RESUMO
Lysergic acid diethylamide (LSD) is a synthetic psychedelic compound with potential therapeutic value for psychiatric disorders. This study aims to establish Caenorhabditis elegans as an in vivo model for examining LSD's effects on locomotor behavior. Our results demonstrate that LSD is absorbed by C. elegans and that the acute treatment reduces animal speed, similar to the role of endogenous serotonin. This response is mediated in part by the serotonergic receptors SER-1 and SER-4. Our findings highlight the potential of this nematode as a new experimental model in psychedelic research.
Assuntos
Caenorhabditis elegans , Alucinógenos , Dietilamida do Ácido Lisérgico , Animais , Caenorhabditis elegans/efeitos dos fármacos , Dietilamida do Ácido Lisérgico/farmacologia , Alucinógenos/farmacologia , Locomoção/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Comportamento Animal/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Serotonina/metabolismoRESUMO
Psychedelic compounds have gained renewed interest for their potential therapeutic applications, but their metabolism and effects on complex biological systems remain poorly understood. Here, we present a systematic characterization of Lysergic Acid Diethylamide (LSD) metabolites in the model organism Caenorhabditis elegans using state-of-the-art analytical techniques. By employing ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry, we putatively identified a range of LSD metabolites, shedding light on their metabolic pathways and offering insights into their pharmacokinetics. Our study demonstrates the suitability of Caenorhabditis elegans as a valuable model system for investigating the metabolism of psychedelic compounds and provides a foundation for further research on the therapeutic potential of LSD.
Assuntos
Caenorhabditis elegans , Alucinógenos , Animais , Cromatografia Líquida de Alta Pressão , Dietilamida do Ácido Lisérgico , Espectrometria de Massas em TandemRESUMO
Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barré syndrome. One of the most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits such as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of coinfections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3913 proteins identified, were also able to quantify 1044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary. Furthermore, from a total of 480 mosquito proteins modulated in our study, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, coinfection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti. The modified aspects mainly were related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganism's infection.
Assuntos
Aedes , Coinfecção , Wolbachia , Infecção por Zika virus , Zika virus , Aedes/microbiologia , Animais , Feminino , Humanos , Recém-Nascido , Mosquitos Vetores , Ovário , ProteômicaRESUMO
Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.
Assuntos
Antígenos de Fungos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Proteínas Fúngicas/metabolismo , Imunoterapia/métodos , Macrófagos/imunologia , Paracoccidioides/fisiologia , Paracoccidioidomicose/imunologia , Animais , Antígenos de Fungos/genética , Proliferação de Células , Células Cultivadas , Resistência à Doença , Proteínas Fúngicas/genética , Humanos , Ativação Linfocitária , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/terapia , Peptídeos/genética , Peptídeos/metabolismoRESUMO
Single-cell analysis came to change the way we look at cell populations. RNA sequencing of single cells allowed us to appreciate the diversity of cell types in the human brain in an unprecedented manner and its power to reveal cell-type specific changes in cell populations has just begun to be explored. In this context, looking at the proteome of single cells promises to bring functional information and contribute to completing the picture. The potential of single cell proteome, in developing a better understanding of the intricate connections between the very diverse cell populations in the brain, is huge. Whereas early approaches to address single-cell proteome have identified hundreds of proteins, today, techniques combining isobaric labelling and LC-MS can lead to the identification of thousands of proteins. In this review, we describe methods which have been used to identify and quantify proteins from single cells and propose that the application of isobaric labeling and label-free quantitative proteomics approach for single-cell analysis is ready to provide useful information for the neurobiology field.
Assuntos
Neurobiologia/tendências , Proteômica/métodos , Análise de Célula Única/métodos , Animais , Cromatografia Líquida/métodos , Humanos , Proteoma/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.
Assuntos
Axônios/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Neurais/metabolismo , Axônios/patologia , Encéfalo/metabolismo , Proliferação de Células/fisiologia , Cromatografia Líquida/métodos , Humanos , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ZIKV in the A. aegypti heads and salivary glands. We show that Wolbachia regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in mosquitoes was determined by MS and corroborates the idea that Wolbachia helps to block ZIKV infections in A. aegypti. The present study offers a rich resource of data that may help to elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in A. aegypti, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and Wolbachia directly in complex tissues.
RESUMO
BACKGROUND: Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined. RESULTS: Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask. CONCLUSION: Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.
Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos/métodos , Organoides/crescimento & desenvolvimento , Estresse Fisiológico/fisiologia , Linhagem Celular , Humanos , Hidrodinâmica , Organoides/citologia , Resistência ao Cisalhamento/fisiologiaRESUMO
Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.