Biossensor baseado em peptídeo mimético ligante de imunoglobulina G: uma plataforma para diagnóstico de artrite idiopática juvenil

Juvenile idiopathic arthritis (JIA) is a wide group of autoimmune and inflammatory diseases that affect children and adolescents under the age of sixteen. In the absence of proper diagnosis and treatment, irreversible damage may occur in the joint tissues. Biosensors emerge in the clinical scenario as promising analytical alternatives to the molecular diagnosis. In this work, an electrochemical biosensor was developed for the diagnosis of JIA using PRF+1 peptide, which has been shown to react against antibody from patients with JIA. The screenprinted electrode surface was activated by chronoamperometry prior to immobilization of the peptide. Electrochemical detections were conducted by differential pulse voltammetry and electrochemical impedance spectroscopy in phosphate buffer and/or potassium ferro/ferricyanide solution. Morphological alterations in the surfaces were studied by atomic force microscopy. The performance of the biosensor was evaluated by testing different serum specimen in different dilutions and stability during storage. The developed biosensor was able to discriminate samples of patients who were diagnosed as having JIA from those that were obtained from healthy individuals. The system presented linear range of response between the dilutions 1:25 and 1:300, sensitivity of 172,8 μC x DF, and limits of detection and quantification of 1:784 and 1:235, respectively. Moreover, the biosensor remained functional for up to 40 days of storage at 8°C. Therefore, a simple, miniaturized, functional, stable, selective and sensitive platform was developed and established as a promising strategy for molecular diagnosis.Conselho Nacional de Desenvolvimento Científico e TecnológicoCoordenação de Aperfeiçoamento de Pessoal de Nível SuperiorFundação de Amparo a Pesquisa do Estado de Minas GeraisDissertação (Mestrado)Artrite idiopática juvenil (JIA) corresponde a um amplo grupo de doenças autoimunes e inflamatórias que se desenvolve em crianças e adolescentes com menos de dezesseis anos de idade. Na ausência de diagnóstico e tratamento adequados, pode haver danos irreversíveis nos tecidos das articulações. Os biossensores inserem-se no cenário clínico como promissoras alternativas analíticas para o diagnóstico molecular. Neste trabalho, foi desenvolvido um biossensor eletroquímico para o diagnóstico de JIA, utilizando o peptídeo denominado de PRF+1, que em estudo anterior mostrou ser reativo com anticorpo de pacientes com JIA. A superfície dos eletrodos impressos foi ativada pelo método cronoamperométrico antes da imobilização do peptídeo. As detecções eletroquímicas foram conduzidas por voltametria de pulso diferencial e espectroscopia de impedância eletroquímica em solução tampão fosfato e/ou solução de ferro/ferricianeto de potássio. Modificações na morfologia da superfície dos sistemas foram estudadas por microscopia de força atômica. A performance do biossensor foi analisada frente a diferentes diluições de amostras de soro, uso de amostras de diferentes pacientes e estabilidade durante o armazenamento. O biossensor produzido foi capaz de discriminar amostras de diferentes pacientes com JIA dos controles saudáveis. O sistema apresentou faixa linear de resposta entre as diluições 1:25 e 1:300, sensibilidade de 172,8 μC x FD, e limites de detecção e quantificação de 1:784 e 1:235, respectivamente. Além disso, permaneceu funcional por até 40 dias de armazenamento a 8°C. Assim, foi desenvolvida uma plataforma simples, miniaturizada, funcional, estável, seletiva e sensível, estabelecendo-se como uma promissora estratégia de diagnóstico molecular

<em>Propionibacterium freudenreichii</em>: General Characteristics and Probiotic Traits

Propionibacterium freudenreichii is a Gram-positive dairy probiotic bacterial species that has been used as a ripening starter in the production of Swiss-type cheese for a long time. It has been exploited for the optimization of cheese production, including ripening capacities and aroma compounds production, but also for the production of vitamin B12 and organic acids. Furthermore, it has emerged in the probiotics landscape owing to several beneficial traits, including tolerance to stress in the gastrointestinal tract, adhesion to host cells, anti-pathogenic activity, anticancer potential and immunomodulatory properties. These beneficial properties have been confirmed with in vitro and in vivo investigations, using several omics approaches that allowed the identification of important molecular actors, such as surface proteins, short-chain fatty acids and bifidogenic factors. The diversity within the species was shown to be an important aspect to take into consideration, since many of these properties were strain-dependent. New studies should dive further into the molecular mechanisms related to the beneficial properties of this species and of its products, while considering the complexities of strain diversity and the interactions with the host and its microbiota. This chapter reviews current knowledge on the possible impact of P. freudenreichii on human health

Preliminary Characterization of Mitochondrial Genome of Melipona scutellaris, a Brazilian Stingless Bee

Bees are manufacturers of relevant economical products and have a pollinator role fundamental to ecosystems. Traditionally, studies focused on the genus Melipona have been mostly based on behavioral, and social organization and ecological aspects. Only recently the evolutionary history of this genus has been assessed using molecular markers, including mitochondrial genes. Even though these studies have shed light on the evolutionary history of the Melipona genus, a more accurate picture may emerge when full nuclear and mitochondrial genomes of Melipona species become available. Here we present the assembly, annotation, and characterization of a draft mitochondrial genome of the Brazilian stingless bee Melipona scutellaris using Melipona bicolor as a reference organism. Using Illumina MiSeq data, we achieved the annotation of all protein coding genes, as well as the genes for the two ribosomal subunits (16S and 12S) and transfer RNA genes as well. Using the COI sequence as a DNA barcode, we found that M. cramptoni is the closest species to M. scutellaris

Impact of Environmental Conditions on the Protein Content of Staphylococcus aureus and Its Derived Extracellular Vesicles

International audienceStaphylococcus aureus, a major opportunistic pathogen in humans, produces extracellular vesicles (EVs) that are involved in cellular communication, the delivery of virulence factors, and modulation of the host immune system response. However, to date, the impact of culture conditions on the physicochemical and functional properties of S. aureus EVs is still largely unexplored. Here, we use a proteomic approach to provide a complete protein characterization of S. aureus HG003, a NCTC8325 derivative strain and its derived EVs under four growth conditions: early- and late-stationary growth phases, and in the absence and presence of a sub-inhibitory concentration of vancomycin. The HG003 EV protein composition in terms of subcellular localization, COG and KEGG categories, as well as their relative abundance are modulated by the environment and differs from that of whole-cell (WC). Moreover, the environmental conditions that were tested had a more pronounced impact on the EV protein composition when compared to the WC, supporting the existence of mechanisms for the selective packing of EV cargo. This study provides the first general picture of the impact of different growth conditions in the proteome of S. aureus EVs and its producing-cells and paves the way for future studies to understand better S. aureus EV production, composition, and roles

Inhibition of Triple-Negative Breast Cancer Cell Aggressiveness by Cathepsin D Blockage: Role of Annexin A1

Triple-negative breast cancers (TNBCs) are more aggressive than other breast cancer (BC) subtypes and lack effective therapeutic options. Unraveling marker events of TNBCs may provide new directions for development of strategies for targeted TNBC therapy. Herein, we reported that Annexin A1 (AnxA1) and Cathepsin D (CatD) are highly expressed in MDA-MB-231 (TNBC lineage), compared to MCF-10A and MCF-7. Since the proposed concept was that CatD has protumorigenic activity associated with its ability to cleave AnxA1 (generating a 35.5 KDa fragment), we investigated this mechanism more deeply using the inhibitor of CatD, Pepstatin A (PepA). Fourier Transform Infrared (FTIR) spectroscopy demonstrated that PepA inhibits CatD activity by occupying its active site; the OH bond from PepA interacts with a CO bond from carboxylic acids of CatD catalytic aspartate dyad, favoring the deprotonation of Asp33 and consequently inhibiting CatD. Treatment of MDA-MB-231 cells with PepA induced apoptosis and autophagy processes while reducing the proliferation, invasion, and migration. Finally, in silico molecular docking demonstrated that the catalytic inhibition comprises Asp231 protonated and Asp33 deprotonated, proving all functional results obtained. Our findings elucidated critical CatD activity in TNBC cell trough AnxA1 cleavage, indicating the inhibition of CatD as a possible strategy for TNBC treatment

Inulin diet uncovers complex diet-microbiota-immune cell interactions remodeling the gut epithelium

Abstract Background The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure. Methods Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment. Results We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk. Conclusion This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstrac

Shotgun metagenomics and systemic targeted metabolomics highlight indole-3-propionic acid as a protective gut microbial metabolite against influenza infection

International audienceThe gut-to-lung axis is critical during respiratory infections, including influenza A virus (IAV) infection. In the present study, we used high-resolution shotgun metagenomics and targeted metabolomic analysis to characterize influenza-associated changes in the composition and metabolism of the mouse gut microbiota. We observed several taxonomic-level changes on day (D)7 post-infection, including a marked reduction in the abundance of members of the Lactobacillaceae and Bifidobacteriaceae families, and an increase in the abundance of Akkermansia muciniphila. On D14, perturbation persisted in some species. Functional scale analysis of metagenomic data revealed transient changes in several metabolic pathways, particularly those leading to the production of short-chain fatty acids (SCFAs), polyamines, and tryptophan metabolites. Quantitative targeted metabolomics analysis of the serum revealed changes in specific classes of gut microbiota metabolites, including SCFAs, trimethylamine, polyamines, and indole-containing tryptophan metabolites. A marked decrease in indole-3-propionic acid (IPA) blood level was observed on D7. Changes in microbiota-associated metabolites correlated with changes in taxon abundance and disease marker levels. In particular, IPA was positively correlated with some Lactobacillaceae and Bifidobacteriaceae species (Limosilactobacillus reuteri, Lactobacillus animalis) and negatively correlated with Bacteroidales bacterium M7, viral load, and inflammation markers. IPA supplementation in diseased animals reduced viral load and lowered local (lung) and systemic inflammation. Treatment of mice with antibiotics targeting IPA-producing bacteria before infection enhanced viral load and lung inflammation, an effect inhibited by IPA supplementation. The results of this integrated metagenomic-metabolomic analysis highlighted IPA as an important contributor to influenza outcomes and a potential biomarker of disease severity