation over the sensor area resulting in less noisy O2 images, and iii) a lower temperature dependency simplifying calibration procedures. We used the new optodes for microscopic imaging of the spatio-temporal O2 dynamics at the base of heterotrophic biofilms in combination with confocal imaging of bacterial biomass and biofilm structure. This allowed us to directly link biomass distribution to O2 distribution under both steady state and non-steady state conditions. We demonstrate that the O2 dynamics in biofilms is governed by a complex interaction between biomass distribution, mass transfer and flow that cannot be directly inferred from structural information on biomass distribution alone.
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Bactérias/química , Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Medições Luminescentes/métodos , Microscopia/métodos , Oxigênio/análise , BiomassaRESUMO
A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.
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Cianobactérias/classificação , Cianobactérias/genética , Ecossistema , Genômica , Meio Ambiente , Genética PopulacionalRESUMO
Bone metastases from a vulvar carcinoma are exceptionally rare with only five reported cases in the literature. We report on a patient who was initially treated with radical vulvectomy and bilateral inguinal lymphadenectomy for a vulvar cancer (pT2, pN2 (6/37), M0; G2). Due to a positive nodal status, adjuvant radiation of the vulva and the pelvis was performed additionally. The patient presented 4 months after initial therapy with severe pain in the right humeral shaft due to a pathologic fracture based on an osteoclastic metastasis. During osteosynthetic stabilization histologic and immunohistochemical stain gave evidence of a metastasis of the known vulvar carcinoma. Bone scan showed enhancements in both humeral heads as well as the right distal femur, whereas plain radiographs confirmed further metastases in all suspected areas. In conclusion, bone metastases should be considered in the differential diagnoses of unclear osseous pain in women with a history of vulvar cancer. Immunohistochemical examinations might be important to depict the epithelial character of the tissue and allude to the metastatic nature of such rare lesions. The atypical location should alert the physician to suspect distant metastasis, rather than locoregional disease.
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Neoplasias Ósseas/patologia , Carcinoma de Células Escamosas/patologia , Neoplasias Vulvares/patologia , Idoso , Neoplasias Ósseas/secundário , Neoplasias Ósseas/terapia , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/terapia , Feminino , Procedimentos Cirúrgicos em Ginecologia , Humanos , Estadiamento de Neoplasias , Radioterapia Adjuvante , Neoplasias Vulvares/terapiaRESUMO
Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to < 1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of > or = 49 degrees C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of < or = 39 degrees C.
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Chlorella/classificação , Ecossistema , Temperatura Alta , Luz , Rodófitas/classificação , Rios/microbiologia , Chlorella/genética , Chlorella/isolamento & purificação , Chlorella/fisiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fotossíntese , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Rodófitas/genética , Rodófitas/isolamento & purificação , Rodófitas/fisiologia , Estações do Ano , Análise de Sequência de DNA , TemperaturaRESUMO
In situ microsensor measurements were combined with biogeochemical methods to determine oxygen, sulfur, and carbon cycling in microbial mats growing in a solar saltern (Salin-de-Giraud, France). Sulfate reduction rates closely followed the daily temperature changes and were highest during the day at 25 degrees C and lowest during the night at 11 degrees C, most probably fueled by direct substrate interactions between cyanobacteria and sulfate-reducing bacteria. Sulfate reduction was the major mineralization process during the night and the contribution of aerobic respiration to nighttime DIC production decreased. This decrease of aerobic respiration led to an increasing contribution of sulfide (and iron) oxidation to nighttime O2 consumption. A peak of elemental sulfur in a layer of high sulfate reduction at low sulfide concentration underneath the oxic zone indicated anoxygenic photosynthesis and/or sulfide oxidation by iron, which strongly contributed to sulfide consumption. We found a significant internal carbon cycling in the mat, and sulfate reduction directly supplied DIC for photosynthesis. The mats were characterized by a high iron content of 56 micromol Fe cm(-3), and iron cycling strongly controlled the sulfur cycle in the mat. This included sulfide precipitation resulting in high FeS contents with depth, and reactions of iron oxides with sulfide, especially after sunset, leading to a pronounced gap between oxygen and sulfide gradients and an unusual persistence of a pH peak in the uppermost mat layer until midnight.
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Carbono/metabolismo , Cianobactérias/metabolismo , Oxigênio/metabolismo , Água do Mar/microbiologia , Enxofre/metabolismo , Fenômenos Bioquímicos , Bioquímica , Cianobactérias/fisiologia , França , Fenômenos Geológicos , Geologia , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Oxirredução , Fotossíntese/fisiologia , TemperaturaRESUMO
Some old persons at risk do develop, but others, at comparable risk, do not develop high-grade pressure sores. To evaluate potentially different risk factors, we performed a post mortem case-control study in old persons who developed high-grade pressure sores within six months until 14 days before death. Consecutive cases with pressure sores grade >/=3 and potential controls at comparably high risk for pressure sores were examined before cremation. After written informed consent had been obtained by the next relatives, all available nursing and medical records of the deceased were thoroughly evaluated. Cases and controls were matched according to age, gender, immobility, and cachexia.A total of 100 cases with 71 pressure sores grade 3 and 29 pressure sores grade 4 were compared to 100 controls with 27 pressure sores grade =2 and maximal risk as assessed by the Norton scale. The mean age was 86 years, 80% were females, 86% were bedridden, and cachexia was found in 66%. The cases were more often seriously disabled, had more often contractions of joints, and were on more drugs with intended or unwanted depressive/sedative effects (neuroleptics, benzodiazepines, centrally acting analgesics). Patient's impaired ability or low intention to comply/cooperate with preventive and/or therapeutic measures was more often recorded in cases than controls.A high degree of disablement and immobility, in particular, are risk factors for high-grade pressure sores in frail older high-risk persons. Sedative drug effects and impaired patient compliance with preventive and therapeutic measures may also be associated with the development of high-grade pressure sores in old persons at high risk.
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Idoso de 80 Anos ou mais/estatística & dados numéricos , Idoso Fragilizado/estatística & dados numéricos , Úlcera por Pressão/diagnóstico , Úlcera por Pressão/epidemiologia , Medição de Risco/métodos , Idoso/estatística & dados numéricos , Estudos de Casos e Controles , Morte , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Úlcera por Pressão/classificação , Valores de Referência , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Mass coral bleaching is linked to elevated sea surface temperatures, 1-2 degrees C above average, during periods of intense light. These conditions induce the expulsion of zooxanthellae from the coral host in response to photosynthetic damage in the algal symbionts. The mechanism that triggers this release has not been clearly established and to further our knowledge of this process, fluorescence rise kinetics have been studied for the first time. Corals that were exposed to elevated temperature (33 degrees C) and light (280 mumol photons m(-2) s(-1)), showed distinct changes in the fast polyphasic induction of chlorophyll-a fluorescence, indicating biophysical changes in the photochemical processes. The fluorescence rise over the first 2000ms was monitored in three species of corals for up to 8 h, with a PEA fluorometer and an imaging-PAM. Pocillopora damicornis showed the least impact on photosynthetic apparatus, while Acropora nobilis was the most sensitive, with Cyphastrea serailia intermediate between the other two species. A. nobilis showed a remarkable capacity for recovery from bleaching conditions. For all three species, a steady decline in the slope of the initial rise and the height of the J-transient was observed, indicating the loss of functional Photosystem II (PS II) centres under elevated-temperature conditions. A significant loss of PS II centres was confirmed by a decline in photochemical quenching when exposed to bleaching stress. Non-photochemical quenching was identified as a significant mechanism for dissipating excess energy as heat under the bleaching conditions. Photophosphorylation could explain this decline in PS II activity. State transitions, a component of non-photochemical quenching, was a probable cause of the high non-photochemical quenching during bleaching and this mechanism is associated with the phosphorylation-induced dissociation of the light harvesting complexes from the PS II reaction centres. This reversible process may account for the coral recovery, particularly in A. nobilis.
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The microenvironment and community composition of microbial mats developing on beaches in Scapa Flow (Orkney Islands) were investigated. Analysis of characteristic biomarkers (major fatty acids, hydrocarbons, alcohols, and alkenones) revealed the presence of different groups of bacteria and microalgae in mats from Waulkmill and Swanbister beach, including diatoms, Haptophyceae, cyanobacteria, and sulfate-reducing bacteria. These analyses also indicated the presence of methanogens, especially in Swanbister beach mats, and therefore a possible role of methanogenesis for the carbon cycle of these sediments. High amounts of algal lipids and slightly higher numbers (genera, abundances) of cyanobacteria were found in Waulkmill Bay mats. However, overall only a few genera and low numbers of unicellular and filamentous cyanobacteria were present in mats from Waulkmill and Swanbister beach, as deduced from CLSM (confocal laser scanning microscopy) analysis. Spectral scalar irradiance measurements with fiber-optic microprobes indicated a pronounced heterogeneity concerning zonation and density of mainly anoxygenic phototrophs in Swanbister Bay mats. By microsensor and T-RFLP (terminal restriction fragment length polymorphism) analysis in Swanbister beach mats, the depth distribution of different populations of purple and sulfate-reducing bacteria could be related to the microenvironmental conditions. Oxygen, but also sulfide and other (inorganic and organic) sulfur compounds, seems to play an important role in the stratification and diversity of these two major bacterial groups involved in sulfur cycling in Swanbister beach mats.
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Bactérias/química , Biodiversidade , Microbiologia Ambiental , Eucariotos/química , Euryarchaeota/química , Cromatografia Gasosa , Análise por Conglomerados , Primers do DNA , Geografia , Microscopia Confocal , Fotossíntese , Polimorfismo de Fragmento de Restrição , Escócia , EspectrofotometriaRESUMO
BACKGROUND: We retrospectively compared the influence of surface finish with respect to the long-term durability of a cemented titanium alloy stem of the same design but different surface finish. METHODS: From 1984 to 1994, the stem was made of a titanium-aluminum-vanadium alloy. A total of 201 patients with 220 smooth stems (mean follow-up 11 years and 4 months) out of 612 implanted between 1984 and 1987 and 319 patients with 343 rough stems (mean follow-up 5 years and 10 months) out of 812 implanted between 1991 and 1993 could be reviewed clinically and radiologically for comparison. The average age of the patients with the smooth stem was 58.1 years and of the patients with the rough stem, 62.2 years. In both groups, 35% of patients were male and 65% female. RESULTS: The mean Merle d'Aubigné hip score increased from 10.5 to 16.0 points in the smooth stem group and from 11.0 to 16.9 points in the rough stem group. The distribution of radiolucent lines, according to the zones of Gruen, was similar in both groups. The smooth stem required revision in 18 cases after a mean follow-up of 11 years and 4 months and the rough stem in 30 cases after a mean follow-up of 5 years and 10 months. The survival analysis (Kaplan-Meier) revealed 95.4% survival after 13 years for the smooth femoral component and 76.7% survival after 8 years for the rough femoral component. CONCLUSIONS: The implantation of titanium alloy stems with a rough surface finish cannot be recommended because of its high aseptic loosening rate.
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Prótese de Quadril , Titânio , Ligas , Artroplastia de Quadril , Cimentos Ósseos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Estudos Retrospectivos , Propriedades de Superfície , Resultado do TratamentoRESUMO
The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an approximately 500-microm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at approximately 10 microM O(2). Flux calculations yielded a molar conversion ratio S(tot)/O(2) of 2.03:1 (S(tot) = [H(2)S] + [HS(-)] + [S(2-)]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 microM O(2). The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 microM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.
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Chromatiaceae/fisiologia , Luz , Oxigênio/farmacologia , Sulfetos/farmacologia , Chromatiaceae/citologia , Chromatiaceae/efeitos dos fármacos , Escuridão , Microscopia de Vídeo , Movimento/efeitos dos fármacos , Movimento/fisiologia , OxirreduçãoRESUMO
Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.
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Chlorobi/classificação , Chlorobi/genética , Ecossistema , Cloreto de Sódio , Microbiologia da Água , Chlorobi/crescimento & desenvolvimento , Meios de Cultura , Primers do DNA , Genes de RNAr , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.
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Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais/genética , Transativadores , Proteínas de Peixe-Zebra , Animais , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Receptores Frizzled , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Relacionadas a Receptor de LDL , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Microinjeções , Modelos Biológicos , Oócitos/fisiologia , Testes de Precipitina , Ligação Proteica , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , beta CateninaRESUMO
Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/beta-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/beta-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/beta-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/beta-catenin pathway upstream of beta-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/beta-catenin signaling cascade downstream of beta-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.