A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates

Funding Information: The authors acknowledge the support from FEDER funds through the POCI-COMPETE 2020–Operational Programme Competitiveness and Internationalisation in Axis I–Strengthening Research, Technological Development and Innovation (Project POCI-01-0145-FEDER-007491), Jorge Barroca-Ferreira's and Ana M. Gonçalves's individual PhD Fellowships (SFRH/BD/130068/2017 and SFRH/BD/147519/2019, respectively), and Luís A. Passarinha's sabbatical fellowship (SFRH/BSAB/150376/2019) from FCT–Fundação para a Ciência e Tecnologia. This work was also supported by the Health Sciences Research Centre CICS-UBI (UIDB/00709/2020 and UIDP/00709/2020), the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy–i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Publisher Copyright: © 2022The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-β-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.publishersversionpublishe

Contribuição dos Sistemas Agrosilvopastoris na Captação de Carbono

The Kyoto Protocol, entered into force in February 2005, emphasizes in Article 12 that projects with Clean Development Mechanism must be chosen to meet both the global concern of climatic changes and the national interests of sustainable development. After more than ten years, however, these goals still need to be adjusted to practice. In view of this necessity, this review seeks to elucidate some aspects related to the use of agroforestry systems associated with carbon sequestration, aiming to extend to farmers the perspectives of sustainable development associated with a new possibility of income source.O Protocolo de Kyoto, que entrou em vigor em fevereiro de 2005, enfatiza em seu 12º artigo que projetos sob o Mecanismo de Desenvolvimento Limpo devem ser selecionados para atender tanto à preocupação internacional com as mudanças climáticas quanto aos interesses nacionais de desenvolvimento sustentável. Passados mais de 10 anos,  entretanto, estes objetivos ainda precisam ser adequados à prática. Tendo em vista essa necessidade a presente revisão busca elucidar alguns aspectos relacionados à utilização de sistemas agrossilvopastoris associados ao seqüestro de carbono, de modo a ampliar ao produtor rural as perspectivas de desenvolvimento sustentável aliadas a uma nova possibilidade de fonte de renda

3-amino-1,2,4-triazole induces quick and strong fat loss in mice with high fat-induced metabolic syndrome

BACKGROUND: Obesity is a growing epidemic with limited effective treatments and an important risk factor for several diseases such as metabolic syndrome (MetS). In this study, we aimed to investigate the effect of 3-amino-1,2,4-triazole (ATZ), an inhibitor of catalase and heme synthesis, in a murine model for MetS. METHODS: Male C57BL/6 mice with high-fat diet-induced MetS received ATZ (500 mg·kg(-1)·24 h(-1)) for 12 weeks. RESULTS: The HFD group showed increased blood pressure and body weight, enhanced fat deposition accompanied by an increase in adipocyte diameter, and decreased lipolysis in white adipose tissue (WAT). The expression of genes related to inflammation was increased in WAT of the HFD group. Concurrently, these mice exhibited an increase in leptin, nonesterified fatty acid (NEFA), insulin, and glucose in plasma, coupled with glucose intolerance and insulin resistance. Strikingly, ATZ prevented the increase in blood pressure and the HFD-induced obesity as observed by lower body weight, WAT index, triglycerides, NEFA, and leptin in plasma. ATZ treatment also prevented the HFD-induced increase in adipocyte diameter and even induced marked atrophy and the accumulation of macrophages in this tissue. ATZ treatment also improved glucose metabolism by increasing glucose tolerance and insulin sensitivity, GLUT4 mRNA expression in WAT in parallel to decreased insulin levels. CONCLUSIONS: In the context of HFD-induced obesity and metabolic syndrome, the fat loss induced by ATZ is probably due to heme synthesis inhibition, which blocks adipogenesis by probably decreased RevErbα activity, leading to apoptosis of adipocytes and the recruitment of macrophages. As a consequence of fat loss, ATZ elicits a beneficial systemic antiobesity effect and improves the metabolic status

Demand and supply of outdoor tourism activities in Northern Portugal: a survey-based approach

This paper focuses on the North of Portugal, as a diversified region with unique natural resources, to create information regarding both resources and equipment and business dynamics; the evolution of tourism supply and demand.This research is a part of a project title “TURNOUT: Desenvolvimento do Turismo Outdoor da Região Norte de Portugal”, with the reference POCI-01-0145-FEDER-032289; funded by the European Regional Development Fund (FEDER) (through the Operational Programme ‘Innovation and competitiveness’) and by the Portuguese Foundation for the Development of Science and Technology (FCT), of the Ministry of Science, Technology and Higher Education. This work is, also, funded by National Funds through the Foundation for Science and Technology under the project UIDB/04752/2020. The SABI database was made available by the Applied Management Research Unit (UNIAG), according to the protocol between UNIAG and COFACE.info:eu-repo/semantics/publishedVersio

Dynamical Breakdown of Symmetry in a (2+1) Dimensional Model Containing the Chern-Simons Field

We study the vacuum stability of a model of massless scalar and fermionic fields minimally coupled to a Chern-Simons field. The classical Lagrangian only involves dimensionless parameters, and the model can be thought as a (2+1) dimensional analog of the Coleman-Weinberg model. By calculating the effective potential, we show that dynamical symmetry breakdown occurs in the two-loop approximation. The vacuum becomes asymmetric and mass generation, for the boson and fermion fields takes place. Renormalization group arguments are used to clarify some aspects of the solution.Comment: Minor modifications in the text and figure

Proteomic landscape of extracellular vesicles for diffuse large b‐cell lymphoma subtyping

Funding Information: R.M. is supported by Funda??o para a Ci?ncia e a Tecnologia (CEEC position, 2019?2025 investigator). This article is a result of the projects (iNOVA4Health?UID/Multi/04462/2013), supported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT?Portuguese Foundation for Science and Technology under the projects number PTDC/BTM?TEC/30087/2017 and PTDC/BTM?TEC/30088/2017. B.C.S. is supported by the Cham-palimaud Foundation and the EMBO Installation Grant 3921. Funding Information: Funding: R.M. is supported by Fundação para a Ciência e a Tecnologia (CEEC position, 2019–2025 investigator). This article is a result of the projects (iNOVA4Health—UID/Multi/04462/2013), sup‐ ported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT—Portuguese Foundation for Science and Technology under the projects number PTDC/BTM‐TEC/30087/2017 and PTDC/BTM‐TEC/30088/2017. B.C.S. is supported by the Cham‐ palimaud Foundation and the EMBO Installation Grant 3921. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.The role of extracellular vesicles (EVs) proteome in diffuse large B‐cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state‐of‐the‐art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole‐cell and secreted EVs proteomes of the two cell‐of‐origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole‐cell proteome and 228/608 proteins from EVs (adjust p‐value < 0.05/p‐value < 0.05). In our preclinical model system, we demonstrated that the EV prote-ome and the whole‐cell proteome possess the capacity to separate cell lines into ABC and GCB sub-types. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B‐cell receptor (BCR), Fc_gamma R‐mediated phagocytosis, ErbB signaling, and endocyto-sis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow‐up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.publishersversionpublishe

Secondary forest fragments offer important carbon‐biodiversity co‐benefits

Tropical forests store large amounts of carbon and high biodiversity, but are being degraded at alarming rates. The emerging global Forest and Landscape Restoration (FLR) agenda seeks to limit global climate change by removing carbon dioxide from the atmosphere through the growth of trees. In doing so, it may also protect biodiversity as a free co‐benefit, which is vital given the massive shortfall in funding for biodiversity conservation. We investigated whether natural forest regeneration on abandoned pastureland offers such co‐benefits, focusing for the first time on the recovery of taxonomic, phylogenetic and functional diversity of trees, including the recovery of threatened and endemic species richness, within isolated secondary forest fragments. We focused on the globally threatened Brazilian Atlantic Forest, where commitments have been made to restore one million hectares under FLR. Three decades after land abandonment, regenerating forests had recovered ~20% (72 Mg/ha−1) of the above‐ground carbon stocks of a primary forest, with cattle pasture containing just 3% of stocks relative to primary forests. Over this period, secondary forest recovered ~76% of taxonomic, 84% of phylogenetic and 96% of functional diversity found within primary forests. In addition, secondary forests had on average recovered 65% of threatened and ~30% of endemic species richness of primary Atlantic forest. Finally, we find positive relationships between carbon stock and tree diversity recovery. Our results emphasize that secondary forest fragments offer co‐benefits under FLR and other carbon‐based payments for ecosystem service schemes (e.g. carbon enhancements under REDD +). They also indicate that even isolated patches of secondary forest could help to mitigate climate change and the biodiversity extinction crisis by recovering species of high conservation concern and improving landscape connectivity