31 research outputs found

    Nutrition Challenges for Middle-Aged and Older Women

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    During the menopausal transition and postmenopausal periods, women are affected by a variety of symptoms, such as hot flashes, night sweats, vaginal dryness, depression, anxiety, and insomnia. Non-specific somatic symptoms are also common, including muscle and joint pain, tiredness, and dizziness. Some of these effects (particularly vasomotor symptoms and vaginal atrophy) are closely associated with estrogen deficiency, but the exact mechanisms underlying the other symptoms are not fully understood. Postmenopausal women are also at increased risk of cardiovascular morbidity as a net effect of central obesity, dyslipidemia, hypertension, and diabetes, as well as for osteoporosis, cognitive decline, and genitourinary syndrome of menopause. Hormone replacement therapy (HRT) has played a central role in improving menopausal symptoms and reducing the disease risks associated with estrogen deficiency. However, due to growing concern for the side effects of HRT, especially in patients with hormone-sensitive cancer such as breast and uterus cancer, research has turned to the effects of nutraceutical approaches to these symptoms and diseases. In this Special Issue of Nutrients, we would like to bring together manuscripts dealing with the topic of “Nutrition Challenges for Middle-Aged and Older Women”. Topics may include dietary interventions with foods, altered nutrient intake or food supplements, and specific dietary pattern interventions, such as the Mediterranean Diet or calorie restriction, in humans

    Diets, Foods and Food Components Effect on Dyslipidemia

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    Dyslipidemia, and particularly hypercholesterolemia, remains a main cardiovascular disease risk factor, partly reversible with the improvement of life-style, including dietary, habits. Even when a pharmacological treatment is begun, dietary support to lipid-lowering is always desired. This book will provide a selection of new evidence on the possible lipid-lowering effects of some dietary and medicinal plant components, reporting some interesting reviews, experimental data and results from clinical trials. The book is adapted for experts in nutrition but also for all scientists involved in cardiovascular disease prevention

    Cellular Oxidative Stress

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    This book collects 17 original research papers and 9 reviews that are part of the Special Issue “Cellular Oxidative Stress”, published in the journal Antioxidants. Oxidative stress on a cellular level affects the function of tissues and organs and may eventually lead to disease. Therefore, a precise understanding of how oxidative stress develops and can be counteracted is of utmost importance. The scope of the book is to emphasize the latest findings on the cellular targets of oxidative stress and the potential beneficial effect of antioxidants on human health

    Phenolic Compounds

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    Phenolics are commonly available compounds in foods, beverages, and spices. They have great importance in all aspects of daily life including industry, health, and research. As such, this book presents a comprehensive overview of phenolic compounds and their potential applications in industry, environment, and public health. Chapters cover such topics as the production of these compounds and their uses in environmental sustainability, climate change, green industry, and treatment of human disease

    Advances in Ophthalmology

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    This book focuses on the different aspects of ophthalmology - the medical science of diagnosis and treatment of eye disorders. Ophthalmology is divided into various clinical subspecialties, such as cornea, cataract, glaucoma, uveitis, retina, neuro-ophthalmology, pediatric ophthalmology, oncology, pathology, and oculoplastics. This book incorporates new developments as well as future perspectives in ophthalmology and is a balanced product between covering a wide range of diseases and expedited publication. It is intended to be the appetizer for other books to follow. Ophthalmologists, researchers, specialists, trainees, and general practitioners with an interest in ophthalmology will find this book interesting and useful

    Nanoparticle-guided brain drug delivery: Expanding the therapeutic approach to neurodegenerative diseases

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    Neurodegenerative diseases (NDs) represent a heterogeneous group of aging-related disorders featured by progressive impairment of motor and/or cognitive functions, often accompanied by psychiatric disorders. NDs are denoted as ‘protein misfolding’ diseases or proteinopathies, and are classified according to their known genetic mechanisms and/or the main protein involved in disease onset and progression. Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) are included under this nosographic umbrella, sharing histopathologically salient features, including deposition of insoluble proteins, activation of glial cells, loss of neuronal cells and synaptic connectivity. To date, there are no effective cures or disease-modifying therapies for these NDs. Several compounds have not shown efficacy in clinical trials, since they generally fail to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells that greatly limits the brain internalization of endogenous substances. By engineering materials of a size usually within 1–100 nm, nanotechnology offers an alternative approach for promising and innovative therapeutic solutions in NDs. Nanoparticles can cross the BBB and release active molecules at target sites in the brain, minimizing side effects. This review focuses on the state-of-the-art of nanoengineered delivery systems for brain targeting in the treatment of AD, PD and HD. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

    Biochemical and molecular basis involved in the synthesis of melatonin and other derivatives of aromatic amino acids in Saccharomyces cerevisiae

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    Recientemente, el metabolismo de los aminoácidos aromáticos en levaduras se ha relacionado con la síntesis de moléculas bioactivas (melatonina, serotonina, tirosol, hidroxitirosol, etc...) que podrían ser relevantes desde diferentes aspectos relacionados tanto con la regulación de las levaduras como con la salud humana. Las actividades de estos compuestos como potenteantioxidante y otros aspectos beneficiosos para la salud del consumidor hacen que sea realmente interesante estudiar su síntesis. Sin embargo, se conoce poca información sobre la síntesis de estas moléculas bioactivas por la levadura porque es un tema de estudio muy reciente. Por tanto, la hipótesis de trabajo de la presente tesis consiste en que a través de una mejor comprensión del amino aromático vías de metabolismo ácido en la levadura S. cerevisiae, podemos aumentar el contenido de moléculas bioactivas en productos de fermentación alcohólica. Por lo tanto, el metabolismo de la levadura puede aumentar el contenido de moléculas bioactivas en bebidas y alimentos fermentados, lo que podría tener un impacto en la salud del consumidor y que sin duda incrementaría el valor agregado de estas bebidas, posicionándolas mejor en un entorno cada vez más competitivo y global. En este contexto, el objetivo principal de este trabajo de tesis fue estudiar los mecanismos moleculares y fisiológicos implicados en la producción de compuestos bioactivos derivados del metabolismo de los aminoácidos aromáticos, principalmente compuestos derivados del triptófano como la melatonina y la serotonina, en S. cerevisiae. Al inicio de la tesis adaptamos y configuramos una técnica simple, rápida y de bajo costo para detectar la presencia demelatonina en muestras intracelulares de S. cerevisiae basadas en voltamperometría, la cual nos ayudó a determinar la producción de melatonina intracelular. Además, evaluamos el posible efecto de la melatonina intracelular en la cepa de laboratorio BY4743 en presencia y ausencia de H2O2 y radiación UV. Nuestros resultados demuestran que la melatonina intracelular es un antioxidante eficaz y una molécula fotoprotectora para la luz UV en S. cerevisiae. Con respecto a la vía biosintética de la melatonina, aún se desconoce qué actividades o genes están involucrados en esta vía en la levadura. Para desvelar la vía biosintética de la melatonina en S. cerevisiae evaluamos los productos generado a partir de diferentes sustratos de la ruta (L-triptófano, 5-hidroxitriptófano, serotonina, nacetilserotonina, triptamina y 5-metoxitriptamina), en la cepa de levadura de vino QA23 en diferentes etapas de crecimiento por HPLC-MS/MS. Además con el fin de identificar los genes que responden a la síntesis de melatonina en levaduras, realizamos un análisis BLAST utilizando las secuencias de proteínas de animales y plantas. Los genes seleccionados como ortólogos putativos, así como un gen positivo como control (de animales o plantas), se sobreexpresaron tanto en S. cerevisiae y E. coli. Es importante destacar que nuestros resultados sugirieron que el único gen que se ha propuesto como homólogo de la arilalquilamina N-acetiltransferasa de vertebrados en levadura, PAA1, puede no ser el exclusive enzima en la acetilación de arilalquilaminas como serotonina, triptamina y 5-metoxitriptamina en S. cerevisiae. Finalmente, construimos una cepa de S. cerevisiae modificada por integración múltiple en el genoma de E. coli complejo de hidroxilasa HpaBC para producir en exceso hidroxitirosol. Posteriormente, varios cambios en la levadura metabolismo (incluida la sobreexpresión de genes ARO, como ARO3, ARO4, ARO7 y ARO10, y estos mismos genes con mutaciones específicas generadas por mutagenesis dirigida) para aumentar el flujo de la ruta hacia la síntesis de tirosol, el alcohol superior derivado del catabolismo de la tirosina. La combinación de la sobreexpresión de ARO4K229L junto con la integración de HpaBC mostró el mayor efecto en la producción de hidroxitirosol alcanzando 374.5 mg/L en experimentos con matraces excediendo la producción en más de 230.000 veces a la de la cepa control.Recently, the metabolism of aromatic amino acids in yeasts has been linked to the synthesis of bioactive molecules (melatonin, serotonin, tyrosol, hydroxytyrosol, etc.) that could be relevant from different aspects related to both yeast regulation and human health. The activities of these compounds as a potent antioxidant and other aspects beneficial to consumer health makes it really interesting to study its synthesis. However, few information is known about the synthesis of these bioactive molecules by yeast because it is a very recent topic of study. Therefore, the working hypothesis of the present thesis is: Through a better understanding of the aromatic amino acid metabolism routes in S. cerevisiae yeast, we can increase the content of bioactive molecules in products of alcoholic fermentation. Thus, yeast metabolism can increase the content of bioactive molecules in fermented beverages and foods, which could have an impact on the health of the consumer and which will undoubtedly increase the added value of these beverages, better positioning them in an increasingly competitive and global market. In this context, the main objective of this thesis work was to study the molecular and physiological mechanisms involved in the production of bioactive compounds derived from the aromatic amino acid metabolism, mainly tryptophan derived compounds such as melatonin and serotonin, in S. cerevisiae. In this thesis we adapt and set up a simple, rapid, and low-cost technique for detecting the presence of melatonin in S. cerevisiae intracellular samples based on voltammetry. Also, we evaluated the possible effect of melatonin on BY4743 laboratory strain in the presence and absence of H2O2 and UV radiation. Our results demonstrate that intracellular melatonin was an efficient antioxidant and UV protector molecule in S. cerevisiae. Regarding melatonin biosynthetic pathway, it is still far unknown which activities or genes are involved in this pathway in yeast. To unveil the melatonin biosynthetic pathway in S. cerevisiae we evaluated the products generated from different substrates of the route (L-tryptophan, 5-hydroxytryptophan, serotonin, Nacetylserotonin, tryptamine, and 5‐methoxytryptamine), in the wine yeast strain QA23 at different stages of growth by HPLC-MS/MS. Besides in order to identify the genes that respond to the synthesis of melatonin in yeasts, we performed a BLAST analysis using the protein sequences of animals and plants. The genes selected as putative orthologs, as well as a positive gene as a control (from animals or plants), were overexpressed both in S. cerevisiae and E. coli. Importantly, our results suggested that the only gene that has been proposed as homolog of the arylalkylamine N-acetyltransferase of vertebrates in yeast, PAA1, may not be the exclusive enzyme in the acetylation of arylalkylamines such as serotonin, tryptamine and 5-methoxytryptamine in S. cerevisiae. Finally, we constructed a S. cerevisiae strain modified by multiple integration into the genome of the E. coli hydroxylase HpaBC complex in order to overproduce hydroxytyrosol. Subsequently, several changes in yeast metabolism (including overexpression of ARO genes, suchas ARO3, ARO4, ARO7 and ARO10 as well as the specific mutations on some of these genes) were made to increase the flow of the route to the synthesis of tyrosol, the higher alcohol derived from tyrosine catabolism. The combination of ARO4K229L overexpression together with HpaBC integration showed the greatest effect on hydroxytyrosol production achieving 374.5 mg/L in shake flask experiments which exceeded the production by more than 230,000 times that of the control strain

    Drug Discovery

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    Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

    HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE: WHEN STRUCTURE AND MOBILITY DRIVE EFFICIENT CATALYSIS. IMPLICATIONS FOR AADC DEFICIENCY

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    L’enzima Decarbossilasi degli L-amino acidi aromatici (AADC) è responsabile della sintesi di due neurotrasmettitori essenziali: la dopamina e la serotonina. AADC deve la sua attività catalitica alla chimica del suo cofattore, il piridossale 5’-fosfato (PLP). La struttura cristallografica dell’enzima da mammifero (precisamente da maiale che ha il 90% di identità con l’enzima umano) nella sua forma olo venne risolta venti anni fa e tale risoluzione aprì la strada ad importanti studi strutturali. Dieci anni dopo venne pubblicata la struttura umana di AADC nella sua forma apo evidenziando quali cambiamenti conformazionali avvengono quando il PLP viene legato dall’enzima. Le strutture apo e olo AADC hanno avuto notevole importanza per la comprensione della patogenicità di varianti enzimatiche associate alla malattia chiamata ‘Deficit da AADC’ (AADCd, OMIM#608643). Questa malattia autosomica recessiva molto rara è dovuta prevalentemente a mutazioni missenso sul gene AADC. I pazienti affetti da AADCd mostrano un’amAromatic L-Amino Acid Decarboxylase (AADC) is the enzyme responsible for the synthesis of two essential neurotransmitter dopamine and serotonin from L-Dopa and L-hydroxytryptophan. AADC owes its specific catalytic activity to the chemistry of its cofactor, pyrydoxal-5’-phosphate (PLP). Almost 20 years ago, the crystal structure of a mammalian holoAADC (porcine, sharing 90% of sequence identity) was solved and the availability of its 3D structure paved the way to structural studies. Moreover, 10 years later, human apoAADC structure was published, shedding light on the conformational rearrangement occurring on the apo enzyme upon addition of PLP. Importantly, apo and holoAADC structures provided crucial insights for the comprehension of the pathogenicity of a number of AADC deficiency associated variants. AADC deficiency (OMIM#608643) is a rare autosomal recessive inborn disease due to missense mutations in the AADC gene. Patients bearing these mutations show mild to severe phenotypes, whose destiny is often fatal. Due to the rarity of the disease and to the heterogeneous response to the treatments, medications are not often satisfactory. In the past years, some efforts on human recombinant AADC pathogenic variants have tried to provide support to the research on AADC deficiency by means of biochemical and biophysical approaches determining the impact of the amino acid substitutions on the enzyme features. Here, a further contribution to the comprehension of the AADC deficiency is provided. The crystal structure of human holoAADC has been solved under different conditions, both in its native and ligand bound form. The combination of crystallographic studies, molecular dynamics simulations (MD) and site directed mutagenesis uncovered novel aspects of the AADC structure-function relationship. Moreover, the characterization of 21 novel identified pathogenic variants (spread on each AADC domain, N-terminal, Large and C-terminal Domains) led to the widening of the range of enzymatic phenotypes associated to AADC deficiency. The proposed combination of biochemical and kinetic studies permitted to determine correlations between structural and functional signals. Enzymatic phenotypes span from variants characterized by a mild phenotypes to variants (mainly located at the NTD-CTD interface) whose dramatic structural defects lead to a catalytic incompetence. In addition, MD simulations and in solutions data point out a critical role for the loop3 element that contains the essential catalytic residue Tyr332. A group of variants affecting loop3 has been identified as catalytically incompetent and their structural features have been dissected thanks also to the solving of the crystal structure of pathogenic variant L353P, which constitutes the first solved structure of an AADC variant. Altogether, this study on human AADC provides new elements for the comprehension of the structure-function relationship of AADC with a particular focus on protein dynamics and mobility. Lastly, structural details might represent the basis for both the designing of novel specific inhibitors and for a better comprehension of the molecular aspects of the variants associated with the AADC deficiency

    Development of new Catechol-Omethyltransferase inhibitors

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    The Catechol-O-methyltransferase (COMT, EC 2.2.1.6) is an enzyme responsible for the Omethylation of catechol substrates, such as catecholamines and catechol estrogens. Considering its physiological functions and the existence of polymorphisms, several studies associate COMT with the pathogenesis of several neurological disorders, especially with Parkinson’s Disease (PD) as well as with cardiovascular and hormone-dependent cancers, like breast cancers. Given the important role that COMT has in the catecholamines and catechol estrogens metabolism, COMT has become a relevant therapeutic target. Currently, the most effective and clinically approved by the Federal Drug Administration and the European Medicines Agency for the PD therapy consists of the use of Levodopa, combined with COMT inhibitors. Since the commercially available inhibitors for this enzyme still display a lot of disadvantages, like hepatoxicity, difficulty to reach the brain, among others, the main goal of this work was to develop new COMT inhibitors with potential clinical interest for the PD therapy. For this, triazolopyrimidinics were prepared through the Biginelli reaction, that can be considered catechol bioisosteres, therefore have a higher potential to interact with COMT. This hypothesis was confirmed through molecular docking, being predicted similar interactions as the ones that the catecholic substrates forms with the COMT active site. Their inhibitory properties were evaluated in human recombinant COMT lysates, after the compounds’ incubation at 10 and 100 µM. Contrary to what was expected, the compounds increased the enzyme specific activity, being considered COMT stabilizers. The compounds cytotoxicity was also evaluated in neural dopaminergic rat cells (N27), in the same concentrations. The vast majority of compounds at 10 µM did not exhibited cytotoxicity, being observed similar values to those of the commercial COMT inhibitors, Entacapone and Tolcapone, in the studied cell line. As expected, with the increase in compounds’ concentration (100 µM) a decrease in the relative cell proliferation was observed, reaching values considered to be cytotoxic. Altogether, the synthesized compounds at the concentration of 10 µM stabilized COMT and did not induce cytotoxicity in the N27 cells. In sum, these compounds may be useful for thermal stability assays, crystallography, structure-activity relationship studies and display potential to be studied in specific breast cancers cell lines.A Catecol-O-metiltransferase (COMT, EC 2.2.1.6)) é a enzima responsável pela O-metilação de substratos catecólicos, como as catecolaminas e os estrogénios com estrutura catecólica. Considerando as suas funções fisiológicas e a existência de polimorfismos, vários estudos associam a COMT com a patogénese de várias desordens neurológicas, especialmente com a Doença de Parkinson (DP) e também com doenças cardiovasculares e cancros hormonodependentes, como cancros da mama. Devido à importância que a COMT tem no metabolismo das catecolaminas e dos estrogénios catecólicos, a COMT tornou-se nas últimas décadas num importante alvo terapêutico. Atualmente, a terapia mais eficaz e clinicamente aprovadas pela Federal Drug Administration e European Medicines Agency para a doença de Parkinson consiste no uso de Levodopa, combinada com inibidores da COMT. Uma vez que os inibidores desta enzima comercialmente disponíveis ainda apresentam diversas desvantagens, como hepatotoxicidade, dificuldade em alcançar o cérebro, entre outras, o objetivo principal deste trabalho foi desenvolver novos inibidores da COMT com potencial clínico para a terapia da DP. Para isto, foram preparados triazolopirimidínicos através da reação de Biginelli, os quais podem ser considerados bioisósteros de catecóis, podendo por isso ter potencial para interagir com a COMT. Esta hipótese foi confirmada através de docking molecular, prevendo-se interações moleculares semelhantes às dos substratos catecóis com o centro ativo da COMT. As suas propriedades inibitórias foram avaliadas em lisados recombinantes da enzima, após incubação dos compostos nas concentrações de 10 e 100 µM. Contrariamente ao expectável, os compostos aumentaram a atividade específica da enzima, podendo ser considerados estabilizadores da COMT. Foi ainda avaliada a citoxicidade dos mesmos em células dopaminérgicas neuronais de rato (N27) nas mesmas concentrações. A grande maioria dos compostos a 10 µM não mostrou citotoxicidade, observando-se valores semelhantes aos dos inibidores comerciais da COMT, Entacapone e Tolcapone, na linha celular N27. Como esperado com o aumento da concentração (100 µM) ocorreu um decréscimo na proliferação celular, atingindo valores já considerados citotóxicos. No geral, os compostos sintetizados, na concentração de 10 µM estabilizaram a COMT e não induziram citoxicidade nas células N27. Em suma, as moléculas sintetizadas podem ser úteis para estudos de estabilidade térmica, de cristalografia, de relação estrutura-atividade e apresentam potencialidade para ser estudados em linhas celulares especificas do cancro da mama
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