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The epigenetic changes associated with melanoma progression to advanced and metastatic stages are still poorly understood. To shed light on the CpG methylation dynamics during melanoma development, we analyzed the methylome profiles of a four-stage cell line model of melanoma progression: non-tumorigenic melanocytes (melan-a), premalignant melanocytes (4C), non-metastatic melanoma cells (4C11−), and metastatic melanoma cells (4C11+). We identified 540 hypo- and 37 hypermethylated gene promoters that together characterized a malignancy signature, and 646 hypo- and 520 hypermethylated promoters that distinguished a metastasis signature. Differentially methylated genes from these signatures were correlated with overall survival using TCGA-SKCM methylation data. Moreover, multivariate Cox analyses with LASSO regularization identified panels of 33 and 31 CpGs, respectively, from the malignancy and metastasis signatures that predicted poor survival. We found a concordant relationship between DNA methylation and transcriptional levels for genes from the malignancy ( Pyroxd2 and Ptgfrn ) and metastasis ( Arnt2 , Igfbp4 and Ptprf ) signatures, which were both also correlated with melanoma prognosis. Altogether, this study reveals novel CpGs methylation markers associated with malignancy and metastasis that collectively could improve the survival prediction of melanoma patients.

Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

A growing body of evidence suggests a key role of tumor microenvironment, especially for bone marrow mesenchymal stem cells (MSC), in the maintenance and progression of multiple myeloma (MM), through direct and indirect interactions with tumor plasma cells. Thus, this study aimed to investigate the gene expression and functional alterations of MSC from MM patients (MM-MSC) in comparison with their normal counterparts from normal donors (ND-MSC). Gene expression analysis (Affymetrix) was performed in MM-MSC and ND-MSC after in vitro expansion. To validate these findings, some genes were selected to be evaluated by quantitative real time PCR (RT-qPCR), and also functional in vitro analyses were performed. We demonstrated that MM-MSC have a distinct gene expression profile than ND-MSC, with 485 differentially expressed genes (DEG) - 280 upregulated and 205 downregulated. Bioinformatics analyses revealed that the main enriched functions among downregulated DEG were related to cell cycle progression, immune response activation and bone metabolism. Four genes were validated by qPCR - ZNF521 and SEMA3A , which are involved in bone metabolism, and HLA-DRA and CHIRL1 , which are implicated in the activation of immune response. Taken together, our results suggest that MM-MSC have constitutive abnormalities that remain present even in the absence of tumors cells. The alterations found in cell cycle progression, immune system activation, and osteoblastogenesis suggest, respectively, that MM-MSC are permanently dependent of tumor cells, might contribute to immune evasion and play an essential role in bone lesions frequently found in MM patients.

Treatment-resistant schizophrenia (TRS) occurs in one-third of the patients, but the molecular determinants of poor antipsychotic response remain unclear. We compared genetic data of patients with TRS ( n  = 63) with non-TRS ( n  = 111) by polygenic risk scores (PRS) calculated by PRSice software using PGC2_SCZ (Psychiatric Genomics Consortium – Schizophrenia) data. TRS criteria followed the International Psychopharmacology Algorithm Project SCZ algorithm. Statistical clustering and functional enrichment analyses of genes harboring TRS-linked variants were performed. Individuals on the top three deciles of schizophrenia PRS distribution exhibited higher odds of being refractory to antipsychotics than those on the bottom three deciles. Clusters of interacting variant-harboring genes were identified among the association signals. They are upregulated in the dorsolateral prefrontal, orbitofrontal, temporal, and inferior parietal areas during adolescence and early adulthood. Similar gene modules were found using transcriptional data from the same brain regions in individuals with schizophrenia. Genes were enriched among markers of cortical interneurons and somatosensory pyramidal cells. Finally, the enrichment of the clustered genes in drug-response expression signatures revealed compounds that could be employed to identify novel antipsychotic targets. In conclusion, we identified variant-harboring genes that may predispose SCZ patients to poor antipsychotic response and found statistically enriched clusters which provided functional and spatiotemporal context for TRS, suggesting that genotypic variation may converge to biological alterations at the interplay between actin dynamics and synaptic organization.

One aspirational goal of computational chemistry is to predict potent and drug-like binders for any protein, such that only those that bind are synthesized. In this Roadmap, we describe the launch of Critical Assessment of Computational Hit-finding Experiments (CACHE), a public benchmarking project to compare and improve small-molecule hit-finding algorithms through cycles of prediction and experimental testing. Participants will predict small-molecule binders for new and biologically relevant protein targets representing different prediction scenarios. Predicted compounds will be tested rigorously in an experimental hub, and all predicted binders as well as all experimental screening data, including the chemical structures of experimentally tested compounds, will be made publicly available and not subject to any intellectual property restrictions. The ability of a range of computational approaches to find novel binders will be evaluated, compared and openly published. CACHE will launch three new benchmarking exercises every year. The outcomes will be better prediction methods, new small-molecule binders for target proteins of importance for fundamental biology or drug discovery and a major technological step towards achieving the goal of Target 2035, a global initiative to identify pharmacological probes for all human proteins. Critical Assessment of Computational Hit-finding Experiments (CACHE) is a public benchmarking project to compare and improve computational small-molecule hit-finding approaches through cycles of prediction, compound synthesis and experimental testing. By that, CACHE will enable a more efficient and effective approach to hit identification and drug discovery.

The topical use of sunscreens is recommended for avoiding the damaging effects of UV radiation. However, improvements are still needed in the existing products to enhance their photoprotection effectiveness and safety. This involves minimizing the use of chemical UV filters while providing enhanced and prolonged photoprotection. This work investigated novel sunscreen formulations and their UV protection effects by encapsulating Uvinul® A, Tinosorb® S, and Uvinul® T150 into nanostructured lipid carriers (NLCs) based on bacuri butter and raspberry seed oil. First, the impact of critical formulation and process parameters on NLCs’ particle size was evaluated using a 22 Face Centered Central Composite Design. Then, formulations were evaluated in terms of critical quality factors, in vitro skin permeation, and in vitro and in vivo photoprotection activities. The developed NLCs-containing formulations exhibited appropriate size (122–135 nm), PdI (<0.3), encapsulation efficiency (>90%), and drug content (>80%), which were preserved for at least 90 days under different stability conditions. Moreover, these NLCs-based formulations had equivalent skin permeation to emulsion-based controls, and the addition of NLCs into sunscreen cream bases in the optimum proportion of 20% (w/w) resulted in enhanced UVA and UVB photoprotection levels, despite a 10% reduction in the total filters content. Altogether, these results describe the application of nanoencapsulated organic UV filters in innovative sunscreen formulations to achieve superior photoprotection and cosmeceutical properties.

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of Angiotensin II (Ang II) receptors has been associated with poor prognosis in human astrocytomas, the most common type of glioma. In this study, we investigated the role of Angiotensin II in glioma malignancy through transcriptional profiling and network analysis of cultured C6 rat glioma cells exposed to Ang II and to inhibitors of its membrane receptor subtypes. C6 cells were treated with Ang II and specific antagonists of AT1 and AT2 receptors. Total RNA was isolated after three and six hours of Ang II treatment and analyzed by oligonucleotide microarray technology. Gene expression data was evaluated through transcriptional network modeling to identify how differentially expressed (DE) genes are connected to each other. Moreover, other genes co-expressing with the DE genes were considered in these analyses in order to support the identification of enriched functions and pathways. A hub-based network analysis showed that the most connected nodes in Ang II-related networks exert functions associated with cell proliferation, migration and invasion, key aspects for glioma progression. The subsequent functional enrichment analysis of these central genes highlighted their participation in signaling pathways that are frequently deregulated in gliomas such as ErbB, MAPK and p53. Noteworthy, either AT1 or AT2 inhibitions were able to down-regulate different sets of hub genes involved in protumoral functions, suggesting that both Ang II receptors could be therapeutic targets for intervention in glioma. Taken together, our results point out multiple actions of Ang II in glioma pathogenesis and reveal the participation of both Ang II receptors in the regulation of genes relevant for glioma progression. This study is the first one to provide systems-level molecular data for better understanding the protumoral effects of Ang II in the proliferative and infiltrative behavior of gliomas.

Complex febrile seizures during infancy constitute an important risk factor for development of epilepsy. However, little is known about the alterations induced by febrile seizures that make the brain susceptible to epileptic activity. In this context, the use of animal models of hyperthermic seizures (HS) could allow the temporal analysis of brain molecular changes that arise after febrile seizures. Here, we investigated temporal changes in hippocampal gene coexpression networks during the development of rats submitted to HS. Total RNA samples were obtained from the ventral hippocampal CA3 region at four time points after HS at postnatal day (P) 11 and later used for gene expression profiling. Temporal endpoints were selected for investigating the acute (P12), latent (P30 and P60) and chronic (P120) stages of the HS model. A weighted gene coexpression network analysis was used to characterize modules of coexpressed genes, as these modules might contain genes with similar functions. The transcriptome analysis pipeline consisted of building gene coexpression networks, identifying network modules and hubs, performing gene-trait correlations and examining changes in module connectivity. Modules were functionally enriched to identify functions associated with HS. Our data showed that HS induce changes in developmental, cell adhesion and immune pathways, such as Wnt, Hippo, Notch, Jak-Stat and Mapk. Interestingly, modules involved in cell adhesion, neuronal differentiation and synaptic transmission were activated as early as 1 day after HS. These results suggest that HS trigger transcriptional alterations that could lead to persistent neurogenesis, tissue remodeling and inflammation in the CA3 hippocampus, making the brain prone to epileptic activity.

The mechanisms triggering renal inflammation in chronic kidney disease (CKD) are unclear. We performed a detailed analysis of the time course of innate and adaptive immunity activation in the 5/6 renal ablation (Nx) model. Munich-Wistar rats undergoing Nx were studied 15, 60 and 120 days after ablation. Hypertension, albuminuria, creatinine retention, interstitial expansion and infiltration by macrophages and T-lymphocytes were already evident 15 days after Nx. PCR-array was used to screen for altered gene expression, whereas gene and protein expressions of TLR4, CASP1, IL-1β and NLRP3 were individually assessed. Tlr4 , Tlr5 , Lbp , Nlrp3 , Casp1 , Irf7 and Il1b were already upregulated 15 days after Nx, while activation of Tlr2 , Tlr7 , Tlr9 , Nod2 , Tnf and Il6 was seen after 60 days post-ablation. The number of genes related to innate or adaptive immunity grew steadily with time. These observations indicate that parallel activation of innate and adaptive immunity antecedes glomerular injury and involves a growing number of intricate signaling pathways, helping to explain the difficulty in detaining renal injury in Nx as CKD advances, and, stressing the need for early treatment. Additionally, these findings may contribute to the search of therapeutic targets specific for advanced phases of CKD.