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A angiogênese é o processo de formação de novos vasos sanguíneos a partir de vasos pré-existentes. A angiogênese fisiológica é bastante restrita durante a fase adulta e ela ocorre sob delicado controle da expressão gênica. Doenças dependentes de angiogênese, como retinopatias e câncer, induzem fatores capazes de alterar a expressão desses genes, promovendo um desenvolvimento desordenado de novos vasos sanguíneos, sendo esse processo caracterizado como angiogênese patológica. Sabe-se que o fator de crescimento endotelial vascular (VEGF) é um dos principais componentes que orquestram a angiogênese e, portanto, esse fator tornou-se um alvo terapêutico para o tratamento de doenças dependentes da angiogênese. Essas terapias são eficazes, mas uma fração importante dos pacientes é refratária ou torna-se resistente à terapia anti-VEGF. Outra dificuldade são os efeitos adversos, uma vez que o VEGF também é importante para a sobrevivência das células endoteliais. Por isso, terapias alternativas mais eficientes e com menos efeitos colaterais são necessárias. Nas últimas décadas, com os avanços das tecnologias genômicas, constatou-se que os RNAs não-codificantes (ncRNAs) têm papel central na regulação gênica, incluindo a angiogênese. Com isso em mente, o presente trabalho buscou identificar ncRNAs importantes na angiogênese para produzir uma rede integrada de mRNAs e ncRNAs da angiogênese patológica. Foram produzidas duas redes de regulação gênica, onde cada uma representa uma fase da neovascularização: (1) a resposta inicial à hipóxia e (2) a consequente proliferação, migração e diferenciação das células endoteliais durante o processo de neovascularização. A medicina de precisão é uma área que busca desenvolver terapias direcionadas para cada paciente. Por exemplo, qual paciente responde ou não a determinado medicamento. Utilizando os genes identificados nas duas redes, desenvolvemos 3 assinaturas gênicas de angiogênese com valor prognóstico para pacientes com câncer de mama, outra doença dependente da angiogênese. Paralelamente, foram identificadas 60 variantes diferencialmente expressas e, portanto, possíveis alvos terapêuticos seletivos da angiogênese patológica. Coletivamente, os resultados desses estudos demonstram o potencial das redes gênicas produzidas para o desenvolvimento de novas terapias ou métodos diagnósticos para as doenças dependentes da angiogênese. Angiogenesis is the process of forming new blood vessels from pre-existing vessels. Physiological angiogenesis is quite restricted during adulthood, and it occurs under the delicate control of gene expression. Angiogenesis-dependent diseases, such as retinopathies and cancer, induce factors capable of altering the expression of these genes, promoting a disordered development of new blood vessels, this process being characterized as pathological angiogenesis. Vascular endothelial growth factor (VEGF) is known to be one of the main components that orchestrate angiogenesis and therefore this factor has become a therapeutic target for the treatment of angiogenesis-dependent diseases. These therapies are effective, but a significant fraction of patients is refractory or resistant to anti-VEGF therapy. Another difficulty is the adverse effects since VEGF is also important for the survival of endothelial cells. Therefore, more efficient alternative therapies with fewer side effects are needed. In recent decades, with advances in genomic technologies, it was found that non-coding RNAs (ncRNAs) play a central role in gene regulation, including angiogenesis. The present work sought to identify ncRNAs important in angiogenesis to produce an integrated network of pathological angiogenesis mRNAs and ncRNAs. Two gene regulatory networks were produced, each representing a phase of neovascularization: (1) the initial response to hypoxia and (2) the consequent proliferation, migration, and differentiation of endothelial cells during the process of neovascularization. Precision medicine is an area that seeks to develop targeted therapies for each patient. For example, which patient responds or does not respond to a particular drug. Using the genes identified in the two networks, we developed 3 angiogenesis gene signatures with prognostic value for patients with breast cancer, another angiogenesis-dependent disease. In parallel, 60 differentially expressed isoforms were identified and, therefore, possible selective therapeutic targets of pathological angiogenesis. Collectively, the results of these studies demonstrate the potential of the gene networks produced for the development of new therapies or diagnostic methods for angiogenesis-dependent diseases.

Background There have been significant improvements in Chagas disease therapy and it is now widely accepted that most patients with chronic disease might benefit from therapy. However, there are challenges to monitor drug efficacy and cure for these patients, which are important impediments for current and future therapies. Trypanosoma cruzi-PCR is highly variable while IgG seroconversion takes decades yielding variable results depending on the antigen(s) used for the assay. Methods and results We used the genomic phage display (gPhage) platform to perform a pairwise comparison of antigens and epitopes recognized by twenty individual patients with chronic Chagas disease before and after treatment with benznidazole. In total, we mapped 54,473 T. cruzi epitopes recognized by IgG from individual patients (N = 20) before benznidazole treatment. After treatment, the number of epitopes recognized by all patients was significantly smaller (21,254), a reduction consistent with a decrease in anti-T. cruzi antibodies. Most of these epitopes represent distinct fragments from the same protein and could, therefore, be grouped into 80 clusters of antigens. After three years of treatment with benznidazole, we observed a 64% reduction in the number of clusters of antigens recognized by patients (59 clusters before versus 21 clusters after treatment). The most abundant antigenic clusters recognized by patients correspond to the surface antigen CA-2 (B13) followed by the microtubule associated antigen, which highlights the value of these epitopes in Chagas disease diagnosis. Most importantly, quantitative pairwise comparison of gPhage data allowed for the prediction of patient response to treatment based on PCR status. Principal finding Here, we compiled a list of antigens and epitopes preferentially recognized by Chagas disease patients before and after benznidazole treatment. Next, we observed that gPhage data correlated with patient PCR-status and could, therefore, predict patient response to treatment. Moreover, gPhage results suggest that overall, independent of PCR status, treatment led to a reduction in the presence of T. cruzi-specific antibody levels and the number of antigens and epitopes recognized by these patients. Conclusion The gPhage platform use of unbiased library of antigens, which is different from conventional serological assays that rely on predetermined antigens, is a contribution for the development of novel diagnostic tools for Chagas disease.

Summary Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens., Graphical abstract, Highlights • Genomic shotgun phage display (gPhage) of eukaryotes is feasible and promising. • gPhage allows rapid antigen ID and epitope mapping, including 3D structures. • Conformation epitopes can be identified and validated by using the gPhage platform. • Most Chagas disease antigens are hypothetical proteins rich in repetitive elements., Parasitology; Sequence analysis; Systems biology

The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10−21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies., Author summary Angiogenesis is the formation of new blood vessels from pre-existing ones and it is the process for revascularization that predominates in adult life. Although angiogenesis is essential in physiological processes, such as wound healing, it also participates in pathological conditions, collectively known as angiogenesis-dependent diseases. Cancer and retinopathies are examples of such diseases. In cancer, angiogenesis is considered one of its hallmarks as tumors depend on a constant supply of oxygen and nutrients to grow. The more angiogenic the tumor, the more aggressive it is for the patient. In the present work, we used a well-suited animal model of retinopathy, an angiogenesis-dependent disease, to study the molecular signature for angiogenesis. We analyzed differences in gene expression from retinas developing under physiological and in retinopathic conditions to identify a molecular gene signature for angiogenesis. Because of the association between these two diseases, we tested our gene signature using data from human cancer. We showed that our gene signature has a robust prognostic value to predict breast cancer patient survival. Our results may lead to a better understanding of molecular mechanisms of pathological angiogenesis and its contribution to breast cancer.

Gills are considered a key player in organism defenses against environmental pollutants. Since it is the major site of uptake of waterborne chemicals, the modulation of important cellular defenses is expected in this tissue. Chlorothalonil, a fungicide presented in herbicides and antifouling paints, might be responsible for toxicity in marine biota. In this context, mussels were exposed to 0.1 μgL−1 and 10 μgL−1 of chlorothalonil for 24 h and 96 h. Genes from biotransformation and antioxidant defense pathways were investigated. Overall, we report, for the first time, an increase in the transcripts of the AhR-like, SULT1A1-like, CYP1A2-like, GSTO-like, MGST-like and SOD-like genes in the gills of the brown mussel Perna perna. This up-regulation was observed mostly after 96 h of exposure to chlorothalonil. Those results reinforce the important role of gills in xenobiotic metabolism and suggest the involvement of the mentioned genes in the detoxification of the compound. Throughout biotransformation and antioxidant defenses pathway, mussels exposed to chlorothalonil are activating mechanisms of defense against this contaminant.

O mexilhão marrom Perna perna (Linnaeus, 1758) auxilia no monitoramento de compostos químicos em ecossistemas marinhos. No entanto, os mecanismos moleculares de detoxificação e resposta ao estresse são desconhecidos. Elucidar esses mecanismos é crucial para entender os efeitos tóxicos dos poluentes químicos e desenvolver biomarcadores para avaliar a qualidade ambiental dos ecossistemas marinhos. No presente estudo, indivíduos da espécie P. perna foram expostos a antraceno (ANT) e os RNAs mensageiros (mRNA) das brânquias foram sequenciados com a plataforma Illumina. A análise química do tecido mole dos animais identificou concentrações de ANT 268 a 715 vezes mais alta no grupo exposto comparado ao grupo controle, demonstrando que a exposição foi realizada com sucesso. O sequenciamento do transcritoma do P. perna gerou 273.152.390 pares de reads, resultando na montagem de 231.728 contigs com tamanho médio de 720 pb e N50 de 1.083 pb, os quais 66.563 contigs (28,7%) pode ser anotado utilizando banco de dados como GenBank, Pfam, Gene Ontology e KEGG. Os resultados obtidos a partir da anotação funcional sugerem que as brânquias tenham papel na biotransformação de xenobióticos, resposta antioxidante, sinalização, resposta imunológica inata, e osmorregulação. Foi possível identificar genes de biotransformação de fase I, II e III, incluindo CYPs e GSTs. Transcritos similares a CYPs e GSTs estavam sendo expressos no grupo exposto, porém nenhum deles foram classificados como diferencialmente expressos. Contudo, muitos genes hipotéticos foram diferencialmente expressos, o que sugere que P. perna utilize mecanismos desconhecidos de biotransformação para lidar com a contaminação de ANT. Genes de sistema imune inato foram regulados tanto positivamente quanto negativamente, assim como observado para Perna viridis exposto a benzo(a)pireno, sugerindo que ANT promove alterações da capacidade de resposta do sistema imune inato do P. perna. The brown mussel Perna perna (Linnaeus, 1758) helps the monitoring of chemical compounds in marine ecosystems. However its molecular mechanisms of detoxification and stress response remain unclear. Elucidating these mechanisms is crucial to understand the toxic effects of chemical pollutants and to develop biomarkers to assess marine ecosystems. In this study, P. perna individuals were exposed to anthracene (ANT) and its mRNA complement was sampled sequenced with Illumina technology. Chemical analysis of the soft tissue identified ANT concentrations 268 - 715 fold higher in the exposed group compared to controls, demonstrating that the exposure procedure was successfully accomplished. Transcriptome sequencing of P. perna generated 273.152.390 paired reads that were assembled in 231.728 contigs of average length 720 bp and N50 1.083 bp , which 66.563 contigs (28,7%) could be annotated using GenBank genes, Pfam domains, Gene Ontology (GO) terms and KEGG pathways. The results obtained from functional annotation suggest gills play a role in xenobiotics biotransformation, antioxidant response, signal transduction, innate immune response, and osmoregulation. It was possible to identify transcripts similar to genes related with biotransformation reactions of phases I, II and III, including CYPs and GSTs. Transcripts similar to CYPs and GSTs isoforms were highly expressed in the group exposed to ANT, however no CYP, GST, or even other genes related with biotransformation reactions were classified as differentially expressed. On the other hand, several hypothetical genes were differentially expressed, which suggests that P. perna uses unknown mechanisms of biotransformation to deal with ANT stress contamination. Immune related-genes were both up and down-regulated, as was also observed for Perna viridis exposed to benzo(a)pyrene, suggesting that ANT promotes alteration in the immune response of P. perna.