13 research outputs found

    Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases

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    Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases‚Äô biodiversity and structure‚Äďfunction relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C2 to C16). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C12 and C16, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure‚Äďfunction analysis. All five enzymes showed a decrease in kcat values with increasing substrate chain length, whereas KM values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low KM values, resulting in high catalytic efficiencies toward pNP-C16. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops

    Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica

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    Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure‚Äďfunction relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme\u27s surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically

    Paraglomus pernambucanum sp. nov. and Paraglomus bolivianum comb. nov., and biogeographic distribution of Paraglomus and Pacispora

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    Paraglomus pernambucanum sp. nov. (Paraglomeromycetes) was found in a tropical dry forest in the semi-arid Caatinga biome of Pernambuco State (NE Brazil), in a cowpea and in two maize production sites. It was characterized by combined morphological and molecular analyses on the spores isolated from field soil samples. Another species, Pacispora boliviana (Glomeromycetes), first described only by spore morphology, had been known from another semi-arid biome in Southern America, the Gran Chaco in Bolivia. We detected this fungus now also at different locations in semi-arid to semi-humid NE Brazil. As for P. pernambucanum phylogenetic analyses were performed on nuclear ribosomal RNA gene sequences of the LSU region. For P. boliviana, the spores for these analyses originated from a trap culture inoculated with soils from the type location. The results now revealed that also P. boliviana belongs to Paraglomus. It grouped in a separate monophyletic cluster adjacent to P. pernambucanum, to P. brasilianum, P. laccatum and the type species P. occultum. Thus, P. boliviana is transferred to Paraglomus, as Paraglomus bolivianum comb. nov. Remarkably, it is the first species known in the Paraglomeromycetes with pigmented spores. Paraglomus pernambucanum and P. bolivianum have several features in common: e.g. bi-walled spores, and densely pitted surface ornamentations on the structural layer of the outer wall. Spores of the two species can be distinguished by color and the diagnostic nature of their pitted ornamentation. The current knowledge about the global distribution of Paraglomus and Pacispora species is summarized and discussed

    Abstracts from the Food Allergy and Anaphylaxis Meeting 2016

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    Immunohistochemical molecular phenotypes of gastric cancer based on SOX2 and CDX2 predict patient outcome

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    Background Gastric cancer remains a serious health concern worldwide. Patients would greatly benefit from the discovery of new biomarkers that predict outcome more accurately and allow better treatment and follow-up decisions. Here, we used a retrospective, observational study to assess the expression and prognostic value of the transcription factors SOX2 and CDX2 in gastric cancer. Methods SOX2, CDX2, MUC5AC and MUC2 expression were assessed in 201 gastric tumors by immunohistochemistry. SOX2 and CDX2 expression were crossed with clinicopathological and follow-up data to determine their impact on tumor behavior and outcome. Moreover, SOX2 locus copy number status was assessed by FISH (N‚ÄČ=‚ÄČ21) and Copy Number Variation Assay (N‚ÄČ=‚ÄČ62). Results SOX2 was expressed in 52% of the gastric tumors and was significantly associated with male gender, T stage and N stage. Moreover, SOX2 expression predicted poorer patient survival, and the combination with CDX2 defined two molecular phenotypes, SOX2+CDX2- versus SOX2-CDX2+, that predict the worst and the best long-term patients‚Äô outcome. These profiles combined with clinicopathological parameters stratify the prognosis of patients with intestinal and expanding tumors and in those without signs of venous invasion. Finally, SOX2 locus copy number gains were found in 93% of the samples reaching the amplification threshold in 14% and significantly associating with protein expression. Conclusions We showed, for the first time, that SOX2 combined with CDX2 expression profile in gastric cancer segregate patients into different prognostic groups, complementing the clinicopathological information. We further demonstrate a molecular mechanism for SOX2 expression in a subset of gastric cancer cases.This work was supported by Project NORTE - 07-0124-FEDER-000024 co-financed by Programa Operacional Regional do Norte (ON.2 ‚Äď O Novo Norte), under Quadro de Refer√™ncia Estrat√©gico Nacional (QREN), by Fundo Europeu de Desenvolvimento Regional (FEDER). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT. V.C. and R.B. acknowledge FCT for financial support (grant numbers SFRH/BD/63300/2009 and SFRH/BPD/68276/2010, respectively)

    AMAZONIA CAMTRAP: A data set of mammal, bird, and reptile species recorded with camera traps in the Amazon forest

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    The Amazon forest has the highest biodiversity on Earth. However, information on Amazonian vertebrate diversity is still deficient and scattered across the published, peer-reviewed, and gray literature and in unpublished raw data. Camera traps are an effective non-invasive method of surveying vertebrates, applicable to different scales of time and space. In this study, we organized and standardized camera trap records from different Amazon regions to compile the most extensive data set of inventories of mammal, bird, and reptile species ever assembled for the area. The complete data set comprises 154,123 records of 317 species (185 birds, 119 mammals, and 13 reptiles) gathered from surveys from the Amazonian portion of eight countries (Brazil, Bolivia, Colombia, Ecuador, French Guiana, Peru, Suriname, and Venezuela). The most frequently recorded species per taxa were: mammals: Cuniculus paca (11,907 records); birds: Pauxi tuberosa (3713 records); and reptiles: Tupinambis teguixin (716 records). The information detailed in this data paper opens up opportunities for new ecological studies at different spatial and temporal scales, allowing for a more accurate evaluation of the effects of habitat loss, fragmentation, climate change, and other human-mediated defaunation processes in one of the most important and threatened tropical environments in the world. The data set is not copyright restricted; please cite this data paper when using its data in publications and we also request that researchers and educators inform us of how they are using these data
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