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BACKGROUND: Peak calling is a fundamental step in the analysis of data generated by ChIP-seq or similar techniques to acquire epigenetics information. Current peak callers are often hard to parameterise and may therefore be difficult to use for non-bioinformaticians. In this paper, we present the ChIP-seq analysis tool available in CLC Genomics Workbench and CLC Genomics Server (version 7.5 and up), a user-friendly peak-caller designed to be not specific to a particular *-seq protocol. RESULTS: We illustrate the advantages of a shape-based approach and describe the algorithmic principles underlying the implementation. Thanks to the generality of the idea and the fact the algorithm is able to learn the peak shape from the data, the implementation requires only minimal user input, while still being applicable to a range of *-seq protocols. Using independently validated benchmark datasets, we compare our implementation to other state-of-the-art algorithms explicitly designed to analyse ChIP-seq data and provide an evaluation in terms of receiver-operator characteristic (ROC) plots. In order to show the applicability of the method to similar *-seq protocols, we also investigate algorithmic performances on DNase-seq data. CONCLUSIONS: The results show that CLC shape-based peak caller ranks well among popular state-of-the-art peak callers while providing flexibility and ease-of-use.
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Algoritmos , Genómica/métodos , Área Bajo la Curva , Inmunoprecipitación de Cromatina , Bases de Datos Genéticas , Humanos , Internet , Curva ROC , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Interfaz Usuario-ComputadorRESUMEN
Pattern recognition receptors (PRRs) induce host defense but can also induce exacerbated inflammatory responses. This raises the question of whether other mechanisms are also involved in early host defense. Using transcriptome analysis of disrupted transcripts in herpes simplex virus (HSV)-infected cells, we find that HSV infection disrupts the hypoxia-inducible factor (HIF) transcription network in neurons and epithelial cells. Importantly, HIF activation leads to control of HSV replication. Mechanistically, HIF activation induces autophagy, which is essential for antiviral activity. HSV-2 infection in vivo leads to hypoxia in CNS neurons, and mice with neuron-specific HIF1/2α deficiency exhibit elevated viral load and augmented PRR signaling and inflammatory gene expression in the CNS after HSV-2 infection. Data from human stem cell-derived neuron and microglia cultures show that HIF also exerts antiviral and inflammation-restricting activity in human CNS cells. Collectively, the HIF transcription factor system senses virus-induced hypoxic stress to induce cell-intrinsic antiviral responses and limit inflammation.
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Encefalitis , Herpes Simple , Humanos , Animales , Ratones , Inflamación , Neuronas , Hipoxia , Antivirales/farmacologíaRESUMEN
SUMMARY: Contact maps are a valuable visualization tool in structural biology. They are a convenient way to display proteins in two dimensions and to quickly identify structural features such as domain architecture, secondary structure and contact clusters. We developed a tool called CMView which integrates rich contact map analysis with 3D visualization using PyMol. Our tool provides functions for contact map calculation from structure, basic editing, visualization in contact map and 3D space and structural comparison with different built-in alignment methods. A unique feature is the interactive refinement of structural alignments based on user selected substructures. AVAILABILITY: CMView is freely available for Linux, Windows and MacOS. The software and a comprehensive manual can be downloaded from http://www.bioinformatics.org/cmview/. The source code is licensed under the GNU General Public License.
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Conformación Proteica , Programas Informáticos , Gráficos por Computador , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. METHODS: We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ("kinobeads"). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. RESULTS: We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. CONCLUSIONS: We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Factor de Crecimiento Transformador beta/metabolismo , Benzoquinonas/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Proteínas HSP90 de Choque Térmico/química , Humanos , Lactamas Macrocíclicas/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteómica , Transducción de Señal/efectos de los fármacosRESUMEN
CONTEXT: Women with Turner syndrome (TS) suffer from hypergonadotropic hypogonadism, causing a deficit in gonadal hormone secretion. As a consequence, these women are treated with estrogen from the age of 12 years, and later in combination with progesterone. However, androgens have been given less attention. OBJECTIVE: To assess sex hormone levels in women with TS, both those treated and those nontreated with hormone replacement therapy (HRT), and investigate the impact of HRT on sex hormone levels. METHODS: At Aarhus University Hospital, 99 women with TS were followed 3 times from August 2003 to February 2010. Seventeen were lost during follow-up. Control group 1 consisted of 68 healthy age-matched control women seen once during this period. Control group 2 consisted of 28 young, eumenorrheic women sampled 9 times throughout the same menstrual cycle. Serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17ß-estradiol, estrone sulfate, DHEAS, testosterone, free androgen index, androstenedione, 17-OH progesterone, and sex hormone-binding globulin (SHBG) were analyzed. RESULTS: All androgens, 17-OH progesterone, and sex hormone-binding globulin (SHBG) were 30% to 50% lower in TS compared with controls (P < 0.01). FSH, LH, and estrone sulfate were more than doubled in women with TS compared with controls (P < 0.02). Using principal component analysis, we describe a positive correlation between women with TS receiving HRT, elevated levels of SHBG, and decreased levels of androgens. CONCLUSION: The sex hormone profile in TS reveals a picture of androgen deficiency, aggravated further by HRT. Conventional HRT does not normalize estradiol levels in TS.
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Andrógenos , Estrógenos , Terapia de Reemplazo de Hormonas , Síndrome de Turner , Andrógenos/deficiencia , Estradiol , Estrógenos/deficiencia , Femenino , Hormona Folículo Estimulante , Hormonas Esteroides Gonadales/uso terapéutico , Humanos , Hormona Luteinizante , Progesterona/uso terapéutico , Globulina de Unión a Hormona Sexual/análisis , Testosterona , Síndrome de Turner/tratamiento farmacológicoRESUMEN
BACKGROUND: Current large-scale cancer sequencing projects have identified large numbers of somatic mutations covering an increasing number of different cancer tissues and patients. However, the characterization of these mutations at the structural and functional level remains a challenge. RESULTS: We present results from an analysis of the structural impact of frequent missense cancer mutations using an automated method. We find that inactivation of tumor suppressors in cancer correlates frequently with destabilizing mutations preferably in the core of the protein, while enhanced activity of oncogenes is often linked to specific mutations at functional sites. Furthermore, our results show that this alteration of oncogenic activity is often associated with mutations at ATP or GTP binding sites. CONCLUSIONS: With our findings we can confirm and statistically validate the hypotheses for the gain-of-function and loss-of-function mechanisms of oncogenes and tumor suppressors, respectively. We show that the distinct mutational patterns can potentially be used to pre-classify newly identified cancer-associated genes with yet unknown function.
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Mutación Missense/genética , Neoplasias/genética , Neoplasias/patología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Bases de Datos Genéticas , Humanos , Modelos Genéticos , Modelos Moleculares , Anotación de Secuencia Molecular , Estructura Molecular , Polimorfismo de Nucleótido Simple/genética , Estabilidad ProteicaRESUMEN
After the completion of the human and other genome projects it emerged that the number of genes in organisms as diverse as fruit flies, nematodes, and humans does not reflect our perception of their relative complexity. Here, we provide reliable evidence that the size of protein interaction networks in different organisms appears to correlate much better with their apparent biological complexity. We develop a stable and powerful, yet simple, statistical procedure to estimate the size of the whole network from subnet data. This approach is then applied to a range of eukaryotic organisms for which extensive protein interaction data have been collected and we estimate the number of interactions in humans to be approximately 650,000. We find that the human interaction network is one order of magnitude bigger than the Drosophila melanogaster interactome and approximately 3 times bigger than in Caenorhabditis elegans.
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Mapeo de Interacción de Proteínas , Animales , Caenorhabditis elegans/metabolismo , Bases de Datos de Proteínas , Drosophila melanogaster/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Contact maps have been extensively used as a simplified representation of protein structures. They capture most important features of a protein's fold, being preferred by a number of researchers for the description and study of protein structures. Inspired by the model's simplicity many groups have dedicated a considerable amount of effort towards contact prediction as a proxy for protein structure prediction. However a contact map's biological interest is subject to the availability of reliable methods for the 3-dimensional reconstruction of the structure. RESULTS: We use an implementation of the well-known distance geometry protocol to build realistic protein 3-dimensional models from contact maps, performing an extensive exploration of many of the parameters involved in the reconstruction process. We try to address the questions: a) to what accuracy does a contact map represent its corresponding 3D structure, b) what is the best contact map representation with regard to reconstructability and c) what is the effect of partial or inaccurate contact information on the 3D structure recovery. Our results suggest that contact maps derived from the application of a distance cutoff of 9 to 11A around the Cbeta atoms constitute the most accurate representation of the 3D structure. The reconstruction process does not provide a single solution to the problem but rather an ensemble of conformations that are within 2A RMSD of the crystal structure and with lower values for the pairwise average ensemble RMSD. Interestingly it is still possible to recover a structure with partial contact information, although wrong contacts can lead to dramatic loss in reconstruction fidelity. CONCLUSIONS: Thus contact maps represent a valid approximation to the structures with an accuracy comparable to that of experimental methods. The optimal contact definitions constitute key guidelines for methods based on contact maps such as structure prediction through contacts and structural alignments based on maximum contact map overlap.
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Biología Computacional/métodos , Proteínas/química , Modelos Moleculares , Conformación Proteica , Pliegue de ProteínaRESUMEN
The network of native non-covalent residue contacts determines the three-dimensional structure of a protein. However, not all contacts are of equal structural significance, and little knowledge exists about a minimal, yet sufficient, subset required to define the global features of a protein. Characterisation of this "structural essence" has remained elusive so far: no algorithmic strategy has been devised to-date that could outperform a random selection in terms of 3D reconstruction accuracy (measured as the Ca RMSD). It is not only of theoretical interest (i.e., for design of advanced statistical potentials) to identify the number and nature of essential native contacts-such a subset of spatial constraints is very useful in a number of novel experimental methods (like EPR) which rely heavily on constraint-based protein modelling. To derive accurate three-dimensional models from distance constraints, we implemented a reconstruction pipeline using distance geometry. We selected a test-set of 12 protein structures from the four major SCOP fold classes and performed our reconstruction analysis. As a reference set, series of random subsets (ranging from 10% to 90% of native contacts) are generated for each protein, and the reconstruction accuracy is computed for each subset. We have developed a rational strategy, termed "cone-peeling" that combines sequence features and network descriptors to select minimal subsets that outperform the reference sets. We present, for the first time, a rational strategy to derive a structural essence of residue contacts and provide an estimate of the size of this minimal subset. Our algorithm computes sparse subsets capable of determining the tertiary structure at approximately 4.8 A Ca RMSD with as little as 8% of the native contacts (Ca-Ca and Cb-Cb). At the same time, a randomly chosen subset of native contacts needs about twice as many contacts to reach the same level of accuracy. This "structural essence" opens new avenues in the fields of structure prediction, empirical potentials and docking.
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Biología Computacional/métodos , Modelos Moleculares , Conformación Proteica , Proteínas/química , Algoritmos , Bases de Datos de Proteínas , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de ProteínasRESUMEN
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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Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.
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Antiinflamatorios/farmacología , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Dimetilfumarato/agonistas , Factor 2 Relacionado con NF-E2/metabolismo , Neumonía Viral/tratamiento farmacológico , Succinatos/agonistas , Adulto , Antioxidantes/farmacología , Betacoronavirus/metabolismo , COVID-19 , Infecciones por Coronavirus/virología , Dimetilfumarato/farmacología , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Interferón Tipo I , Pulmón/patología , Masculino , Factor 2 Relacionado con NF-E2/genética , Pandemias , Neumonía Viral/virología , SARS-CoV-2 , Transducción de Señal/efectos de los fármacos , Succinatos/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Multi-omics approaches use a diversity of high-throughput technologies to profile the different molecular layers of living cells. Ideally, the integration of this information should result in comprehensive systems models of cellular physiology and regulation. However, most multi-omics projects still include a limited number of molecular assays and there have been very few multi-omic studies that evaluate dynamic processes such as cellular growth, development and adaptation. Hence, we lack formal analysis methods and comprehensive multi-omics datasets that can be leveraged to develop true multi-layered models for dynamic cellular systems. Here we present the STATegra multi-omics dataset that combines measurements from up to 10 different omics technologies applied to the same biological system, namely the well-studied mouse pre-B-cell differentiation. STATegra includes high-throughput measurements of chromatin structure, gene expression, proteomics and metabolomics, and it is complemented with single-cell data. To our knowledge, the STATegra collection is the most diverse multi-omics dataset describing a dynamic biological system.
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Linfocitos B , Diferenciación Celular , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Línea Celular , Genómica , Metabolómica , Ratones , ProteómicaRESUMEN
BACKGROUND: Identifying the active site of an enzyme is a crucial step in functional studies. While protein sequences and structures can be experimentally characterized, determining which residues build up an active site is not a straightforward process. In the present study a new method for the detection of protein active sites is introduced. This method uses local network descriptors derived from protein three-dimensional structures to determine whether a residue is part of an active site. It thus does not involve any sequence alignment or structure similarity to other proteins. A scoring function is elaborated over a set of more than 220 proteins having different structures and functions, in order to detect protein catalytic sites with a high precision, i.e. with a minimal rate of false positives. RESULTS: The scoring function was based on the counts of first-neighbours on side-chain contacts, third-neighbours and residue type. Precision of the detection using this function was 28.1%, which represents a more than three-fold increase compared to combining closeness centrality with residue surface accessibility, a function which was proposed in recent years. The performance of the scoring function was also analysed into detail over a smaller set of eight proteins. For the detection of 'functional' residues, which were involved either directly in catalytic activity or in the binding of substrates, precision reached a value of 72.7% on this second set. These results suggested that our scoring function was effective at detecting not only catalytic residues, but also any residue that is part of the functional site of a protein. CONCLUSION: As having been validated on the majority of known structural families, this method should prove useful for the detection of active sites in any protein with unknown function, and for direct application to the design of site-directed mutagenesis experiments.
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Biología Computacional/métodos , Algoritmos , Animales , Catálisis , Dominio Catalítico , Humanos , Modelos Biológicos , Modelos Estadísticos , Conformación Molecular , Conformación Proteica , Pliegue de Proteína , Reproducibilidad de los Resultados , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , Ubiquitina/químicaRESUMEN
BACKGROUND: For over 30 years potentials of mean force have been used to evaluate the relative energy of protein structures. The most commonly used potentials define the energy of residue-residue interactions and are derived from the empirical analysis of the known protein structures. However, single-body residue 'environment' potentials, although widely used in protein structure analysis, have not been rigorously compared to these classical two-body residue-residue interaction potentials. Here we do not try to combine the two different types of residue interaction potential, but rather to assess their independent contribution to scoring protein structures. RESULTS: A data set of nearly three thousand monomers was used to compare pairwise residue-residue 'contact-type' propensities to single-body residue 'contact-count' propensities. Using a large and standard set of protein decoys we performed an in-depth comparison of these two types of residue interaction propensities. The scores derived from the contact-type and contact-count propensities were assessed using two different performance metrics and were compared using 90 different definitions of residue-residue contact. Our findings show that both types of score perform equally well on the task of discriminating between near-native protein decoys. However, in a statistical sense, the contact-count based scores were found to carry more information than the contact-type based scores. CONCLUSION: Our analysis has shown that the performance of either type of score is very similar on a range of different decoys. This similarity suggests a common underlying biophysical principle for both types of residue interaction propensity. However, several features of the contact-count based propensity suggests that it should be used in preference to the contact-type based propensity. Specifically, it has been shown that contact-counts can be predicted from sequence information alone. In addition, the use of a single-body term allows for efficient alignment strategies using dynamic programming, which is useful for fold recognition, for example. These facts, combined with the relative simplicity of the contact-count propensity, suggests that contact-counts should be studied in more detail in the future.
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Aminoácidos/química , Proteínas/química , Bases de Datos de Proteínas , TermodinámicaRESUMEN
In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.
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The functional characterization of genes and their gene products is the main challenge of the genomic era. Examining interaction information for every gene product is a direct way to assemble the jigsaw puzzle of proteins into a functional map. Here we demonstrate a method in which the information gained from pull-down experiments, in which single proteins act as baits to detect interactions with other proteins, is maximized by using a network-based strategy to select the baits. Because of the scale-free distribution of protein interaction networks, we were able to obtain fast coverage by focusing on highly connected nodes (hubs) first. Unfortunately, locating hubs requires prior global information about the network one is trying to unravel. Here, we present an optimized 'pay-as-you-go' strategy that identifies highly connected nodes using only local information that is collected as successive pull-down experiments are performed. Using this strategy, we estimate that 90% of the human interactome can be covered by 10,000 pull-down experiments, with 50% of the interactions confirmed by reciprocal pull-down experiments.
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Biología Computacional/métodos , Proteínas/fisiología , Algoritmos , Bases de Datos como Asunto , Humanos , Modelos Biológicos , Modelos Teóricos , Unión Proteica , Proteoma , Saccharomyces cerevisiae/metabolismo , Programas Informáticos , Factores de TiempoRESUMEN
A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).
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Sistemas de Administración de Bases de Datos/normas , Bases de Datos de Proteínas/normas , Almacenamiento y Recuperación de la Información/normas , Mapeo de Interacción de Proteínas/normas , Proteínas/clasificación , Proteómica/normas , Interfaz Usuario-Computador , Guías como Asunto , Almacenamiento y Recuperación de la Información/métodos , Internacionalidad , Procesamiento de Lenguaje Natural , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteómica/métodos , Estándares de Referencia , Programas InformáticosRESUMEN
Protein sequence alignments are more reliable the shorter the evolutionary distance. Here, we align distantly related proteins using many closely spaced intermediate sequences as stepping stones. Such transitive alignments can be generated between any two proteins in a connected set, whether they are direct or indirect sequence neighbors in the underlying library of pairwise alignments. We have implemented a greedy algorithm, MaxFlow, using a novel consistency score to estimate the relative likelihood of alternative paths of transitive alignment. In contrast to traditional profile models of amino acid preferences, MaxFlow models the probability that two positions are structurally equivalent and retains high information content across large distances in sequence space. Thus, MaxFlow is able to identify sparse and narrow active-site sequence signatures which are embedded in high-entropy sequence segments in the structure based multiple alignment of large diverse enzyme superfamilies. In a challenging benchmark based on the urease superfamily, MaxFlow yields better reliability and double coverage compared to available sequence alignment software. This promises to increase information returns from functional and structural genomics, where reliable sequence alignment is a bottleneck to transferring the functional or structural characterization of model proteins to entire protein superfamilies.
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Secuencias de Aminoácidos , Biología Computacional , Análisis de Secuencia de Proteína , Actinas/genética , Algoritmos , Secuencia de Aminoácidos , ADN Polimerasa beta/genética , Datos de Secuencia Molecular , Familia de Multigenes , Alineación de Secuencia , Ureasa/genéticaRESUMEN
Recultivation of disturbed oil sand mining areas is an issue of increasing importance. Nevertheless only little is known about the fate of organic matter, cell abundances and microbial community structures during oil sand processing, tailings management and initial soil development on reclamation sites. Thus the focus of this work is on biogeochemical changes of mined oil sands through the entire process chain until its use as substratum for newly developing soils on reclamation sites. Therefore, oil sand, mature fine tailings (MFTs) from tailings ponds and drying cells and tailings sand covered with peat-mineral mix (PMM) as part of land reclamation were analyzed. The sample set was selected to address the question whether changes in the above-mentioned biogeochemical parameters can be related to oil sand processing or biological processes and how these changes influence microbial activities and soil development. GC-MS analyses of oil-derived biomarkers reveal that these compounds remain unaffected by oil sand processing and biological activity. In contrast, changes in polycyclic aromatic hydrocarbon (PAH) abundance and pattern can be observed along the process chain. Especially naphthalenes, phenanthrenes and chrysenes are altered or absent on reclamation sites. Furthermore, root-bearing horizons on reclamation sites exhibit cell abundances at least ten times higher (10(8) to 10(9) cells g(-1)) than in oil sand and MFT samples (10(7) cells g(-1)) and show a higher diversity in their microbial community structure. Nitrate in the pore water and roots derived from the PMM seem to be the most important stimulants for microbial growth. The combined data show that the observed compositional changes are mostly related to biological activity and the addition of exogenous organic components (PMM), whereas oil extraction, tailings dewatering and compaction do not have significant influences on the evaluated compounds. Microbial community composition remains relatively stable through the entire process chain.
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Consorcios Microbianos , Minería , Yacimiento de Petróleo y Gas/microbiología , Residuos Industriales/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Contaminantes Químicos del Agua/análisisRESUMEN
Hydrocarbons can be found in many different habitats and represent an important carbon source for microbes. As fossil fuels, they are also an important economical resource and through natural seepage or accidental release they can be major pollutants. DNA-specific stains and molecular probes bind to hydrocarbons, causing massive background fluorescence, thereby hampering cell enumeration. The cell extraction procedure of Kallmeyer et al. (2008) separates the cells from the sediment matrix. In principle, this technique can also be used to separate cells from oily sediments, but it was not originally optimized for this application. Here we present a modified extraction method in which the hydrocarbons are removed prior to cell extraction. Due to the reduced background fluorescence the microscopic image becomes clearer, making cell identification, and enumeration much easier. Consequently, the resulting cell counts from oily samples treated according to our new protocol are significantly higher than those treated according to Kallmeyer et al. (2008). We tested different amounts of a variety of solvents for their ability to remove hydrocarbons and found that n-hexane and - in samples containing more mature oils - methanol, delivered the best results. However, as solvents also tend to lyse cells, it was important to find the optimum solvent to sample ratio, at which hydrocarbon extraction is maximized and cell lysis minimized. A volumetric ratio of 1:2-1:5 between a formalin-fixed sediment slurry and solvent delivered highest cell counts. Extraction efficiency was around 30-50% and was checked on both oily samples spiked with known amounts of E. coli cells and oil-free samples amended with fresh and biodegraded oil. The method provided reproducible results on samples containing very different kinds of oils with regard to their degree of biodegradation. For strongly biodegraded oil MeOH turned out to be the most appropriate solvent, whereas for less biodegraded samples n-hexane delivered best results.