A versatile nanocarrierCubosomes, characterization, and applications

The impact of nanotechnology on the exponential growth of several research areas, particularly nanomedicine, is undeniable. The ability to deliver active molecules to the desired site could significantly improve the efficiency of medical treatments. One of the nanocarriers developed which has drawn researchers’ attention are cubosomes, which are nanosized dispersions of lipid bicontinuous cubic phases in water, consisting of a lipidic interior and aqueous domains folded in a cubic lattice. They stand out due to their ability to incorporate hydrophobic, hydrophilic, and amphiphilic compounds, their tortuous internal configuration that provides a sustained release, and the capacity to protect and safely deliver molecules. Several approaches can be taken to prepare this structure, as well as different lipids like monoolein or phytantriol. This review paper describes the different methods to prepare nanocarriers. As it is known, the physicochemical properties of nanocarriers are very important, as they influence their pharmacokinetics and their ability to incorporate and deliver active molecules. Therefore, an extensive characterization is essential to obtain the desired effect. As a result, we have extensively described the most common techniques to characterize cubosomes, particularly nanocarriers. The exceptional properties of the cubosomes make them suitable to be used in several applications in the biomedical field, from cancer therapeutics to imaging, which will be described. Taking in consideration the outstanding properties of cubosomes, their application in several research fields is envisaged.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, by Marie Skłodowska Curie grant (MSCA-RISE; FODIAC; 778388) and by European Regional Development Fund (ERDF) through the Competitiveness factors Operational program—Norte 2020, COMPETE and National Funds through the FCT—under the project AgriFood XXI (NORTE- 01-0145-FEDER-000041). J.L.P. acknowledge the Instituto de Salud Carlos III for a “Sara Borrell” grant (CD19/00250), cofounded by European Social Fund (“El FSE invierte en futuro”). C.J.O.F. acknowledge the FCT for the grant SFRH/149/BD/14199/2019.info:eu-repo/semantics/publishedVersio

Microglial depletion has no impact on disease progression in a mouse model of machado–joseph disease

Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor.Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/127828/2016). B.C. was also supported by FCT (2020.03898.CEECIND). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Genetic diversity of Brazilian isolates of feline immunodeficiency virus

We isolated Feline immunodeficiency virus (FIV) from three adult domestic cats, originating from two open shelters in Brazil. Viruses were isolated from PBMC following co-cultivation with the feline T-lymphoblastoid cell line MYA-1. All amplified env gene products were cloned directly into pGL8MYA. The nucleic acid sequences of seven clones were determined and then compared with those of previously described isolates. The sequences of all of the Brazilian virus clones were distinct and phylogenetic analysis revealed that all belong to subtype B. Three variants isolated from one cat and two variants were isolated from each of the two other cats, indicating that intrahost diversity has the potential to pose problems for the treatment and diagnosis of FIV infection

Alginate-lysozyme nanofibers hydrogels with improved rheological behavior, printability and biological properties for 3D bioprinting applications

In this study, alginate nanocomposite hydrogel bioinks reinforced with lysozyme nanofibers (LNFs) were developed. Alginate-LNF (A-LNF) suspensions with different LNF contents (1, 5 and 10 wt.%) were prepared and pre-crosslinked with 0.5% (w/v) CaCl2 to formulate A-LNF inks. These inks exhibit proper shear-thinning behavior and good recovery properties (~90%), with the pre-crosslinking step playing a crucial role. A-LNF fully crosslinked hydrogels (with 2% (w/v) CaCl2) that mimic 3D printing scaffolds were prepared, and it was observed that the addition of LNFs improved several properties of the hydrogels, such as the morphology, swelling and degradation profiles, and mechanical properties. All formulations are also noncytotoxic towards HaCaT cells. The printing parameters and 3D scaffold model were then optimized, with A-LNF inks showing improved printability. Selected A-LNF inks (A-LNF0 and A-LNF5) were loaded with HaCaT cells (cell density 2 × 106 cells mL-1), and the cell viability within the bioprinted scaffolds was evaluated for 1, 3 and 7 days, with scaffolds printed with the A-LNF5 bioink showing the highest values for 7 days (87.99 ± 1.28%). Hence, A-LNF bioinks exhibited improved rheological performance, printability and biological properties representing a good strategy to overcome the main limitations of alginate-based bioinks.publishe

Intracellular autofluorescence as a new biomarker for cancer stem cells in glioblastoma

The identification of cancer stem cells (CSCs), which are implicated in tumor initiation, progression, therapy resistance, and relapse, is of great biological and clinical relevance. In glioblastoma (GBM), this is still a challenge, as no single marker is able to universally identify populations of GBM cancer stem cells (GSCs). Indeed, there is still controversy on whether biomarker-expressing cells fulfill the functional criteria of bona fide GSCs, despite being widely used. Here, we describe a novel subpopulation of autofluorescent (Fluo+) cells in GBM that bear all the functional characteristics of GSCs, including higher capacity to grow as neurospheres, long-term self-renewal ability, increased expression of stem cell markers, and enhanced in vivo tumorigenicity. Mechanistically, the autofluorescent phenotype is largely due to the intracellular accumulation of riboflavin, mediated by the ABC transporter ABCG2. In summary, our work identifies an intrinsic cellular autofluorescent phenotype enriched in GBM cells with functional stem cells features that can be used as a novel, simple and reliable biomarker to target these highly malignant tumors, with implications for GBM biological and clinical research.This research was funded by FEDER funds through the Operational Programme Competitiveness Factors–COMPETE and National Funds through FCT under the projects UIDB/50026/2020, UIDP/50026/2020, and POCI-01-0145-FEDER-007038; by the project NORTE-01-0145-FEDER-000013, NORTE-01-0246-FEDER-000012, and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.V.d.C., C.S.G., E.P.M., and B.M.C. was funded by FCT-Foundation for Science and Technology (SFRH/BD/88121/2012 to J.V.d.C.; SFRH/BD/92786/2013 to C.S.G.; PD/BDE/143154/2019 to E.P.M.; and PTDC/SAUGMG/113795/2009, IF/00601/2012 and CEECIND/00072/2018 to B.M.C.). B.M.C. was also funded by Fundação Calouste Gulbenkian and Liga Portuguesa Contra o Cancro

Coleção de microrganismos de interesse para a agroindústria tropical (CMIAT): implementação de requisitos da qualidade.

A criação da Rede de Recursos Genéticos Microbianos (Rede Microbiana), em 2009, permitiu a integração de coleções de culturas microbianas das UDs, por meio da proposição de novos modelos de organização, com fusões de coleções que apresentavam escopo semelhante e a sua distribuição em três categorias: (1) Centro de Recursos Biológicos (CRB); (2) Coleção Institucional (CI); e Coleção de Trabalho (CT), quanto às suas características e ao seu potencial para prestação de serviços (Pontes et al., 2015). s. Nesse contexto, o objetivo deste trabalho foi relatar as atividades desenvolvidas na Coleção de Microrganismos de Interesse para a Agroindústria Tropical (CMIAT) no escopo da implementação do modelo de gestão de coleções de microrganismos da Embrapa.bitstream/item/215782/1/CT-268.pd

Global Analyses Of Ceratocystis Cacaofunesta Mitochondria: From Genome To Proteome.

The ascomycete fungus Ceratocystis cacaofunesta is the causal agent of wilt disease in cacao, which results in significant economic losses in the affected producing areas. Despite the economic importance of the Ceratocystis complex of species, no genomic data are available for any of its members. Given that mitochondria play important roles in fungal virulence and the susceptibility/resistance of fungi to fungicides, we performed the first functional analysis of this organelle in Ceratocystis using integrated omics approaches. The C. cacaofunesta mitochondrial genome (mtDNA) consists of a single, 103,147-bp circular molecule, making this the second largest mtDNA among the Sordariomycetes. Bioinformatics analysis revealed the presence of 15 conserved genes and 37 intronic open reading frames in C. cacaofunesta mtDNA. Here, we predicted the mitochondrial proteome (mtProt) of C. cacaofunesta, which is comprised of 1,124 polypeptides - 52 proteins that are mitochondrially encoded and 1,072 that are nuclearly encoded. Transcriptome analysis revealed 33 probable novel genes. Comparisons among the Gene Ontology results of the predicted mtProt of C. cacaofunesta, Neurospora crassa and Saccharomyces cerevisiae revealed no significant differences. Moreover, C. cacaofunesta mitochondria were isolated, and the mtProt was subjected to mass spectrometric analysis. The experimental proteome validated 27% of the predicted mtProt. Our results confirmed the existence of 110 hypothetical proteins and 7 novel proteins of which 83 and 1, respectively, had putative mitochondrial localization. The present study provides the first partial genomic analysis of a species of the Ceratocystis genus and the first predicted mitochondrial protein inventory of a phytopathogenic fungus. In addition to the known mitochondrial role in pathogenicity, our results demonstrated that the global function analysis of this organelle is similar in pathogenic and non-pathogenic fungi, suggesting that its relevance in the lifestyle of these organisms should be based on a small number of specific proteins and/or with respect to differential gene regulation. In this regard, particular interest should be directed towards mitochondrial proteins with unknown function and the novel protein that might be specific to this species. Further functional characterization of these proteins could enhance our understanding of the role of mitochondria in phytopathogenicity.149