Coronavirus disease 2019 (COVID-19) is an acute viral disease with millions of cases worldwide. Although the number of daily new cases and deaths has been dropping, there is still a need for therapeutic alternatives to deal with severe cases. A promising strategy to prospect new therapeutic candidates is to investigate the regulatory mechanisms involved in COVID-19 progression using integrated transcriptomics approaches. In this work, we aimed to identify COVID-19 Master Regulators (MRs) using a series of publicly available gene expression datasets of lung tissue from patients which developed the severe form of the disease. We were able to identify a set of six potential COVID-19 MRs related to its severe form, namely TAL1, TEAD4, EPAS1, ATOH8, ERG, and ARNTL2. In addition, using the Connectivity Map drug repositioning approach, we identified 52 different drugs which could be used to revert the disease signature, thus being candidates for the design of novel clinical treatments. Furthermore, we compared the identified signature and drugs with the ones obtained from the analysis of nasopharyngeal swab samples from infected patients and preclinical cell models. This comparison showed significant similarities between them, although also revealing some limitations on the overlap between clinical and preclinical data in COVID-19, highlighting the need for careful selection of the best model for each disease stage.

Neurodevelopmental disorders (NDDs) present symptoms associated with abnormal brain development, onset of which typically occurs during infancy or childhood. Here, we discuss the current knowledge regarding isolated cannabinoids and Cannabis treatment during infancy and adolescence in NDDs. Endogenous and plant-derived cannabinoids target the G-protein-coupled cannabinoid receptors (CB1 and 2). The cannabinoid receptors, endocannabinoids, and their related enzymes constitute the endocannabinoid system (ECS). The ECS is active early in brain development, undergoing neurodevelopment until reaching adolescence, a period associated with increased vulnerability to cannabinoid exposure. The deleterious effects of Cannabis are mostly related to delta-9-tetrahydrocannabinol (Δ9-THC), which has psychoactive properties. In contrast, cannabidiol (CBD) is devoid of psychotomimetic effects, being beneficial toward a number of brain functions. The ECS might be involved either in the pathophysiology of NDDs and in protective mechanisms. Cannabinoid exposure can potentially disrupt the neurodevelopmental processes mediated by the ECS. Indeed, cannabinoids have shown to produce both improvements and adverse effects depending on the condition. Cannabis is associated with neurodevelopmental impairments in bipolar disorder and schizophrenia. CBD presents a general safer profile and is effective for some infantile epileptic syndromes and for the most common comorbidities of NDDs such as sleep disorder, anxiety, and seizures. For psychosis, addictive behavior, mood or cognitive disorders, and aggressiveness, the evidence of CBD's effectiveness is limited. Side effects occur, though severe ones are rare. To provide reliable treatment recommendations, more high-quality clinical trials are required to address the beneficial and adverse effects of cannabinoids in young patients.

Parkinson's disease (PD) is among the most prevalent neurodegenerative diseases. Available evidences support the view of PD as a complex disease, being the outcome of interactions between genetic and environmental factors. In face of diagnosis and therapy challenges, and the elusive PD etiology, the use of alternative methodological approaches for the elucidation of the disease pathophysiological mechanisms and proposal of novel potential therapeutic interventions has become increasingly necessary. In the present study, we first reconstructed the transcriptional regulatory networks (TN), centered on transcription factors (TF), of two brain regions affected in PD, the substantia nigra pars compacta (SNc) and the frontal cortex (FCtx). Then, we used case-control studies data from these regions to identify TFs working as master regulators (MR) of the disease, based on region-specific TNs. Twenty-nine regulatory units enriched with differentially expressed genes were identified for the SNc, and twenty for the FCtx, all of which were considered MR candidates for PD. Three consensus MR candidates were found for SNc and FCtx, namely ATF2, SLC30A9, and ZFP69B. In order to search for novel potential therapeutic interventions, we used these consensus MR candidate signatures as input to the Connectivity Map (CMap), a computational drug repositioning webtool. This analysis resulted in the identification of four drugs that reverse the expression pattern of all three MR consensus simultaneously, benperidol, harmaline, tubocurarine chloride, and vorinostat, thus suggested as novel potential PD therapeutic interventions.

BACKGROUND:Alzheimer's disease (AD) is a multifactorial and complex neuropathology that involves impairment of many intricate molecular mechanisms. Despite recent advances, AD pathophysiological characterization remains incomplete, which hampers the development of effective treatments. In fact, currently, there are no effective pharmacological treatments for AD. Integrative strategies such as transcription regulatory network and master regulator analyses exemplify promising new approaches to study complex diseases and may help in the identification of potential pharmacological targets.METHODS:In this study, we used transcription regulatory network and master regulator analyses on transcriptomic data of human hippocampus to identify transcription factors (TFs) that can potentially act as master regulators in AD. All expression profiles were obtained from the Gene Expression Omnibus database using the GEOquery package. A normal hippocampus transcription factor-centered regulatory network was reconstructed using the ARACNe algorithm. Master regulator analysis and two-tail gene set enrichment analysis were employed to evaluate the inferred regulatory units in AD case-control studies. Finally, we used a connectivity map adaptation to prospect new potential therapeutic interventions by drug repurposing.RESULTS:We identified TFs with already reported involvement in AD, such as ATF2 and PARK2, as well as possible new targets for future investigations, such as CNOT7, CSRNP2, SLC30A9, and TSC22D1. Furthermore, Connectivity Map Analysis adaptation suggested the repositioning of six FDA-approved drugs that can potentially modulate master regulator candidate regulatory units (Cefuroxime, Cyproterone, Dydrogesterone, Metrizamide, Trimethadione, and Vorinostat).CONCLUSIONS:Using a transcription factor-centered regulatory network reconstruction we were able to identify several potential molecular targets and six drug candidates for repositioning in AD. Our study provides further support for the use of bioinformatics tools as exploratory strategies in neurodegenerative diseases research, and also provides new perspectives on molecular targets and drug therapies for future investigation and validation in AD.

Research on Parkinson’s disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host–parasite relationship. In this work, we performed an LC–MS/MS proteomic analysis of the excretory–secretory products obtained from the first 48h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory–secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory–secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory–secretory products, revealing a set of identified proteins not previously thought to be exposed at the host–parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory–secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory–secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.

BACKGROUND:Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.METHODOLOGY/PRINCIPAL FINDINGS:The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.CONCLUSIONS/SIGNIFICANCE:For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.