Human phenylalanine hydroxylase as the case study

Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. The authors would like to thank Luís Miguel Ramos and Cátia Nascimento who contributed to the exploratory research that culminated in the work herein presented. Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. Publisher Copyright: © 2023 The Author(s)The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.publishersversionpublishe

Metabolic coupling in urothelial bladder cancer compartments and its correlation to tumour aggressiveness

Monocarboxylate transporters (MCTs) are vital for intracellular pH homeostasis by extruding lactate from highly glycolytic cells. These molecules are key players of the metabolic reprogramming of cancer cells, and evidence indicates a potential contribution in urothelial bladder cancer (UBC) aggressiveness and chemoresistance. However, the specific role of MCTs in the metabolic compartmentalization within bladder tumors, namely their preponderance on the tumor stroma, remains to be elucidated. Thus, we evaluated the immunoexpression of MCTs in the different compartments of UBC tissue samples (n = 111), assessing the correlations among them and with the clinical and prognostic parameters. A significant decrease in positivity for MCT1 and MCT4 occurred from normoxic toward hypoxic regions. Significant associations were found between the expression of MCT4 in hypoxic tumor cells and in the tumor stroma. MCT1 staining in normoxic tumor areas, and MCT4 staining in hypoxic regions, in the tumor stroma and in the blood vessels were significantly associated with UBC aggressiveness. MCT4 concomitant positivity in hypoxic tumor cells and in the tumor stroma, as well as positivity in each of these regions concomitant with MCT1 positivity in normoxic tumor cells, was significantly associated with an unfavourable clinicopathological profile, and predicted lower overall survival rates among patients receiving platinum-based chemotherapy. Our results point to the existence of a multi-compartment metabolic model in UBC, providing evidence of a metabolic coupling between catabolic stromal and cancer cells' compartments, and the anabolic cancer cells. It is urgent to further explore the involvement of this metabolic coupling in UBC progression and chemoresistance.This study was supported by the Life and Health Sciences Research Institute (ICVS) from the School of Health Sciences of the University of Minho. JA received a postdoctoral fellowship from ICVS (reference ICVS-SSRL: ON.2 SR&TD Integrated Program, NORTE-07-0124-FEDER-000017).info:eu-repo/semantics/publishedVersio

Organic Farming e-book

A agricultura biológica está-se a tornar cada vez mais importante como um caminho preferencial para a produção de produtos agrícolas, face à crescente procura do mercado mundial. A relevância da agricultura biológica é ainda maior, devido à necessidade e procura de produtos agrícolas de origem biológica, que são isentos de produtos químicos, saudáveis e amigos do ambiente. Atualmente, a agricultura biológica resulta em produtos de valor acrescentado, mas estes sistemas de produção exigem abordagens especializadas. Verifica-se uma lacuna de conhecimento especializado para enfrentar os desafios e exigências da agricultura biológica. Além disso, um número crescente de pessoas com níveis elevados de educação está a mudar a sua atividade para a agricultura sem qualquer tipo de formação nesta área técnica, principalmente em países com dificuldades económicas, como Portugal. Há, portanto, necessidade de desenvolver a capacidade de pessoas com algum tipo de qualificação prévio, a fim de melhorar suas competências agrícolas e facilitar a sua capacidade de desempenho e inovação, para que possam contribuir para a Estratégia Europeia (CE) de Desenvolvimento Rural. Este e-book foi concebido para melhorar as competências desses agricultores. O seu objectivo geral é dotar os novos agricultores com conhecimentos e capacidades necessários para o desenvolvimento da cadeia de valor dos produtos da agricultura biológica. Os objectivos específicos são: i) Fornecer conhecimento básico em vários aspectos da agricultura biológica e áreas afins, tais como a gestão de recursos naturais (solo, água, plantas, ambiente) e desenvolvimento rural (conservação, agricultura biológica e familiar, multifuncionalidade). ii) Facilitar a troca efetiva de conhecimento e experiências em agricultura biológica, desenvolvimento rural e ambiente. iii) Oferecer suporte técnico e conhecimento em agricultura biológica num contexto de mobilidade e em ambiente de trabalho. Este e-livro, produzido em sete idiomas diferentes (Português, Inglês, Espanhol, Italiano, Eslovaco, Turco e Húngaro) também contribui para preservar línguas e culturas Europeias e, assim, melhorar a comunicação entre os diferentes intervenientes e grupos-alvo. O e-book inclui os princípios e técnicas da agricultura biológica, com base no triângulo planta-solo-ambiente e nas relações entre a produção animal e o ambiente. A preparação de alimentos e rotulagem, marketing e conversão à agricultura biológica são também abordados. Os princípios e as técnicas apresentadas são explicados com base em regras e diretrizes (normas), baseados numa abordagem logística que garante o equilíbrio e integridade do sistema. O e-book apresenta também os regulamentos e normas nacionais e Europeias que são obrigatórias para os agricultores biológicos.info:eu-repo/semantics/publishedVersio

Quality of life and social support of the patients of continued care units of Algarve

This study assessed the relations between quality of life and social support network of patients in continuing care units in Algarve, using WHOQOL-BREF and ASSIS. Participants were 92 users of ages between 34-101 years: very elderly (40.2%), female (58.7%), widows (40.2%), upper-middle class (46.8%), cohabiting with spouse (43.2%), and partly dependent (72.5%). Emotional, tangible and informational support networks were mainly composed by family. We found significant associations between informative social support and physical domains, social relations and environment of quality of life (p< 0.05). The results suggest middle levels in self-perception and satisfaction with health, and lower levels in physical domain. Informational support needs were negatively associated with psychological and social relations dimensions of quality of life

Gi/o-protein coupled receptors in the aging brain

Cells translate extracellular signals to regulate processes such as differentiation, metabolism and proliferation, via transmembranar receptors. G protein-coupled receptors (GPCRs) belong to the largest family of transmembrane receptors, with over 800 members in the human species. Given the variety of key physiological functions regulated by GPCRs, these are main targets of existing drugs. During normal aging, alterations in the expression and activity of GPCRs have been observed. The central nervous system (CNS) is particularly affected by these alterations, which results in decreased brain functions, impaired neuroregeneration, and increased vulnerability to neuropathologies, such as Alzheimer's and Parkinson diseases. GPCRs signal via heterotrimeric G proteins, such as Go, the most abundant heterotrimeric G protein in CNS. We here review age-induced effects of GPCR signaling via the Gi/o subfamily at the CNS. During the aging process, a reduction in protein density is observed for almost half of the Gi/o-coupled GPCRs, particularly in age-vulnerable regions such as the frontal cortex, hippocampus, substantia nigra and striatum. Gi/o levels also tend to decrease with aging, particularly in regions such as the frontal cortex. Alterations in the expression and activity of GPCRs and coupled G proteins result from altered proteostasis, peroxidation of membranar lipids and age-associated neuronal degeneration and death, and have impact on aging hallmarks and age-related neuropathologies. Further, due to oligomerization of GPCRs at the membrane and their cooperative signaling, down-regulation of a specific Gi/o-coupled GPCR may affect signaling and drug targeting of other types/subtypes of GPCRs with which it dimerizes. Gi/o-coupled GPCRs receptorsomes are thus the focus of more effective therapeutic drugs aiming to prevent or revert the decline in brain functions and increased risk of neuropathologies at advanced ages.This work was supported by Fundação para a Ciência e Tecnologia, Centro 2020 and Portugal 2020, the COMPETE program, QREN, and the European Union (FEDER program) via the GoBack project (PTDC/CVT-CVT/32261/2017), the pAGE program (Centro-01-0145-FEDER-000003), and Institute for Biomedicine iBiMED (UID/BIM/04501/2013; UID/BIM/04501/2019).publishe

The Dual Paradigm of Mining Waste: &ldquo;From Ecotoxicological Sources to Potential Polymetallic Resources&rdquo;&mdash;An Example from Iberian Pyrite Belt (Portugal)

The main goal of this study was to identify potential chemical elements present in three types of polymetallic mine waste, stored in the old mine site of S&atilde;o Domingos, located in the Iberian Pyrite Belt, Alentejo, Southern Region of Portugal. This study involves the characterization of potential resources in those mine residues, bearing in mind that its reprocessing can facilitate the environmental remediation and rehabilitation activities which are underway at the site. X-ray Fluorescence (XRF) and micro (&mu;)-XRF 2D mapping surveys were performed. Univariate and multivariate data analysis reveal that differences in compositions are mainly related with element concentration per type of waste. Image processing and clustering analysis allowed the recognition of distinct elemental spatial distribution patterns. Some of these residues, although classified as archeological-industrial heritage materials may present toxicity to the ecological environment and to human health. This fact enhances, therefore, geoethical doubts regarding its remining and exploitability. In this context, a multi-criteria decision analysis considering two geoethical alternatives was performed

The Dual Paradigm of Mining Waste: “From Ecotoxicological Sources to Potential Polymetallic Resources”—An Example from Iberian Pyrite Belt (Portugal)

The main goal of this study was to identify potential chemical elements present in three types of polymetallic mine waste, stored in the old mine site of São Domingos, located in the Iberian Pyrite Belt, Alentejo, Southern Region of Portugal. This study involves the characterization of potential resources in those mine residues, bearing in mind that its reprocessing can facilitate the environmental remediation and rehabilitation activities which are underway at the site. X-ray Fluorescence (XRF) and micro (μ)-XRF 2D mapping surveys were performed. Univariate and multivariate data analysis reveal that differences in compositions are mainly related with element concentration per type of waste. Image processing and clustering analysis allowed the recognition of distinct elemental spatial distribution patterns. Some of these residues, although classified as archeological-industrial heritage materials may present toxicity to the ecological environment and to human health. This fact enhances, therefore, geoethical doubts regarding its remining and exploitability. In this context, a multi-criteria decision analysis considering two geoethical alternatives was performed

In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process