Exploring the physiological role of transthyretin in glucose metabolism in the liver

Transthyretin (TTR), a 55 kDa evolutionarily conserved protein, presents altered levels in several conditions, including malnutrition, inflammation, diabetes, and Alzheimer’s Disease. It has been shown that TTR is involved in several functions, such as insulin release from pancreatic ß-cells, recovery of blood glucose and glucagon levels of the islets of Langerhans, food intake, and body weight. Here, the role of TTR in hepatic glucose metabolism was explored by studying the levels of glucose in mice with different TTR genetic backgrounds, namely with two copies of the TTR gene, TTR+/+; with only one copy, TTR+/-; and without TTR, TTR-/-. Results showed that TTR haploinsufficiency (TTR+/-) leads to higher glucose in both plasma and in primary hepatocyte culture media and lower expression of the influx glucose transporters, GLUT1, GLUT3, and GLUT4. Further, we showed that TTR haploinsufficiency decreases pyruvate kinase M type (PKM) levels in mice livers, by qRT-PCR, but it does not affect the hepatic production of the studied metabolites, as determined by 1H NMR. Finally, we demonstrated that TTR increases mitochondrial density in HepG2 cells and that TTR insufficiency triggers a higher degree of oxidative phosphorylation in the liver. Altogether, these results indicate that TTR contributes to the homeostasis of glucose by regulating the levels of glucose transporters and PKM enzyme and by protecting against mitochondrial oxidative stress.This work was supported by Norte-01-0145-FEDER-000008-Porto Neurosciences and Neurologic Disease Research Initiative at I3S, and Pest-OE/SAU/UI0215/2014 at UMIB, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, by COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, through the European Regional Development Fund (FEDER), by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and by a grant from Fundação Millennium bcp. Alemi M was a recipient of fellowship by Norte-01-0145-FEDER-000008. Oliveira  was a recipient of fellowship by Norte-01-0145-FEDER-000008. Cardoso I, Oliveira PF, and Alves MG work under the Investigator FCT Program, which is financed by national funds through the Foundation for Science and Technology and co-financed by the European Social Fund (ESF) through the Human Potential Operational Programme (HPOP), type 4.2—Promotion of Scientific Employment

Screening of Dementia in Portuguese Primary Care: Methodology, Assessment Tools, and Main Results

The objectives of this article are as follows: (1) to describe the assessment protocol used to outline people with probable dementia in Primary Health Care; (2) to show the methodological design and procedure to obtain a representative sample of patients with probable dementia; and (3) to report the main characteristics of the sample collected in the context of the study “Characteristics and needs of people with probable dementia.” The study protocol was based on the “Community Assessment of Risk and Treatment Strategies (CARTS) Program” and is composed by a set of instruments that allow the assessment of older adults with probable dementia in several areas (health, psychological, functionality, and other). Descriptive analysis was used to characterize the final sample (n = 436). The study protocol as well as the methodological procedure to obtain the referral of research participants and data collection on the condition of people with probable dementia in Primary Health Care proved to be a valuable tool to obtain a sample of patients distributed by the full range of probable dementia in a large geographical area. Results may allocate the design of care pathways for old people with cognitive disorders to prevent, delay impairment, and/or optimize quality of life of patients

Sperm, metabolic memory and echoes from Lamarck

There is a widespread misconception that only maternal variables affect in utero development.Epigenetic markers carried by the spermatozoon are transmitted to the zygote.Sperm-born epigenetic factors influence in utero development, for various generations.Acquired traits of metabolic disease can be inherited by the offspring via the male gamete.Health assessment of future fathers is essential to predict the offspring's health.</ul

A switch from high-fat to normal diet does not restore sperm quality but prevents metabolic syndrome

In recent decades, the prevalence of metabolic diseases has concomitantly increased with a decline on fertility rates and sperm quality. High-fat diets (HFD) are seldom considered part of the problem, but the molecular mechanisms underlying its effects on male fertility remain poorly understood. Herein we postulated that HFD alter sperm quality. We evaluated the effects of switching from a HFD to a normal diet in early adulthood on metabolic disease onset, testicular metabolism and sperm quality. Thirty-six male C57BL6/J mice were divided in: a control group fed with standard chow; a group fed with HFD for 200 days; and a group fed with HFD for 60 days and then with standard chow (HFDt). Biometric data and whole-body metabolism were assessed. Epididymal sperm was studied for concentration, motility, viability and morphology. 1H-NMR metabolomics approach was performed on testicular extracts to trace the metabolic changes. Diet switch reduced body weight and fat mass, preventing metabolic syndrome onset. However, sperm viability, motility and morphology were deteriorated by HFD consumption and not restored by diet switch. HFD induced irreversible changes in pyruvate and glutamate metabolism, ethanol degradation and ammonia recycling in testis. Furthermore, HFDt changed purine and cysteine metabolism, urea cycle, and glutathione content. Overall, HFD caused irreversible changes in testicular metabolism even after switching to normal diet. HFD feeding until early adulthood decreases sperm quality, which cannot be restored by diet switch or weight loss, even when development of metabolic syndrome is avoided

H-Ferritin Is Essential for Macrophages' Capacity to Store or Detoxify Exogenously Added Iron

Macrophages are central cells both in the immune response and in iron homeostasis. Iron is both essential and potentially toxic. Therefore, iron acquisition, transport, storage, and release are tightly regulated, by several important proteins. Cytosolic ferritin is an iron storage protein composed of 24 subunits of either the L- or the H-type chains. H-ferritin differs from L-ferritin in the capacity to oxidize Fe2+ to Fe3+. In this work, we investigated the role played by H-ferritin in the macrophages' ability to respond to immune stimuli and to deal with exogenously added iron. We used mice with a conditional deletion of the H-ferritin gene in the myeloid lineage to obtain bone marrow-derived macrophages. These macrophages had normal viability and gene expression under basal culture conditions. However, when treated with interferon-gamma and lipopolysaccharide they had a lower activation of Nitric Oxide Synthase 2. Furthermore, H-ferritin-deficient macrophages had a higher sensitivity to iron-induced toxicity. This sensitivity was associated with a lower intracellular iron accumulation but a higher production of reactive oxygen species. These data indicate that H-ferritin modulates macrophage response to immune stimuli and that it plays an essential role in protection against iron-induced oxidative stress and cell death.Tis work was fnanced by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/IMI-MIC/1683/2014 (POCI-01-0145-FEDER-016590). PFO and MGA acknowledge FCT for the Investigador FCT 2015. We thank the valuable collaboration of the following i3S Scientifc Platforms: Cell Culture and Genotyping Core Facility (CCGen), [Histology and Electron Microscopy Service (HEMS), and BioSciences Screening], member of the PPBI (PPBI-POCI-01-0145-FEDER-022122)], Animal Facility, and Flow Cytometry Unit (TraCy). We acknowledge Lukas Kuhn (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) for kindly providing the frst breeding pairs of Fth1−/− mice. Te authors also acknowledge Marisa Castro, from Departamento de Biologia Molecular from ICBAS, Clara Bento, from i3S, and Edgar Pinto from LAQV – REQUIMTE for technical assistance at diferent stages of the project

Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.The authors acknowledge funding from the European Union (EDUFLUVAC project FP7-HEALTH-2013-INNOVATION), the Fundação para a Ciência e Tecnologia (FCT, Portugal; project HIVERA/0002/2013 and FCT Investigator to G.J.L.B.), EPSRC (to G.J.L.B.), the European Commission, Marie Skłodowska-Curie Actions (MSCA), and RISE project grant 644167. S. B. C., J. M. F., F. M., and D. G. acknowledge FCT for fellowships SFRH/BD/52302/2013, SFRH/BD/70423/2010, SFRH/BD/70139/2010, and SFRH/BPD/73500/2010, respectively. The authors acknowledge Ricardo Silva for all his help in fluorescence analysis implementation and fruitful discussions. The authors also acknowledge Patrícia Gomes-Alves for her help for mass spectrometry analysis. Mass spectrometry data was obtained by the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal. G. J. L. B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt)

Hyperoside Supplementation in Preservation Media Surpasses Vitamin C Protection Against Oxidative Stress-Induced Damages in Human Spermatozoa

Background/Aims: Oxidative Stress (OS) is reported as one of the main causes of male infertility. Infertile couples often resort to assisted reproductive technology (ART) to achieve parenthood. However, preparation for ART protocols increases the exposer of gametes to OS. Thus, it is crucial to find suitable preservation media that can counteract the OS-induced damages in spermatozoa. In this work, we tested and compared the efficiency of vitamin C (VC) and hyperoside (HYP) as potential antioxidant supplements for sperm preservation media. Methods: We evaluated the cytotoxicity of HYP (0, 5, 50, 100, and 500 µM) in spermatozoa. After incubation of sperm cells with VC (600 µM) and HYP (100 and 500 µM), in the presence and absence of H2O2 (300 µM), the following parameters were assessed: total sperm motility and vitality, OS biomarkers expression, total antioxidant capacity (TAC) of the media, percentage of DNA fragmentation, mitochondrial membrane potential (MMP), and metabolite quantification of the media by proton nuclear magnetic resonance (1H-NMR). Results: The supplementation with VC (600 µM) and HYP (100 and 500 µM) did not induce any deleterious effects to the physiology and metabolism of the spermatozoa, after 1-hour of treatment. In the presence of H2O2 (300 µM), both VC and HYP were able to prevent some of the deleterious effects of H2O2 in sperm, which were represented by an increase in sperm motility, a decrease in DNA fragmentation, and a decreasing trend in lipid peroxidation levels. However, these antioxidants were not able to prevent the decrease of MMP associated with H2O2 treatment, nor were able to prevent the conversion of pyruvate into acetate (a reaction promoted by H2O2). Conclusion: The supplementation of sperm preservation media with VC and HYP could be beneficial for the preservation of sperm physiology. From the antioxidant conditions tested, the supplementation of media with HYP (100 µM) demonstrated the best results regarding sperm preservation, evidencing the higher antioxidant capacity of HYP compared to VC. Nevertheless, none of the antioxidants used was able to prevent the metabolic alterations promoted by H2O2 in spermatozoa.This work was supported by Fundação para a Ciência e a Tecnologia - FCT to Sara C. Pereira (2021.05487.BD); David F. Carrageta (SFRH/BD/136779/2018); Marco G. Alves (IFCT2015 and PTDC/MEC-AND/28691/2017); LAQV-REQUIMTE (UIDB/50006/2020); UMIB (UIDB/00215/2020, and UIDP/00215/2020); ITR - Laboratory for Integrative and Translational Research in Population Health (LA/P/0064/2020). Pedro F. Oliveira was funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Scientific Employment Stimulus - Institutional Call - reference CEECINST/00026/2018

Diet during early life defines testicular lipid content and sperm quality in adulthood

Childhood obesity is a serious concern associated with ill health later in life. Emerging data suggest that obesity has long-term adverse effects upon male sexual and reproductive health but few studies addressed this issue. We hypothesized that exposure to high-fat diet during early life alters testicular lipid content and metabolism leading to permanent damage to sperm parameters. After weaning (day 21 after birth), 36 male mice were randomly divided into 3 groups and fed with different diet regimen for 200 days: CTRL-standard chow; HFD-high-fat diet (Carbohydrate: 35.7%, Protein: 20.5%, Fat: 36.0%); HFDt-high-fat diet for 60 days then replaced by standard chow. Biometric and metabolic data were monitored. Animals were then sacrificed, and tissues collected. Epididymal sperm parameters and endocrine parameters were evaluated. Testicular metabolites were extracted and characterized by 1H-NMR and GC-MS. Testicular mitochondrial and antioxidant activity were evaluated. Our results show that mice fed with high-fat diet, even if only until early adulthood, had lower sperm viability and motility, and higher incidence of head and tail defects. Although diet reversion with weight loss during adulthood prevents the progression of metabolic syndrome, testicular content in fatty acids is irreversibly affected. Excessive fat intake promoted an over-accumulation of pro-inflammatory n-6 polyunsaturated fatty acids in testis, which are strongly correlated with negative effects upon sperm quality. Therefore, the adoption of high-fat diets during early life correlates to irreversible changes in testicular lipid content and metabolism, which are related to permanent damage to sperm quality later in life

A participação do investigado no Inquérito Policial

Busca-se por meio deste artigo fazer uma análise crítica sobre a participação do investigado no Inquérito Policial à luz da Constituição Federal de 1988. Para tanto, faz-se necessário perpassar pelos principais sistemas processuais adotados no ordenamento jurídico diante do atual Estado Democrático de Direito, bem como analisar a abrangência do art. 5°, inciso LV da CRFB/88 e do art. 14 do Código de Processo Penal, visando propiciar uma reflexão sobre a atual forma de participação do investigado na fase pré processual Palavras-chaves: DIREITOS FUNDAMENTAIS, INQUÉRITO POLICIAL, PARTICIPAÇÃO DO INVESTIGADO THE INVESTIGATED PARTICIPATION IN POLICE INVESTIGATION ABSTRACT The aim is to through this article make a critical analysis of the participation of the police inquiry investigated in the light of the 1988 Federal Constitution Therefore, it is necessary to pervade the main procedural systems adopted in the legal system before the current democratic state and to examine the scope of art. 5 °, item LV CRFB / 88 and the art. 14 of the Criminal Procedure Code, in order to provide a reflection on the current form of participation of the investigation in the pre procedural stage. Keywords: FUNDAMENTAL RIGHTS, POLICE INVESTIGATION, INVESTIGATED PARTICIPATION Data da submissão: 31/03/2015 Data da aceitação: 27/07/201

Metabolic diseases affect male reproduction and induce signatures in gametes that may compromise the offspring health

The most prevalent diseases worldwide are non-communicable such as obesity and type 2 diabetes. Noteworthy, the prevalence of obesity and type 2 diabetes is expected to steadily increase in the next decades, mostly fueled by bad feeding habits, stress, and sedentarism. The reproductive function of individuals is severely affected by abnormal metabolic environments, both at mechanical and biochemical levels. Along with mechanical dysfunctions, and decreased sperm quality (promoted both directly and indirectly by metabolic abnormalities), several studies have already reported the potentially harmful effects of metabolic disorders in the genetic and epigenetic cargo of spermatozoa, and the epigenetic inheritance of molecular signatures induced by metabolic profile (paternal diet, obesity, and diabetes). The inheritance of epigenetic factors towards the development of metabolic abnormalities means that more people in reproductive age can potentially suffer from these disorders and for longer periods. In its turn, these individuals can also transmit this (epi)genetic information to future generations, creating a vicious cycle. In this review, we collect the reported harmful effects related to acquired metabolic disorders and diet in sperm parameters and male reproductive potential. Besides, we will discuss the novel findings regarding paternal epigenetic inheritance, particularly the ones induced by paternal diet rich in fats, obesity, and type 2 diabetes. We analyze the data attained with in vitro and animal models as well as in long-term trans generational population studies. Although the findings on this topic are very recent, epigenetic inheritance of metabolic disease has a huge societal impact, which may be crucial to tackle the `fat epidemic' efficiently