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ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes , herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector. During microbial infection, proteins are modified by the ubiquitin-like protein ISG15. Here, the authors uncover RNF213 as a sensor for ISGylated proteins on the surface of lipid droplets, showing that RNF213 has antiviral properties but also directly targets intracellular bacteria in infected cells.

ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity. Here, we map the endogenous in vivo ISGylome in the liver following Listeria mono-cytogenes infection by combining murine models of reduced or enhanced ISGylation with quantitative proteomics. Our method identifies 930 ISG15 sites in 434 proteins and also detects changes in the host ubiquitylome. The ISGylated targets are enriched in proteins which alter cellular metabolic processes, including upstream modulators of the catabolic and antibacterial pathway of autophagy. Computational analysis of substrate structures reveals that a number of ISG15 modifications occur at catalytic sites or dimerization interfaces of enzymes. Finally, we demonstrate that animals and cells with enhanced ISGylation have increased basal and infection-induced autophagy through the modification of mTOR, WIPI2, AMBRA1, and RAB7. Taken together, these findings ascribe a role of ISGylation to temporally reprogram organismal metabolism following infection through direct modification of a subset of enzymes in the liver.

Objectives This study aimed to estimate diarrheagenic Escherichia coli (DEC) prevalence among pediatric patients with diarrhea at the Costa Rican National Children’s Hospital-Social Security Service (Hospital Nacional de Niños-Caja Costarricense del Seguro Social; HNN-CCSS). DEC variations with respect to rainfall, presence of coinfections, and DEC antimicrobial resistance were also investigated. Results A retrospective observational study from January 2008 to December 2016 was conducted. A total of 12 247 gastroenteritis records were analyzed. Annual DEC prevalence ranged from 2.7% (2008) to 9.0% (2013). The most prevalent pathotypes were enteroaggregative E. coli (EAEC) [n = 189 (31%)], enteropathogenic E. coli (EPEC) [n = 145 (24%)] and enteroinvasive E . coli (EIEC) [n = 91 (15%)]. A reduction in the probability of EAEC gastroenteritis was detected as rainfall rose above 200 mm/mo. [(Generalized Additive Model (GAM), p = 0.04)]. Coinfections were observed mainly between EPEC and Campylobacter spp. (10%). Antimicrobial resistance occurred in 0.6%, 29%, and 42% of DEC for ciprofloxacin, trimethoprim/sulfamethoxazole, and ampicillin, respectively.

Alzheimer’s disease is a challenge in modern healthcare due to its complex etiology and increasing prevalence. Despite advances, further understanding of Alzheimer’s disease pathophysiology is needed, particularly the role of Aβ neurotoxic peptide. Fourier transform infrared spectroscopy (FTIR) has shown potential as a screening tool for several pathologies, including Alzheimer’s disease. Nonetheless, limited research has explored Aβ direct effects on neurons and extracellular vesicles metabolic profiles. Hence, this study aims to investigate Aβ impact on the spectroscopic profiles of neuronal-like cells (N2a) and N2a-derived extracellular vesicles, employing FTIR spectroscopy and focusing on the 1280–900 cm−1 region. A comprehensive analysis of spectral data was carried out, including multivariate partial least squares (PLS) analysis and peak intensities analysis. PLS analysis revealed moderate to strong correlations within this spectral region for both N2a and N2a-derived extracellular vesicles. The peak intensity analysis revealed additional peaks with significant differences in EVs’ spectra relative to N2a, following Aβ treatment. Specifically, Aβ seems to cause alterations in protein phosphorylation and in the nucleic acids content of extracellular vesicles. These findings support that Aβ’s role in Alzheimer’s disease pathology may be mediated by extracellular vesicles and highlight FTIR’s potential for advancing Alzheimer’s disease research and clinical applications.

Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E‐NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR‐Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high affinity for mCherry. FLAG‐tag‐based immunocapture from cell conditioned media allowed selection of cargo‐loaded E‐NoMi‐EVs, and tobacco etch virus (TEV) protease cleavage sites were used to remove the 3xFLAG‐tag from the surface of E‐NoMi‐EVs after capture. For functional payload delivery to recipient cells, the vesicular stomatitis virus G (VSV‐G) fusogenic protein was incorporated into E‐NoMi‐EVs to form fusogenic EV‐based vectors (EVVs) and proved to be 10‐fold more effective at cargo delivery than EVs generated by size‐exclusion chromatography. Functional delivery of cargo with E‐NoMi‐EVVs was validated in two mouse brain models in vivo.

Phosphorylation plays a key role in Alzheimer’s disease (AD) pathogenesis, impacting distinct processes such as amyloid-beta (Aβ) peptide production and tau phosphorylation. Impaired phosphorylation events contribute to senile plaques and neurofibrillary tangles’ formation, two major histopathological hallmarks of AD. Blood-derived extracellular particles (bdEP) can represent a disease-related source of phosphobiomarker candidates, and hence, in this pilot study, bdEP of Control and AD cases were analyzed by a targeted phosphoproteomics approach using a high-density microarray that featured at least 1145 pan-specific and 913 phosphosite-specific antibodies. This approach, innovatively applied to bdEP, allowed the identification of 150 proteins whose expression levels and/or phosphorylation patterns were significantly altered across AD cases. Gene Ontology enrichment and Reactome pathway analysis unraveled potentially relevant molecular targets and disease-associated pathways, and protein-protein interaction networks were constructed to highlight key targets. The discriminatory value of both the total proteome and the phosphoproteome was evaluated by univariate and multivariate approaches. This pilot experiment supports that bdEP are enriched in phosphotargets relevant in an AD context, holding value as peripheral biomarker candidates for disease diagnosis.

No âmbito da unidade curricular “Estágio” do Mestrado Integrado em Ciências Farmacêuticas da Faculdade de Farmácia da Universidade de Coimbra, o presente documento apresenta, sob a forma de uma análise SWOT(Pontos Fortes, Pontos Fracos, Oportunidades e Ameaças), o relatório de estágio na Farmácia Universal, em Coimbra, com início a 16 de Abril e término a 17 de Agosto de 2018, sob orientação do Dr. Pedro Baptista e o relatório de estágio na Direção de Avaliação de Tecnologias de Saúde do INFARMED - Autoridade Nacional do Medicamento e Produtos de Saúde, I.P., em Lisboa, com início a 11 de Janeiro e término a 30 de Março de 2018 , sob orientação da professora Cláudia Furtado. Este documento inclui ainda uma monografia intitulada “Drug-resistant Epilepsy: An overview on the relevance of ABC Transporters Superfamily”A epilepsia fármaco-resistente afeta aproximadamente um terço dos doentes epiléticos. Das várias teorias subjacentes à epilepsia fármaco-resistente, a teoria dos transportadores de efluxo é uma das mais extensivamente estudadas. Consequentemente, o aumento da expressão de transportadores de efluxo na barreira hemato-encefálica, principalmente da superfamília de transportadores ATP binding cassette(ABC), é responsável por dificultar o acesso de fármacos antiepiléticos ao cérebro. Sabe-se que existe expressão exacerbada de glicoproteína P em células endoteliais da barreira hemato-encefálica, astrócitos e neurónios da unidade neurovascular, células predominantemente envolvidas na penetração cerebral de fármacos. A progressão patológica da epilepsia, as crises recorrentes não controladas, a indução de transportadores de efluxo pelos próprios fármacos e os polimorfismos genéticos nestes transportadores estão entre as possíveis causas da expressão acentuada dos transportadores ABC em doentes com epilepsia fármaco- resistente. Para evitar a ação dos transportadores de efluxo, várias abordagens estão sob investigação: a inibição direta destes transportadores, o uso da tecnologia de RNA de interferência ou a inibição do eixo glutamato/receptor NMDA/COX-2.

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominant neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats in the ATXN3 gene. This mutation induces a toxic gain-of-function of the ATXN3 protein, leading to neurodegeneration, particularly in the cerebellum and brainstem. Despite extensive research, no disease-modifying treatments are available for SCA3 patients. In this study, we developed and tested a novel therapeutic strategy using recombinant adeno-associated virus (rAAV) to deliver bicistronic artificial microRNAs designed to selectively silence the mutant ATXN3 allele. Through in vitro screening, we identified a lead construct (miATXN3-10x2) that effectively and specifically silenced the mutant allele by targeting of a single nucleotide polymorphism (SNP) associated with the repeat expansion. This construct was packaged into rAAV9 and delivered via intra-cerebellar administration into two mouse models of SCA3, resulting in robust suppression of mutant ATXN3 in the cerebellum. To assess long-term efficacy, we performed intra-cisterna magna (ICM) injections of rAAV9-miATXN3-10x2 in a severe SCA3 transgenic mouse model. Widespread distribution of viral vectors and miATXN3 copies was observed in disease-relevant brain regions. Treated animals exhibited significant and sustained improvements in motor function at 5, 8, and 11 weeks post-injection. Histological analyses showed a reduction in mutant ATXN3 aggregates and a trend toward preventing shrinkage of cerebellar molecular layer. These findings were supported by dose-dependent reductions in mutant ATXN3 mRNA levels and decreased expression of neuroinflammatory markers in the cerebellum. Additionally, a significant increase of the neuronal marker NeuN was also observed in treated animals. Finally, transcriptomic profiling of the cerebellum demonstrated that treated transgenic animals exhibited an improved transcriptomic signature, shifting toward a wild-type profile. In conclusion, our findings highlight the therapeutic potential of a single administration of rAAVs encoding bicistronic artificial microRNAs for allele-specific gene silencing in SCA3. This study provides compelling preclinical evidence supporting the translation of this approach into clinical applications for SCA3 patients.

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named “EV-AAVs”. EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.