Mechanism of replication origin melting nucleated by CMG helicase assembly.

The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library.

Xylanases are key biocatalysts in the degradation of the ß-1,4-glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-ß-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. KEY POINTS: • A GH10 endo-1,4-ß-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB's in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis.

Effect of the alveolate parasite Perkinsus olseni infection on sexual maturation and spawning efficiency of the clam Ruditapes decussatus.

The effect of Perkinsus olseni infection on the reproduction ability of clams has been underestimated so far. Although some studies found evidence of reduction of egg production and delay in gonad maturation after infection, the total effect of the infection is still unclear. In this study, Ruditapes decussatus clams from a naïve population were injected with two different doses of P. olseni parasites, a low dose leading to a light infection and a high dose leading to a heavy infection. Clams were maintained during 2 months for maturation, and at the end of the experiment, the spawning was induced, the number of larvae release and mortality were evaluated. During the maturation period, infection level, gonadal stage, condition index, gross biochemical composition and oxidative status of progenitors were evaluated at days 0, 30 and 60 post-injection. The effects of P. olseni infection on clams showed alterations on biochemical parameters, namely lipid peroxidation, a significant mortality and a delayed gonad maturation, with a greater effect in the highly infected individuals. The reproductive capacity of the clams was impaired in both infected groups showing a lower production and a higher mortality rate of larvae. Finally, this study indicates that the production of natural beds with a high prevalence of P. olseni could be compromised by a deregulation of the natural reproduction cycle and a decrease in larvae production by infected animals, probably due to a combination of lower egg production and lower lipid reserves in larvae from infected clams.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples.

The renewable, abundant , and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples' microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited ß-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples. KEY POINTS: • Sequence- and function-based metagenomics were used to find CAZymes in compost samples. • Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes. • Compost-derived fosmid libraries are enriched in clones with ß-glucosidase activity.

Single Modality vs. Multimodality: What Works Best for Lung Cancer Screening?

In a clinical context, physicians usually take into account information from more than one data modality when making decisions regarding cancer diagnosis and treatment planning. Artificial intelligence-based methods should mimic the clinical method and take into consideration different sources of data that allow a more comprehensive analysis of the patient and, as a consequence, a more accurate diagnosis. Lung cancer evaluation, in particular, can benefit from this approach since this pathology presents high mortality rates due to its late diagnosis. However, many related works make use of a single data source, namely imaging data. Therefore, this work aims to study the prediction of lung cancer when using more than one data modality. The National Lung Screening Trial dataset that contains data from different sources, specifically, computed tomography (CT) scans and clinical data, was used for the study, the development and comparison of single-modality and multimodality models, that may explore the predictive capability of these two types of data to their full potential. A ResNet18 network was trained to classify 3D CT nodule regions of interest (ROI), whereas a random forest algorithm was used to classify the clinical data, with the former achieving an area under the ROC curve (AUC) of 0.7897 and the latter 0.5241. Regarding the multimodality approaches, three strategies, based on intermediate and late fusion, were implemented to combine the information from the 3D CT nodule ROIs and the clinical data. From those, the best model-a fully connected layer that receives as input a combination of clinical data and deep imaging features, given by a ResNet18 inference model-presented an AUC of 0.8021. Lung cancer is a complex disease, characterized by a multitude of biological and physiological phenomena and influenced by multiple factors. It is thus imperative that the models are capable of responding to that need. The results obtained showed that the combination of different types may have the potential to produce more comprehensive analyses of the disease by the models.

Reduced Chitosan as a Strategy for Removing Copper Ions from Water.

Toxic heavy metals are priority pollutants in wastewater, commonly present in dangerous concentrations in many places across the globe. Although in trace quantities copper is a heavy metal essential to human life, in excess it causes various diseases, whereby its removal from wastewater is a necessity. Among several reported materials, chitosan is a highly abundant, non-toxic, low-cost, biodegradable polymer, comprising free hydroxyl and amino groups, that has been directly applied as an adsorbent or chemically modified to increase its performance. Taking this into account, reduced chitosan derivatives (RCDs 1-4) were synthesised by chitosan modification with salicylaldehyde, followed by imine reduction, characterised by RMN, FTIR-ATR, TGA and SEM, and used to adsorb Cu(II) from water. A reduced chitosan (RCD3), with a moderate modification percentage (43%) and a high imine reduction percentage (98%), proved to be more efficient than the remainder RCDs and even chitosan, especially at low concentrations under the best adsorption conditions (pH 4, RS/L = 2.5 mg mL-1). RCD3 adsorption data were better described by the Langmuir-Freundlich isotherm and the pseudo-second-order kinetic models. The interaction mechanism was assessed by molecular dynamics simulations, showing that RCDs favour Cu(II) capture from water compared to chitosan, due to a greater Cu(II) interaction with the oxygen of the glucosamine ring and the neighbouring hydroxyl groups.

Corema album Leaves Mediate DNA Damage in Triple-Negative Breast Cancer Cells.

Corema (C.) album is a shrub endemic to the Atlantic coast and has been described as yielding beneficial effects for human health. Nevertheless, studies concerning the bioactivity of C. album leaves are scarce. This study aims at investigating the anticancer potential and mode of action, of an hydroethanolic extract of C. album leaves (ECAL) on triple-negative breast cancer. This is a poor survival breast cancer subtype, owing to its high risk of distant reappearance, metastasis rates and the probability of relapse. The ECAL ability to prevent tumor progression through (i) the inhibition of cell proliferation (cell viability); (ii) the induction of apoptosis (morphological changes, TUNEL assay, caspase-3 cleaved) and (iii) the induction of DNA damage (PARP1 and γH2AX) with (iv) the involvement of NF-κB and of ERK1/2 pathways (AlphaScreen assay) was evaluated. ECAL activated the apoptotic pathway (through caspase-3) along with the inhibition of ERK and NF-κB pathways causing DNA damage and cell death. The large polyphenolic content of ECAL was presumed to be accountable for these effects. The extract of C. album leaves can target multiple pathways and, thus, can block more than one possible means of disease progression, evidencing the anticancer therapeutic potential from a plant source.

Sintra Grows Healthy: development and implementation of a food literacy curriculum for primary schools.

OBJECTIVE: Describe the process of development and implementation of Health at the Table - a food literacy curriculum for primary school aged children. DESIGN: Through a community-based research process, Health at the Table development and implementation took place in four stages: exploratory study, production, implementation and monitoring. SETTING: Primary schools of Sintra's municipality, Portugal. PARTICIPANTS: Children (6-10 years), teachers, school staff and children's legal guardians of three primary schools during the pilot project and eight primary schools in the second year. RESULTS: During the needs assessment phase, 99·1 % (n 341) of the children's legal guardians, 100 % (n 34) of the teachers and 100 % (n 19) of the school staff considered that the school plays an important or very important role in children's food literacy (stage 1). During the pilot project, a manual with sixty session plans was developed (stage 2). In the second year, Health at the Table was implemented by seventy-two trained teachers during one school year (stage 3). Most of the teachers agreed that the curriculum was appropriate (69·2 %) and that children developed health, wellness/well-being and environmental skills (83·1 %). Most of the children said they had learned about healthy eating (86·3 %) and claimed to eat healthier since the Health at the Table implementation (58·9 %) (stage 4). CONCLUSIONS: Health at the Table is a food literacy curriculum that can be reproduced in similar contexts in a sustainable way. The need to combine educational strategies with a healthy school food environment is reinforced to increase effectiveness in tackling childhood obesity.

The Influence of a Coherent Annotation and Synthetic Addition of Lung Nodules for Lung Segmentation in CT Scans.

Lung cancer is a highly prevalent pathology and a leading cause of cancer-related deaths. Most patients are diagnosed when the disease has manifested itself, which usually is a sign of lung cancer in an advanced stage and, as a consequence, the 5-year survival rates are low. To increase the chances of survival, improving the cancer early detection capacity is crucial, for which computed tomography (CT) scans represent a key role. The manual evaluation of the CTs is a time-consuming task and computer-aided diagnosis (CAD) systems can help relieve that burden. The segmentation of the lung is one of the first steps in these systems, yet it is very challenging given the heterogeneity of lung diseases usually present and associated with cancer development. In our previous work, a segmentation model based on a ResNet34 and U-Net combination was developed on a cross-cohort dataset that yielded good segmentation masks for multiple pathological conditions but misclassified some of the lung nodules. The multiple datasets used for the model development were originated from different annotation protocols, which generated inconsistencies for the learning process, and the annotations are usually not adequate for lung cancer studies since they did not comprise lung nodules. In addition, the initial datasets used for training presented a reduced number of nodules, which was showed not to be enough to allow the segmentation model to learn to include them as a lung part. In this work, an objective protocol for the lung mask's segmentation was defined and the previous annotations were carefully reviewed and corrected to create consistent and adequate ground-truth masks for the development of the segmentation model. Data augmentation with domain knowledge was used to create lung nodules in the cases used to train the model. The model developed achieved a Dice similarity coefficient (DSC) above 0.9350 for all test datasets and it showed an ability to cope, not only with a variety of lung patterns, but also with the presence of lung nodules as well. This study shows the importance of using consistent annotations for the supervised learning process, which is a very time-consuming task, but that has great importance to healthcare applications. Due to the lack of massive datasets in the medical field, which consequently brings a lack of wide representativity, data augmentation with domain knowledge could represent a promising help to overcome this limitation for learning models development.

Fetal Undernutrition Modifies Vascular RAS Balance Enhancing Oxidative Damage and Contributing to Remodeling.

Fetal stress is known to increase susceptibility to cardiometabolic diseases and hypertension in adult age in a process known as fetal programming. This study investigated the relationship between vascular RAS, oxidative damage and remodeling in fetal programming. Six-month old Sprague-Dawley offspring from mothers that were fed ad libitum (CONTROL) or with 50% intake during the second half of gestation (maternal undernutrition, MUN) were used. qPCR or immunohistochemistry were used to obtain the expression of receptors and enzymes. Plasma levels of carbonyls were measured by spectrophotometry. In mesenteric arteries from MUN rats we detected an upregulation of ACE, ACE2, AT1 receptors and NADPH oxidase, and lower expression of AT2, Mas and MrgD receptors compared to CONTROL. Systolic and diastolic blood pressure and plasma levels of carbonyls were higher in MUN than in CONTROL. Vascular morphology evidenced an increased media/lumen ratio and adventitia/lumen ratio, and more connective tissue in MUN compared to CONTROL. In conclusion, fetal undernutrition indices RAS alterations and oxidative damage which may contribute to the remodeling of mesenteric arteries, and increase the risk of adverse cardiovascular events and hypertension.

Decoration of A-Ring of a Lupane-Type Triterpenoid with Different Oxygen and Nitrogen Heterocycles.

Betulinic acid (BA) was used as starting building block to create a library of novel BA-derived compounds containing O- and N-heterocycles. Firstly, BA was converted into methyl betulonate (BoOMe), which was used as intermediate in the developed methodologies. 1,2-Oxazine-fused BoOMe compounds were obtained in 12-25% global yields through a Michael addition of nitromethane to methyl (E)-2-benzylidenebetulonate derivatives, followed by nitro group reduction and intramolecular cyclization. Remarkably, the triterpene acts as a diastereoselective inducer in the conjugate addition of nitromethane, originating only one diastereomer out of four possible ones. Furthermore, other oxygen and nitrogen-containing heterocycles were installed at the A-ring of BoOMe, affording 2-amino-3-cyano-4H-pyran-fused BoOMe, diarylpyridine-fused BoOMe and 1,2,3-triazole-BoOMe compounds, using simple and straightforward synthetic methodologies. Finally, BA was revealed to be a versatile starting material, allowing the creation of a molecular diversification of compounds containing a triterpenic scaffold and O- and N-heterocycles.

Identification of the 5-HT1A serotonin receptor as a novel therapeutic target in a C. elegans model of Machado-Joseph disease.

Machado-Joseph disease (MJD) or Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder that affects movement coordination leading to a premature death. Despite several efforts, no disease-modifying treatment is yet available for this disease. Previous studies pinpointed the modulation of serotonergic signaling, through pharmacological inhibition of the serotonin transporter SERT, as a promising therapeutic approach for MJD/SCA3. Here, we describe the 5-HT1A receptor as a novel therapeutic target in MJD, using a C. elegans model of ATXN3 proteotoxicity. Chronic and acute administration of befiradol (also known as NLX-112), a highly specific 5-HT1A agonist, rescued motor function and suppressed mutant ATXN3 aggregation. This action required the 5-HT1A receptor orthologue in the nematode, SER-4. Tandospirone, a clinically tested 5-HT1A receptor partial agonist, showed a limited impact on animals' motor dysfunction on acute administration and a broader receptor activation profile upon chronic treatment, its effect depending on 5-HT1A but also on the 5-HT6/SER-5 and 5-HT7/SER-7 receptors. Our results support high potency and specificity of befiradol for activation of 5-HT1A/SER-4 receptors and highlight the contribution of the auto- and hetero-receptor function to the therapeutic outcome in this MJD model. Our study deepens the understanding of serotonergic signaling modulation in the suppression of ATXN3 proteotoxicity and suggests that a potent and selective 5-HT1A receptor agonist such as befiradol could constitute a promising therapeutic agent for MJD.

Mesenchymal stem cell secretome protects against alpha-synuclein-induced neurodegeneration in a Caenorhabditis elegans model of Parkinson's disease.

BACKGROUND AIMS: The capacity of the secretome from bone marrow-derived mesenchymal stem cells (BMSCs) to prevent dopaminergic neuron degeneration caused by overexpression of alpha-synuclein (α-syn) was explored using two Caenorhabditis elegans models of Parkinson's disease (PD). METHODS: First, a more predictive model of PD that overexpresses α-syn in dopamine neurons was subjected to chronic treatment with secretome. This strain displays progressive dopaminergic neurodegeneration that is age-dependent. Following chronic treatment with secretome, the number of intact dopaminergic neurons was determined. Following these initial experiments, a C. elegans strain that overexpresses α-syn in body wall muscle cells was used to determine the impact of hBMSC secretome on α-syn inclusions. Lastly, in silico analysis of the components that constitute the secretome was performed. RESULTS: The human BMSC (hBMSC) secretome induced a neuroprotective effect, leading to reduced dopaminergic neurodegeneration. Moreover, in animals submitted to chronic treatment with secretome, the number of α-syn inclusions was reduced, indicating that the secretome of MSCs was possibly contributing to the degradation of those structures. In silico analysis identified possible suppressors of α-syn proteotoxicity, including growth factors and players in the neuronal protein quality control mechanisms. CONCLUSIONS: The present findings indicate that hBMSC secretome has the potential to be used as a disease-modifying strategy in future PD regenerative medicine approaches.

Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5-13.8 µg g-1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory. KEY POINTS/HIGHLIGHTS: • Metagenomic DNA was successfully extracted from compost samples rich in humic acids • The improved protocol was established by optimizing the cell lysis method and buffer • Complete removal of humic acids was achieved through the use of activated charcoal • The suitability of the DNA was proven by the construction of a metagenomic library.

The Diversity, Metabolomics Profiling, and the Pharmacological Potential of Actinomycetes Isolated from the Estremadura Spur Pockmarks (Portugal).

The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat.

Analyses of competent and non-competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence.

Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.

Structural Specificity of Flavonoids in the Inhibition of Human Fructose 1,6-Bisphosphatase.

Liver fructose 1,6-bisphosphatase (FBPase) is a recognized regulatory enzyme of the gluconeogenesis pathway, which has emerged as a valid target to control gluconeogenesis-mediated overproduction of glucose. As such, the management of diabetes with FBPase inhibitors represents a potential alternative for the currently used antidiabetic agents. In this study, the FBPase inhibition of a panel of 55 structurally related flavonoids was tested, through a microanalysis screening system. Then, a subset of seven active inhibitors and their close chemical relatives were further evaluated by molecular dynamics (MD) simulations using a linear interaction energy (LIE) approach. The results obtained showed that D14 (herbacetin) was the most potent inhibitor, suggesting that the presence of -OH groups at the C-3, C-4', C-5, C-7, and C-8 positions, as well as the double bond between C-2 and C-3 and the 4-oxo function at the pyrone ring, are favorable for the intended effect. Furthermore, D14 (herbacetin) is stabilized by a strong interaction with the Glu30 side chain and the Thr24 backbone of FBPase. This is the first investigation studying the in vitro inhibitory effect of a panel of flavonoids against human liver FBPase, thus representing a potentially important step for the search and design of novel inhibitors of this enzyme.

Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.

A school-based intervention for a better future: study protocol of Sintra Grows Healthy.

BACKGROUND: Preventing childhood obesity is a public health challenge of the twenty-first century and it must be a priority. Governments play a major role in creating and supporting a healthy school environment and should prioritise actions to improve children's health. Sintra Grows Healthy aims to promote healthy lifestyles to prevent childhood obesity and improve children's health-related quality of life and social and emotional skills, through the development of a school evidence-based and sustainable model. METHODS: This protocol describes a quasi-experimental design and community-based participatory research. The participants included in the study are the school community of Portuguese public primary schools from the municipality of Sintra. Data will be collected on demographic and socio-economic characterization, nutritional status, eating habits and behaviours, physical activity, sedentary behaviours and sleep, health-related quality of life, and social and emotional skills. DISCUSSION: There is evidence to support interventions in school settings as strategies for obesity prevention. Up-to-date homogeneous and community-based interventions for preventing childhood obesity are lacking, therefore Sintra Grows Healthy intends to fill this gap. Furthermore, Sintra Grows Healthy aims to contribute with relevant scientific findings that will allow the development of better strategies for policymakers and society to manage this major public health problem.