Targeting cells with cathelicidin nanomedicines improves insulin function and pancreas regeneration in type 1 diabetic rats

Type 1 diabetes (T1D) is an incurable condition with an increasing incidence worldwide, in which the hallmark is the autoimmune destruction of pancreatic insulin-producing β cells. Cathelicidin-based peptides have been shown to improve β cell function and neogenesis and may thus be relevant while developing T1D therapeutics. In this work, a cathelicidin-derived peptide, LLKKK18, was loaded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), surface-functionalized with exenatide toward a GLP-1 receptor, aiming the β cell-targeted delivery of the peptide. The NPs present a mean size of around 100 nm and showed long-term stability, narrow size distribution, and negative ζ-potential (−10 mV). The LLKKK18 association efficiency and loading were 62 and 2.9%, respectively, presenting slow and sustained in vitro release under simulated physiologic fluids. Glucose-stimulated insulin release in the INS-1E cell line was observed in the presence of the peptide. In addition, NPs showed a strong association with β cells from isolated rat islets. After administration to diabetic rats, NPs induced a significant reduction of the hyperglycemic state, an improvement in the pancreatic insulin content, and glucose tolerance. Also remarkable, a considerable increase in the β cell mass in the pancreas was observed. Overall, this novel and versatile nanomedicine showed glucoregulatory ability and can pave the way for the development of a new generation of therapeutic approaches for T1D treatment.C.C., S.P., and J.M. acknowledge FCT for the granted scholarships SFRH/BD/139402/2018, SFRH/BD/144719/2019, and PD/BD/145149/2019, respectively. The authors acknowledge the support of the i3S Scientific Platform Histology and Electron Microscopy (HEMS), member of the national infrastructure PPBI─Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122), in particular, Nuno Mendes for performing the immunohistochemistry assay, and Tiago Bordeira Gaspar for the blind analysis of the tissue sections and for the antiglucagon antibody used in immunohistochemistry analysis.info:eu-repo/semantics/publishedVersio

Membrane and nuclear estrogen receptors in sea bass provide insight to explore genomic and non-genomic estrogenic actions: the mineralized scale example

The numerous estrogen functions across vertebrates have been classically explained by binding to nuclear estrogen receptors (ERs) regulating the transcription of responsive genes. It is now known that estrogenic compounds can also produce rapid non-genomic actions initiated by binding to plasma estrogen membrane receptors, such as the recently identified G protein-coupled estrogen receptor1 (GPER). Sea bass (Dicentrarchus labrax) express three ER subtype genes, one esr1 and two esr2 genes that appear to have been originated from the original esr2 gene in the teleost-specific whole genome duplication. We have recently identified two genes for GPER in the sea bass genome and phylogenetic analyses also suggest they are teleost-specific gene duplicates. Quantitative PCR revealed a wide tissue distribution for the five receptors in both male and female sea bass and expression throughout the reproductive cycle in brain and pituitary, although with subtype-specific and seasonal differences. When analyzing the sea bass scales, mineralized structures previously shown to be estrogen-responsive, a different receptor repertoire and regulation was detected compared to liver, a classical target gene. In juvenile sea bass scales, the main forms expressed were esr2a and gperb, which were also up regulated after injection with the natural estrogen estradiol (E2) and the phytoestrogen genistein (Gen). Both rapid (30 min) and slow (1 day or more) changes in the activities of enzymes related to mineral turnover were detected in fish scales in response to E2, Gen and xenoestrogens and the gene networks activated 1-5 days after injection of E2 and Gen are being characterized by transcriptomics, revealing both common and compound-specific effects at the transcriptional level. Functional characterization of the three sea bass ER subtypes and two GPERs is underway in mammalian cells, to allow to compare their signaling to different estrogenic compounds. These studies will help to understand the normal estrogen regulation of fish scale functions as well as its possible disruption by phytoestrogens and other xenoestrogens and the relative importance of genomic and non-genomic mechanisms of action of the five receptors.info:eu-repo/semantics/publishedVersio

Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroids

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotrophin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisseptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Additionally, there is evidence that the galaninergic system in the brain and testis of pre-pubertal male sea bass is a possible mediator involved in the translation of somatic signals leading to gonadal maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologues. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes 15 and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17β-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog in fish reproductive function

Mutations in the Schmallenberg virus Gc glycoprotein facilitate cellular protein synthesis shutoff and restore pathogenicity of NSs deletion mutants in mice

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach higher titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild type SBV as has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an IFN antagonist. Therefore SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by the infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins while the ability to control the production of IFN maps to the NSs protein. Importance The identification of viral determinants of pathogenesis is key to the development of prophylactic and interventions measures. In this study we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore pathogenicity of attenuated mutants resulting from deletions or mutations in the non-structural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of non-structural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes

Effects of mutations in the effector domain of influenza A virus NS1 protein.

OBJECTIVE: The multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs. RESULTS: The NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1's effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment

Patient-physician discordance in assessment of adherence to inhaled controller medication: a cross-sectional analysis of two cohorts

We aimed to compare patient's and physician's ratings of inhaled medication adherence and to identify predictors of patient-physician discordance.(SFRH/BPD/115169/2016) funded by Fundação para a Ciência e Tecnologia (FCT); ERDF (European Regional Development Fund) through the operations: POCI-01-0145-FEDER-029130 ('mINSPIRERS—mHealth to measure and improve adherence to medication in chronic obstructive respiratory diseases—generalisation and evaluation of gamification, peer support and advanced image processing technologies') cofunded by the COMPETE2020 (Programa Operacional Competitividade e Internacionalização), Portugal 2020 and by Portuguese Funds through FCT (Fundação para a Ciência e a Tecnologia).info:eu-repo/semantics/publishedVersio

Temas de investigação em direitos humanos para o século XXI

Edição comemorativa do 10.º aniversário do Mestrado em Direitos Humanos da Universidade do Minho.Este livro é uma celebração do ensino e da investigação em direitos humanos que têm vindo a ser desenvolvidos, na Escola de Direito da Universidade do Minho, há já mais de uma década. A sua publicação num momento em que se avolumam os riscos para valores fundamentais subjacentes à proteção dos direitos humanos – como a igualdade e a não discriminação, a proibição da escravatura e de tratamentos cruéis, desumanos e degradantes, a liberdade de religião ou crença, entre muitos outros –, torna-o especialmente oportuno. Os sinais de aparente retrocesso no consenso das nossas sociedades a respeito desses valores – visíveis no triunfo político de discursos abertamente racistas, xenófobos, sexistas, etc. – recordam-nos que, também no mundo ocidental, os direitos humanos são um work in progress, não um dado adquirido. Os novos riscos para a dignidade da pessoa humana associados aos avanços tecnológicos andam de par com velhas formas de subalternização e de opressão. O campo para a reflexão crítica é muito vasto. Os temas que hoje (pre)ocupam académicos, decisores políticos e ativistas de direitos humanos são também aqueles que estruturam o plano de estudos do Mestrado em Direitos Humanos da Universidade do Minho. Todos estes temas surgem ao longo do presente livro, que reúne contributos de muitos dos membros da comunidade científica e académica que o Mestrado em Direitos Humanos mobilizou e ajudou a dinamizar ao longo da última década, entre docentes do Mestrado, colaboradores em júris de provas públicas e/ou na orientação de mestrandos, oradores convidados e estudantes. Os textos aqui reunidos refletem bem as sinergias interdisciplinares, interinstitucionais e inter-nacionais que o Mestrado em Direitos Humanos foi capaz de criar, não apenas pela variedade de campos disciplinares representados – Direito, Filosofia, Relações Internacionais, Antropologia –, mas também pela participação de autores que são docentes e/ou investigadores em diversas instituições nacionais e estrangeiras, como a Faculdade de Direito da Universidade do Porto, a Faculdade de Economia da Universidade de Coimbra, a Universidade Federal da Paraíba e a Pontifícia Universidade Católica de São Paulo.info:eu-repo/semantics/publishedVersio

IL-6 is constitutively expressed during lung morphogenesis and enhances fetal lung explant branching

Previous studies have shown that chorioamnionitis, with increased IL-6, promotes fetal lung maturation and decreases the incidence of respiratory distress syndrome in premature neonates. However, the expression pattern and the effects of IL-6 on fetal lung growth mechanisms remain unknown. IL-6 expression was assessed by in situ hybridization and by real-time PCR between 14.5 and 21.5 d postconception. Normal and nitrofen-induced hypoplastic lung explants were cultured with increasing IL-6 doses or IL-6 neutralizing antibodies. Branching, cellular proliferation (Ki-67) and MAPK phosphorylation in fetal lung explants were analyzed. Pulmonary primitive epithelium expressed IL-6 constitutively throughout all gestational ages, displaying highest levels during earliest stages. In normal and hypoplastic lung explants, IL-6 neutralizing antibodies significantly reduced, whereas IL-6 supplementation induced a biphasic effect (lower doses increased, while the highest dose did not accomplish additional effect) on branching and cellular proliferation. IL-6 enhanced p38-MAPK phosphorylation without changing MEK1/2 and JNK pathways. The present study suggests a physiological role for IL-6 on pulmonary branching mechanisms most likely involving p38-MAPK intracellular signalling pathway