Heterologous Expression of Immature Forms of Human Islet Amyloid Polypeptide in Yeast Triggers Intracellular Aggregation and Cytotoxicity

Funding: iNOVA4Health – UID/Multi/04462/2019, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is acknowledged. Funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), is gratefully acknowledged. This study was also supported by FCT via PTDC/BIA-MOL31104/2017, UID/Multi/04462/2013- SubProj iNOVA4Health 44, and UID/Multi/04462/2019-SubProj iNOVA4Health C44 to RM, PD/BD/135504/2018 to AR. Sociedade Portuguesa de Diabetologia for the Nuno CasteloBranco Prize – 2016 attributed to RM is also acknowledged.Diabetes is a major public health issue that has attained alarming levels worldwide. Pancreatic aggregates of human islet amyloid polypeptide (IAPP) represent a major histopathological hallmark of type 2 diabetes. IAPP is expressed in β-cells as pre-pro-IAPP (ppIAPP) that is first processed to pro-IAPP (pIAPP) and finally to its mature form (matIAPP), being released upon glucose stimulation together with insulin. Impairment and overload of the IAPP processing machinery seem to be associated with the accumulation of immature IAPP species and the formation of toxic intracellular oligomers, which have been associated with β-cell dyshomeostasis and apoptosis. Nevertheless, the pathological importance of these immature IAPP forms for the assembly and cytotoxicity of these oligomers is not completely understood. Here, we describe the generation and characterization of unprecedented Saccharomyces cerevisiae models recapitulating IAPP intracellular oligomerization. Expression of green fluorescent protein (GFP) fusions of human ppIAPP, pIAPP, and matIAPP proved to be toxic in yeast cells at different extents, with ppIAPP exerting the most deleterious effect on yeast growth and cell viability. Although expression of all IAPP constructs induced the formation of intracellular aggregates in yeast cells, our data point out the accumulation of insoluble oligomeric species enriched in immature ppIAPP as the trigger of the high toxicity mediated by this construct in cells expressing ppIAPP-GFP. In addition, MS/MS analysis indicated that oligomeric species found in the ppIAPP-GFP lysates contain the N-terminal sequence of the propeptide fused to GFP. These models represent powerful tools for future research focused on the relevance of immature forms in IAPP-induced toxicity. Furthermore, they are extremely useful in high-throughput screenings for genetic and chemical modulators of IAPP aggregation.publishersversionpublishe

Antioxidant power of small fruits and its beneficial effects to human health

The fruits as well as other organs of plants in its constitution have various phytochemicals, including polyphenols, the antioxidant molecules highly abundant in foods. Despite its ubiquity, its benefits to human health only start to be evaluated in 90’s, and it continues to reveal a growing interest, mainly due to epidemiological studies suggesting a link between the consumption of food and beverages rich in polyphenols and the reducing of the incidence and/or initiation of chronic and degenerative diseases such as cardiovascular disease, atherosclerosis, and some cancers. The investigation of its neuroprotective effects have being developed in the past 10 years, and it have been clearly demonstrated their ability to protect the cells either in neuronal models in vitro and their in vivo intracellular effects. A review of the evidences will be presented and in particular the work being conducted in the laboratory of Disease and Stress Biology on the antioxidant power of berries such as wild blackberries, raspberry and strawberry tree fruit as well as their leave

Ethyl 3,5-dimethyl-1H-pyrrole-2-carboxyl­ate

In the title compound, C9H13NO2, there are two independent mol­ecules per asymmetric unit. The mol­ecules are very similar and almost planar, with the ethoxy­carbonyl group anti to the pyrrole N atom. The two independent mol­ecules are joined into dimeric units by strong hydrogen bonds between NH groups and carbonyl O atoms

Two-way attack on IAPP proteotoxicity with implications for diabetes

Funding Information: This study was supported by FCT–Fundação para a Ciência e a Tecnologia (grants UIDB/04567/2020 and UIDP/ 04567/2020 to CBIOS, PTDC/BIA-MOL/31104/2017, and PhD grants PD/BD/135504/2018 to AFR and UI/BD/151421/2021 to SF. RM is funded by FCT Scientific Employment Stimulus contract with the reference number CEEC/04567/ CBIOS/2020. Authors also acknowledge COFAC/ILIND – Cooperativa De Formação e Animação Cultural CRL/Instituto Lusófono de Investigação e Desenvolvimento (grant COFAC/ILIND/CBIOS/2/2021). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia (FCT) / Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged (UIDB/04462/2020 and UIDP/04462/2020). CNS acknowledge the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 804229. JAB gratefully acknowledges FCT-Fundação para a Ciência e a Tecnologia, I.P. through MOSTMICRO-ITQB R&D Unit-UIDB/04612/2020 and LS4FUTURE Associated Laboratory-LA/P/0087/2020, and by the framework of Article 23 of Decree-Law No.57/2017 of August 29. Publisher Copyright: Copyright © 2022 Raimundo, Ferreira, Pobre, Lopes-da-Silva, Brito, dos Santos, Saraiva, dos Santos and Menezes.Introduction: Diabetes is one of the major metabolic diseases worldwide. Despite being a complex systemic pathology, the aggregation and deposition of Islet Amyloid Polypeptide (IAPP), or amylin, is a recognized histopathological marker of the disease. Although IAPP proteotoxicity represents an important trigger of β-cell dysfunction and ultimately death, its exploitation as a therapeutic tool remains underdeveloped. The bioactivity of (poly)phenols towards inhibition of pathological protein aggregation is well known, however, most of the identified molecules have limited bioavailability. Methods: Using a strategy combining in silico, cell-free and cell studies, we scrutinized a unique in-house collection of (poly)phenol metabolites predicted to appear in the human circulation after (poly)phenols ingestion. Results: We identified urolithin B as a potent inhibitor of IAPP aggregation and a powerful modulator of cell homeostasis pathways. Urolithin B was shown to affect IAPP aggregation pattern, delaying the formation of amyloid fibrils and altering their size and morphology. The molecular mechanisms underlying urolithin B-mediated protection include protein clearance pathways, mitochondrial function, and cell cycle ultimately rescuing IAPP-mediated cell dysfunction and death. Discussion: In brief, our study uncovered urolithin B as a novel small molecule targeting IAPP pathological aggregation with potential to be exploited as a therapeutic tool for mitigating cellular dysfunction in diabetes. Resulting from the colonic metabolism of dietary ellagic acid in the human body, urolithin B bioactivity has the potential to be explored in nutritional, nutraceutical, and pharmacological perspectives.publishersversionpublishe

Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases

Arthritic diseases are disabling conditions affecting millions of patients worldwide. Pro-inflammatory cytokines, particularly interleukin-6 (IL-6), plays a crucial role in inflammation and cartilage destruction. Although the beneficial effects of antibody therapy, its efficacy is limited. Therefore, this work proposes the immobilization of antibodies at the surface of biodegradable polymeric nanoparticles (NPs) to capture and neutralize IL-6. Our system is intended to protect, extend and enhance the therapeutic efficacy after delivery. Chitosanâ hyaluronic acid NPs are synthesized as a stable monodisperse population. After determining the maximum immobilization capacity (10 μg/mL), the capture ability was confirmed. Biological assays demonstrate the NPs cytocompatibility with human articular chondrocytes (hACs) and human macrophages. hACs stimulated with macrophage conditioned medium shows the beneficial role of IL-6 capture and neutralization. Biofunctionalized NPs exhibit a prolonged action and stronger efficacy than the free antibody. In conclusion, this system can be an effective and long lasting treatment for arthritic diseases.FCT/MCTES (Portuguese Foundation for Science and Technology/Ministry of Science, Technology and Higher Education) and the FSE/ POCH (European Social Fund through the Operational Program of Human Capital), for the PhD scholarship PD/ BD/11384/2015 of A. C. Lima (PD/59/2013), the FCT for the grant of A. Carvalho (IF/00735/2014) and C. Carvalho (SFRH/BPD/96176/2013). Authors would also like to acknowledge FCT for the project PTDC/CTM-BIO/4388/2014SPARTAN, and the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER- 000023-FROnTHERA and NORTE-01-0145-FEDER-000013- PersonalizedNOS)info:eu-repo/semantics/publishedVersio

Chemical characterization and bioactivity of phytochemicals from Iberian endemic Santolina semidentata and strategies for ex situ propagation

Asteraceae family members are well-known for their medicinal potential, comprising several properties that make them unique among plants. Here we focus on Santolina semidentata, an endemic plant from the Iberian Peninsula, not yet described for its medicinal properties. Phytochemical characterization of S. semidentata was performed, concerning total phenol content, flavonoid content, antioxidant capacity, HPLC-DAD profile, acetylcholinesterase inhibitory capacity, cytotoxicity and neuroprotective effect in a human neurodegeneration cell model. Moreover, essential oil composition and antifungal activity were also analised. This oil might be useful for therapeutical purposes, particularly in the treatment of dermatophytosis. S. semidentata potential for neuroprotection was revealed by acetylcholinesterase inhibitory capacity and also by an effective protective effect in human neuronal cells. Furthermore, different seed conservation protocols, as well as successful in vitro propagation were established which may be useful when integrated in a broad strategy for the conservation of these endemic plants and their sustainable use for potential biotechnological applications. The results presented here greatly contribute to value this species regarding its potential as a source of phytochemicals with prospective neuroprotective health benefits, either as alternative neuroprotective drugs or as leads for synthetizing more effective molecules.The authors wish to thank to “Fundo EDP para a Biodiversidade” for financial support. This work was also supported by “Fundação para a Ciência e a Tecnologia” through grant PEst-OE/EQB/LA0004/2011, BGCT/33418/2008, Green-it: UID/Multi/04551/2013, iNOVA4Health: UID/Multi/04462/2013 and financial support to CNS (IF/01097/20132), RP (SFRH/BD/63615/2009), IF (SFRH/BD/86584/2012) and AG (SFRH/BD/103155/2014).info:eu-repo/semantics/publishedVersio

Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii

AbstractThis work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR–RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge

Pulmonary hemorrhage syndrome associated with dengue fever, High-resolution computed tomography findings: a case report

Dengue hemorrhagic fever is an acute infectious disease caused by dengue virus. We described the high-resolution CT findings in a 70-year-old male with the disease, which was diagnosed by clinical examination and confirmed by serological methods. High-resolution CT demonstrated bilateral areas of consolidation with air bronchogram and ground glass opacities, as well as small bilateral pleural effusions. Dengue hemorrhagic fever should be considered in the differential diagnosis of diffuse pulmonary hemorrhage

Acidity and characterization of 12-Tungstophosphoric acid supported on Silica-alumina

This work deals with preparation and characterization of H3PW12O40 (H3PW) supported on silica-alumina. Impregnation of H3PW (15, 20, 30 and 40 wt.%) on commercial silica-alumina support in acidic aqueous solution is effective for preparing this catalyst keeping its Keggin structure, according to different methods of characterization. The catalysts were tested in a model reaction of acetic acid with ethanol and 30 wt.% H3PW/SiO2-Al2O3 had the highest activity under the conditions: catalyst calcination at 300 ºC, temperature of 100 ºC, acetic acid:ethanol molar ratio of 2:1 and catalyst:acetic acid mass ratio of 10 wt.%. The reaction yield was 79 and 100% selectivity for ethyl acetate over three reutilizations, for reaction time of 2 h. The calculated total acid site distribution was 0.299 mmol g-1 (97% of the theoretical probed by pyridine), and most of these (0.236 mmol g-1) were Brønsted weak-medium strength (pyridine desorption between 300 and 500 ºC)