Quantification and Localization of Intracellular Free Mg2+ in Bovine Chromaffin Cells

Magnesium is an essential element for all living systems. The quantification of free intracellular Mg2+ concentration ([Mg2+]i) is of utmost importance since changes in its basal value may be an indication of different pathologies due to abnormalities of Mg2+ metabolism. In this work we used 31P NMR and fluorescence spectroscopy to determine the resting [Mg2+]i in bovine chromaffin cells, a neuron-like cellular model, as well as confocal laser scanning microscopy to study the free Mg2+ spatial distribution in these cells. 31P NMR spectroscopy did not prove to be effective for the determination of [Mg2+]i in this particular case due to some special morphological and physiological properties of this cell type. A basal [Mg2+]i value of 0.551 ± 0.008 mM was found for these cells using fluorescence spectroscopy and the Mg2+-sensitive probe furaptra; this value falls in the concentration range reported in the literature for neurons from different sources. This technique proved to be an accurate and sensitive tool to determine the [Mg2+]i

Li+ Influx and Binding, and Li+/Mg2+ Competition in Bovine Chromaffin Cell Suspensions as Studied by 7Li NMR and Fluorescence Spectroscopy

Li+ influx by bovine chromaffin cells, obtained from bovine adrenal medulla, was studied in intact cell suspensions using 7Li NMR spectroscopy with the shift reagent [Tm(HDOTP)]4-. The influx rate constants, ki, were determined in the absence and in the presence of two Na+ membrane transport inhibitors. The values obtained indicate that both voltage sensitive Na+ channels and (Na+/K+)-ATPase play an important role in Li+ uptake by these cells. 7Li NMR T1 and T2 relaxation times for intracellular Li+ in bovine chromaffin cells provided a T1/T2 ratio of 305, showing that Li+ is highly, immobilized due to strong binding to intracellular structures. Using fluorescence spectroscopy and the Mg2+ fluorescent probe, furaptra, the free intracellular Mg2+ concentration in the bovine chromaffin cells incubated with 15 mM LiCl was found to increase by about mM after the intracellular Li+ concentration reached a steady state. Therefore, once inside the cell, Li+ is able to displace Mg2+ from its binding sites

Valorization of brewery waste through Polyhydroxyalkanoates production supported by a metabolic specialized microbiome

Raw brewers’ spent grain (BSG), a by-product of beer production and produced at a large scale, presents a composition that has been shown to have potential as feedstock for several biological processes, such as polyhydroxyalkanoates (PHAs) production. Although the high interest in the PHA production from waste, the bioconversion of BSG into PHA using microbial mixed cultures (MMC) has not yet been explored. This study explored the feasibility to produce PHA from BSG through the enrichment of a mixed microbial culture in PHA-storing organisms. The increase in organic loading rate (OLR) was shown to have only a slight influence on the process performance, although a high selectivity in PHA-storing microorganisms accumulation was reached. The culture was enriched on various PHA-storing microorganisms, such as bacteria belonging to the Meganema, Carnobacterium, Leucobacter, and Paracocccus genera. The enrichment process led to specialization of the microbiome, but the high diversity in PHA-storing microorganisms could have contributed to the process stability and efficiency, allowing for achieving a maximum PHA content of 35.2 ± 5.5 wt.% (VSS basis) and a yield of 0.61 ± 0.09 CmmolPHA/CmmolVFA in the accumulation assays. Overall, the production of PHA from fermented BSG is a feasible process confirming the valorization potential of the feedstock through the production of added-value products.info:eu-repo/semantics/publishedVersio

Comparing trawl and creel fishing for Norway lobster (Nephrops norvegicus): biological and economic considerations

This study compares the fishing activity and landings of the trawl and creel fisheries for Norway lobster (Nephrops norvegicus (L.)) off the Portuguese coast, and evaluates the financial viability of two vessels typical of each fleet. Crustacean trawlers are part of an industrial fleet that, besides Nephrops, targets deep water shrimps. Creels are used by a multi-gear, multi-target artisanal fleet, fishing only in areas unavailable to trawlers and, when catching Nephrops, set specifically to target this species. Trawlers have in recent years contributed with 85% of the landings in weight, but only 74% in value (2005-2009 average). Despite smaller landings, the Nephrops creel fishery provides individuals of larger size and in better condition, thereby obtaining higher unit prices. Economic viability was also higher for the creel vessel, with trawling being only viable if major costs (such as labor and fuel) are covered by the revenue from other target species (e. g., the rose shrimp). At present, Nephrops populations on the South and SW coast are subject to intense fishing and to a recovery plan. The possibility of reallocation of some of the fishing effort directed at Nephrops from trawlers to creels is discussed in terms of the conservation of the resource and economic return.Foundation for Science and Technology, Portugal: Project "Nephrops survival when escaping from trawls nets and by-catch escaping devices" [PDCT/MAR/59366/2004]; Foundation for Science and Technology, Portugal: Project "Environmental impact of fixing gears in the southwest coast of Portugal. Combine fisheries and marine ecosystem conservation" [POCTI/CTA/49248/2002]info:eu-repo/semantics/publishedVersio

Atherosclerosis and Bone Loss in Humans–Results From Deceased Donors and From Patients Submitted to Carotid Endarterectomy

Funding: We wish to thank all the collaborators (administrative staff, nurses, etc.) of the surgery block, as well as the doctors of the vascular surgery and transplantation departments of the Hospital of Santa Maria for the availability and assistance in the collection of the samples. We also thank Sociedade Portuguesa de Reumatologia for funding with two fellowships: Fundo de Apoio à Investigação 2014 and SPR/MSD 2015. DC-F received funding from a PhD grant from Fundação para a Ciência e a Tecnologia (SFRH/BD/80940/2011).Background and Aims: Atherosclerosis and osteoporosis share common risk factors, as well as inflammatory mechanisms. Our aim was to understand how atherosclerotic lesions are related with disturbances in bone. Methods: Gene expression of pro-inflammatory and bone metabolism related proteins (IL-1β, IL-6, IL-17A, TNF, RANKL, OPG, COL1, CTSK, OCL, TRAP, CBFA1, DKK1, SOST, ADIPOQ, and ADIPOR1) were analyzed in arteries and bones from 45 deceased donors and adipose tissue was used as control. Additionally, in 139 patients with advanced atherosclerosis submitted to carotid endarterectomy we compared calcium content (Alizarin red) and plaque inflammatory scores (CD3+, CD68+, and adiponectin) of patients with normal bone mineral density (BMD) with those with low BMD and explored the associations between gene expression in atherosclerotic plaques and BMD. Serum levels of pro-inflammatory and bone related proteins were measured both in donors and patients. Associations were investigated by the Pearson or Spearman correlation tests, and multivariate regression analyzes were performed when justified. Results: Gene expression of bone remodeling and pro-inflammatory proteins correlated positively in bone and aorta, independently of age and sex of donors, but not in adipose tissue. The expression of bone formation genes was significantly higher in atheroma plaques from endarterectomized patients with normal vs. low BMD as well as inflammatory CD68+ scores, regardless of patients' age and sex, but not of body mass index. No relationship was observed between serum levels and gene expression levels of pro-inflammatory or bone remodeling proteins. Conclusions: Our results suggest that the relationship between bones and vessels in the context of atherosclerotic disease and osteoporosis may rely on the intrinsic connection between the tissues involved, independently of disease stage. Serum measurements of pro-inflammatory and bone-remodeling proteins do not accurately translate tissue pathologic processes.publishersversionpublishe

Conventional and molecular cytogenetics of human non-medullary thyroid carcinoma: characterization of eight cell line models and review of the literature on clinical samples

<p>Abstract</p> <p>Background</p> <p>Cell lines are often poorly characterized from a genetic point of view, reducing their usefulness as tumor models. Our purpose was to assess the genetic background of eight commonly used human thyroid carcinoma models and to compare the findings with those reported for primary tumors of the gland.</p> <p>Methods</p> <p>We used chromosome banding analysis and comparative genomic hybridization to profile eight non-medullary thyroid carcinoma cell lines of papillary (TPC-1, FB2, K1 and B-CPAP), follicular (XTC-1) or anaplastic origin (8505C, C643 and HTH74). To assess the representativeness of the findings, we additionally performed a thorough review of cytogenetic (n = 125) and DNA copy number information (n = 270) available in the literature on clinical samples of thyroid carcinoma.</p> <p>Results</p> <p>The detailed characterization of chromosomal markers specific for each cell line revealed two cases of mistaken identities: FB2 was shown to derive from TPC-1 cells, whereas K1 cells have their origin in cell line GLAG-66. All cellular models displayed genomic aberrations of varying complexity, and recurrent gains at 5p, 5q, 8q, and 20q (6/7 cell lines) and losses at 8p, 13q, 18q, and Xp (4/7 cell lines) were seen. Importantly, the genomic profiles were compatible with those of the respective primary tumors, as seen in the meta-analysis of the existing literature data.</p> <p>Conclusion</p> <p>We provide the genomic background of seven independent thyroid carcinoma models representative of the clinical tumors of the corresponding histotypes, and highlight regions of recurrent aberrations that may guide future studies aimed at identifying target genes. Our findings further support the importance of routinely performing cytogenetic studies on cell lines, to detect cross-contamination mishaps such as those identified here.</p

Tauroursodeoxycholic Acid Improves Motor Symptoms in a Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is characterized by severe motor symptoms, and currently there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Tauroursodeoxycholic acid (TUDCA) is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD; however, its effect in PD motor symptoms has never been addressed. In the present work, an extensive behavior analysis was performed to better characterize the MPTP model of PD and to evaluate the effects of TUDCA in the prevention/improvement of mice phenotype. MPTP induced significant alterations in general motor performance paradigms, including increased latency in the motor swimming, adhesive removal and pole tests, as well as altered gait, foot dragging, and tremors. TUDCA administration, either before or after MPTP, significantly reduced the swimming latency, improved gait quality, and decreased foot dragging. Importantly, TUDCA was also effective in the prevention of typical parkinsonian symptoms such as spontaneous activity, ability to initiate movement and tremors. Accordingly, TUDCA prevented MPTP-induced decrease of dopaminergic fibers and ATP levels, mitochondrial dysfunction and neuroinflammation. Overall, MPTP-injected mice presented motor symptoms that are aggravated throughout time, resembling human parkinsonism, whereas PD motor symptoms were absent or mild in TUDCA-treated animals, and no aggravation was observed in any parameter. The thorough demonstration of improvement of PD symptoms together with the demonstration of the pathways triggered by TUDCA supports a subsequent clinical trial in humans and future validation of the application of this bile acid in PD.National funds, through the Foundation for Science and Technology (Portugal) (FCT), under the scope of the projects PTDC/NEU-NMC/0248/2012, UID/DTP/04138/2013 and POCI-01-0145-FEDER-007038, and post-doctoral grants SFRH/BPD72891/2010 (to A.I.R.), SFRH/BPD/95855/2013 (to M.J.N.), SFRH/BPD/98023/2013 (to A.N.C.), SFRH/BPD/91562/2012 (to A.S.F.) and UMINHO/BI/248/2016 (to S.D.S.). This work has also been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and by FEDER funds, through the Competitiveness Factors Operational Program (COMPETE)info:eu-repo/semantics/publishedVersio

Characterization of Apoptosis-Related Oxidoreductases from Neurospora crassa

The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins

Toxoplasmosis-associated IRIS involving the CNS: a case report with longitudinal analysis of T cell subsets

Background: HIV-infected patients may present an unforeseen clinical worsening after initiating antiretroviral therapy known as immune reconstitution inflammatory syndrome (IRIS). This syndrome is characterized by a heightened inflammatory response toward infectious or non-infectious triggers, and it may affect different organs. Diagnosis of IRIS involving the central nervous system (CNS-IRIS) is challenging due to heterogeneous manifestations, absence of biomarkers to identify this condition, risk of long-term sequelae and high mortality. Hence, a deeper knowledge of CNS-IRIS pathogenesis is needed. Case presentation: A 37-year-old man was diagnosed with AIDS and cerebral toxoplasmosis. Anti-toxoplasma treatment was initiated immediately, followed by active antiretroviral therapy (HAART) 1 month later. At 2 months of HAART, he presented with progressive hyposensitivity of the right lower limb associated with brain and dorsal spinal cord lesions, compatible with paradoxical toxoplasmosis-associated CNS-IRIS, a condition with very few reported cases. A stereotactic biopsy was planned but was postponed based on its inherent risks. Patient showed clinical improvement with no requirement of corticosteroid therapy. Routine laboratorial analysis was complemented with longitudinal evaluation of blood T cell subsets at 0, 1, 2, 3 and 6 months upon HAART initiation. A control group composed by 9 HIV-infected patients from the same hospital but with no IRIS was analysed for comparison. The CNS-IRIS patient showed lower percentage of memory CD4(+) T cells and higher percentage of activated CD4(+) T cells at HAART initiation. The percentage of memory CD4(+) T cells drastically increased at 1 month after HAART initiation and became higher in comparison to the control group until clinical recovery onset; the percentage of memory CD8(+) T cells was consistently lower throughout follow-up. Interestingly, the percentage of regulatory T cells (Treg) on the CNS-IRIS patient reached a minimum around 1 month before symptoms onset. Conclusion: Although both stereotactic biopsies and steroid therapy might be of use in CNS-IRIS cases and should be considered for these patients, they might be unnecessary to achieve clinical improvement as shown in this case. Immunological characterization of more CNS-IRIS cases is essential to shed some light on the pathogenesis of this condition.Portuguese Foundation for Science and Technology (FCT; PIC/IC/83313/2007) and co-financed by the Portuguese North Regional Operational Program (ON.2 - O Novo Norte) under the National Strategic Reference Framework (QREN) through the European Regional Development Fund (FEDER). A FCT fellowship was attributed to RRS (PD/BD/106047/2015; Inter-University Doctoral Program in Ageing and Chronic Disease) and to CN [SFRH/BPD/65380/2009; Programa Operacional Potencial Humano (POPH) through the Fundo Social Europeu (FSE)]info:eu-repo/semantics/publishedVersio

A randomized controlled trial to evaluate self-determination theory for exercise adherence and weight control: rationale and intervention description

<p>Abstract</p> <p>Background</p> <p>Research on the motivational model proposed by Self-Determination Theory (SDT) provides theoretically sound insights into reasons why people adopt and maintain exercise and other health behaviors, and allows for a meaningful analysis of the motivational processes involved in behavioral self-regulation. Although obesity is notoriously difficult to reverse and its recidivism is high, adopting and maintaining a physically active lifestyle is arguably the most effective strategy to counteract it in the long-term. The purposes of this study are twofold: i) to describe a 3-year randomized controlled trial (RCT) aimed at testing a novel obesity treatment program based on SDT, and ii) to present the rationale behind SDT's utility in facilitating and explaining health behavior change, especially physical activity/exercise, during obesity treatment.</p> <p>Methods</p> <p>Study design, recruitment, inclusion criteria, measurements, and a detailed description of the intervention (general format, goals for the participants, intervention curriculum, and main SDT strategies) are presented. The intervention consists of a 1-year group behavioral program for overweight and moderately obese women, aged 25 to 50 (and pre-menopausal), recruited from the community at large through media advertisement. Participants in the intervention group meet weekly or bi-weekly with a multidisciplinary intervention team (30 2 h sessions in total), and go through a program covering most topics considered critical for successful weight control. These topics and especially their delivery were adapted to comply with SDT and Motivational Interviewing guidelines. Comparison group receive a general health education curriculum. After the program, all subjects are follow-up for a period of 2 years.</p> <p>Discussion</p> <p>Results from this RCT will contribute to a better understanding of how motivational characteristics, particularly those related to physical activity/exercise behavioral self-regulation, influence treatment success, while exploring the utility of Self-Determination Theory for promoting health behavior change in the context of obesity.</p> <p>Trial Registration</p> <p><b>Clinical Trials Gov. Identifier </b>NCT00513084</p