Design and synthesis of new inhibitors of p53–MDM2 interaction with a chalcone scaffold

The virtual screening of a library of chalcone derivatives led us to the identification of potential new MDM2 ligands. The chalcones with the best docking scores obeying the Lipinski rule of five were subsequently prepared by base-catalyzed aldol reactions. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast-based screening assay. Using this approach two chalcones (3 and 4) were identified as putative small molecule inhibitors of p53–MDM2 interaction. The activity of both chalcones was further investigated in a panel of human tumor cells. Chalcones 3 and 4 revealed a pronounced tumor cell growth inhibitory effect on tumor cell lines. Additionally, chalcone 4 caused alterations in the cell cycle profile, induced apoptosis and increased the levels of p53, p21 and PUMA proteins in NCI-H460 cells. Computational docking studies allowed to predict that, like nutlin-3A (a well-known small-molecule inhibitor of p53–MDM2 interaction), chalcones 3 and 4 bind to the p53-binding site of MDM2. The results here presented will be valuable for the structure-based design of novel and potent p53–MDM2 inhibitors.This research was partially supported by the Strategic Funding UID/Multi/04423/2013 , ERDF , COMPETE , and FCT under the projects PTDC/MAR-BIO/4694/2014, and INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035 , Research Line NOVELMAR . This work also received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013 and the FCT project PTDC/DTP-FTO/1981/2014, “PEst-C/SAU/LA0003/2013”, “NORTE-07-0162-FEDER-00018 – Contributos para o reforço da capacidade do IPATIMUP enquanto actor do sistema regional de inovação” and NORTE-07-0162-FEDER-000067 – Reforço e consolidação da capacidade infraestrutural do IPATIMUP para o sistema regional de inovação”, both supported by ON.2 – O Novo Norte, through FEDER funds under the QREN. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. The authors also thank FCT for the grants of R.T. Lima ( SFRH/BPD/68787/2010 ), J. Soares ( SFRH/BD/78971/2011 ), and S. Gomes ( SFRH/BD/96189/2013 ; Doctoral Programme BiotechHealth), L. Raimundo ( PD/BI/113926/2015 , Doctoral Programme BiotechHealth)

Electrochemical miRNA-34a-based biosensor for the diagnosis of Alzheimer’s disease

Alzheimer's disease (AD) is the most common dementia type and a leading cause of death and disability in the elderly. Diagnosis is expensive and invasive, urging the development of new, affordable, and less invasive diagnostic tools. The identification of changes in the expression of non-coding RNAs prompts the development of diagnostic tools to detect disease-specific blood biomarkers. Building on this idea, this work reports a novel electrochemical microRNA (miRNA) biosensor for the diagnosis of AD, based on carbon screen-printed electrodes (C-SPEs) modified with two gold nanostructures and a complementary anti-miR-34a oligonucleotide probe. This biosensor showed good target affinity, reflected on a 100 pM to 1 M linearity range and a limit of detection (LOD) of 39 pM in buffer and 94 aM in serum. Moreover, the biosensors response was not affected by serum compounds, indicating selectivity for miR-34a. The biosensor also detected miR-34a in the cell culture medium of a common AD model, stimulated with a neurotoxin to increase miR-34a secretion. Overall, the proposed biosensor makes a solid case for the introduction of a novel, inexpensive, and minimally invasive tool for the early diagnosis of AD, based on the detection of a circulating miRNA overexpressed in this pathology.This work was supported by 0624_2IQBIONEURO_6_E, 2IQBioneuro, Promotion of an R&I network in biological chemistry for the diagnosis and treatment of neurological diseases EP-INTERREG V Spain Portugal (POCTEP), and by Portuguese funds through FCT in the framework of the project PTDC/BTM-MAT/4156/2021. S.D.S acknowledges FCT (Fundação para a Ciência e a Tecnologia, I.P.) for her contract under the Norma Transitória – DL57/2016/CP/CP1360/CT0013.info:eu-repo/semantics/publishedVersio

Multivariate geostatistical analysis of stable isotopes in Portuguese varietal extra virgin olive oils

Stable isotope contents of carbon, hydrogen and oxygen are known to reflect the geo-climatic conditions under which olives grown. This study aims to unravel the correlation between some of the main geographic variables and the isotopic composition of different Portuguese varietal extra virgin olive oil (EVOO) samples. Thus, the isotopic composition (δ13C, δ18O and δ2H) of 38 EVOO samples from 11 olive varieties from 2 Portuguese regions (Alentejo and Trás-os-Montes) was studied using an elemental analyzer coupled to an isotope ratio mass spectrometry. Multivariate analysis indicated that bulk δ13C, δ2H and δ18O values were enough to significantly (P < 0.05) predict altitude, latitude, longitude, temperature, rainfall, and sea distance. This work showed that the assessment of EVOO isotopic composition give information not only on the geographic origin, but also on the environmental conditions. To the best of our knowledge, this is the first report on bulk isotopic composition of Portuguese EVOOs.This work was funded by European Regional Development Fund (FEDER) and National Funds through Foundation for Science and Technology (FCT) under Project “Por3O - Portuguese Olive Oil Omics for traceability and authenticity - PTDC/AGRPRO/2003/2014, and by National Funds through FCT - Foundation for Science and Technology under the Projects UIDB/05183/2020 and UID/AGR/00690/2019. Pedro N. Jiménez-Morillo is acknowledged for statistical assistance.info:eu-repo/semantics/publishedVersio

Biological and immunological characterization of recombinant Yellow Fever 17D Viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome

<p>Abstract</p> <p>Background</p> <p>The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a <it>Trypanosoma cruzi </it>CD8⁺T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8⁺T cell epitope was cloned and expressed based on two different genomic insertion sites: in the <it>fg </it>loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.</p> <p>Results</p> <p>Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by <it>T. cruzi </it>with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8⁺T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of <it>T. cruzi</it>.</p> <p>Conclusions</p> <p>We conclude that the YF 17D platform is useful to express <it>T. cruzi </it>(Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model <it>T. cruzi </it>epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.</p

Assessing the antitumor potential of variants of the extracellular carbohydrate polymer from synechocystis ΔsigF mutant

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.info:eu-repo/semantics/publishedVersio

The grieving process inherent to death from childhood to old age

Revista de Psicologia da Criança e do Adolescente. - ISSN 1647-4120. - V. 5, n. 2 (Julho-Dezembro 2014). - p. 31-42.Identificar as crenças da população face à morte e ao respetivo processo de luto e compará-las em 3 grupos etários: adolescentes, adultos e idosos. Amostra: 20 indivíduos de cada uma das faixas etárias, adolescentes, adultos e idosos (n=60), com uma média de idades de 16.5, 39.6 e 69.7 anos, respetivamente, sendo 39 dos sujeitos do género feminino. Resultados: As variáveis mais identificadas foram apoio familiar e/ou social (63,3%), evitar isolamento/ocupar o tempo e reorganizar a vida que favorece positivamente o luto (43,3%), resiliência e aceitação (33,3%), falta de resiliência e aceitação (36,7%), laços com a pessoa que faleceu que favorece negativamente o luto (28,3%) e falar e recordar que favorece negativamente o luto (26,7%). Conclusões: As reações e as formas de lidar com o luto variam de sujeito para sujeito, podendo ser observadas isoladamente ou em combinação e sendo influenciadas por fatores intrapessoais, interpessoais e extrapessoais, com variações ao longo da vida

Study of the cell growth inhibitory effect of aqueous extracts from Tuberaria lignosa in human tumor cell lines

Tuberaria lignosa (Sweet) Samp. (“alcária”), a plant mostly found in Western regions of the Iberian Peninsula, has antioxidant properties due to its composition in ascorbic acid and phenolic compounds [1]. Given these antioxidant properties, together with the traditional use of the plant to treat several diseases, the aims of this work were to: i) investigate if aqueous extracts from this plant, obtained by infusion and decoction, were inhibitors of cell growth in three human tumor cell lines; ii) study the cellular mechanism of action of the most potent extract. A screening of tumor cell growth inhibitory potential was performed with the Sulforhodamine B (SRB) assay using three different human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and HCT-15 (colorectal adenocarcinoma). Results showed that both aqueous extracts of T. lignosa decreased the growth of the cell lines tested and that the T. lignosa infusion extract was the most potent one, particularly in the NCI-H460 and HCT-15 cells. The T. lignosa infusion extract was further tested in the NCI-H460 cells. A determination of its effect on cell cycle profile was carried out, by analyzing cellular DNA content by flow cytometry following incubation with propidium iodide. Determination of cellular apoptosis was also performed, with the Annexin V-FICT and propidium iodide assay, and analyzed by flow cytometry. Preliminary results showed that the selected extract promoted a slight increase in the percentage of cells in the G1 phase of the cell cycle and induced cellular apoptosis. In conclusion, the T. lignosa extract decreased growth of the human tumor cell lines tested and the most potent effect was observed for the T. lignosa infusion extract. Future work will confirm if this effect is due mainly to induction of apoptosis

COVID-19 and hospitalizations for SARI in Brazil: a comparison up to the 12th epidemiological week of 2020.

Surveillance of the severe acute respiratory illness (SARI) in Brazil aims to characterize the circulation of the Influenza A and B viruses in hospitalized cases and deaths, having been expanded in 2012 to include other respiratory viruses. COVID-19 was detected in Brazil for the time in the 9th epidemiological week of 2020, and the test for the SARS-CoV-2 virus was included in the surveillance protocol starting in the 12th epidemiological week. This study's objective was to investigate the pattern of hospitalizations for SARI in Brazil since the entry of SARS-CoV-2, comparing the temporal and age profiles and laboratory results to the years 2010 through 2019. In 2020, hospitalizations for SARI, compiled from the date of the first confirmed case of COVID-19 up to the 12th week, exceeded the numbers observed during the same period in each of the previous 10 years. The age bracket over 60 years was the most heavily affected, at higher than historical levels. There was a considerable increase in negative laboratory tests, suggesting circulation of a different virus from those already present in the panel. We concluded that the increase in hospitalizations for SARI, the lack of specific information on the etiological agent, and the predominance of cases among the elderly during the same period in which there was an increase in the number of new cases of COVID-19 are all consistent with the hypothesis that severe cases of COVID-19 are already being detected by SARI surveillance, placing an overload on the health system. The inclusion of testing for SARS-CoV-2 in the SARI surveillance protocol and the test's effective nationwide deployment are extremely important for monitoring the evolution of severe COVID-19 cases in Brazil

Lateral hypothalamic activity indicates hunger and satiety states in humans

Lateral hypothalamic area (LHA) local field potentials (LFPs) were recorded in a Prader–Willi patient undergoing deep brain stimulation (DBS) for obesity. During hunger, exposure to food-related cues induced an increase in beta/ low-gamma activity. In contrast, recordings during satiety were marked by prominent alpha rhythms. Based on these findings, we have delivered alphafrequency DBS prior to and during food intake. Despite reporting an early sensation of fullness, the patient continued to crave food. This suggests that the pattern of activity in LHA may indicate hunger/satiety states in humans but attest to the complexity of conducting neuromodulation studies in obesity

Effects of orofacial myofunctional therapy on masticatory function in individuals submitted to orthognathic surgery: a randomized trial

Abstract Objectives: The esthetic and functional results of orthognathic surgery of severe dentofacial deformities are predictable, however there are differences regarding the effects on stomatognathic system. The aim was to investigate the effects of orofacial myofunctional therapy (OMT) on the masticatory function in individuals with dentofacial deformity submitted to orthognathic surgery (OGS). Material and Methods: Forty-eight individuals (18-40 years) were evaluated, 14 undergoing OMT (treated group-TG), 10 without this treatment (untreated group-UTG) and 24 in a control group with normal occlusion; for clinical aspects the data of an individual was missed (n=46). Chewing was performed using the Expanded protocol of orofacial myofunctional evaluation with scores (OMES-E). Muscle tone and mobility were also analyzed before (P0), three (P1) and six months (P2) after OGS. Surface electromyography of the masseter and temporalis muscles was performed, considering the parameters amplitude and duration of act and cycle, and the number of masticatory cycles. The OMT consisted of ten therapeutic sessions along the postoperative period. The results were compared using parametric and non-parametric tests. Results: TG showed higher scores in P1 and P2 than P0; for the masticatory type the scores in P2 were significantly higher than P0. In addition, the proportion of individuals with adequate tone of lower lip and adequate tongue mobility for TG increased significantly from P1 and P2 in relation to P0. The EMG results showed a decrease in act and cycle duration in P2 in relation to P0 and P1 for the TG; furthermore the values were close to controls. An increase in the number of cycles from P0 to P2 was also observed, indicating faster chewing, which may be attributed to an improvement of balanced occlusion associated with OMT. Conclusion: There were positive effects of OMT on the clinical and electromyography aspects of chewing in individual submitted to orthognathic surgery