Evolução dos genes da rede OXT - AVP - PRL: Aspectos moleculares, fisiológicos e comportamentais em mamíferos

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos.The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades

FOXP in tetrapoda : intrinsically disordered regions, short linear motifs and their evolutionary significance

The FOXP subfamily is probably the most extensively characterized subfamily of the forkhead superfamily, playing important roles in development and homeostasis in vertebrates. Intrinsically disorder protein regions (IDRs) are protein segments that exhibit multiple physical interactions and play critical roles in various biological processes, including regulation and signaling. IDRs in proteins may play an important role in the evolvability of genetic systems. In this study, we analyzed 77 orthologous FOXP genes/proteins from Tetrapoda, regarding protein disorder content and evolutionary rate. We also predicted the number and type of short linear motifs (SLIMs) in the IDRs. Similar levels of protein disorder (approximately 70%) were found for FOXP1, FOXP2, and FOXP4. However, for FOXP3, which is shorter in length and has a more specific function, the disordered content was lower (30%). Mammals showed higher protein disorders for FOXP1 and FOXP4 than non-mammals. Specific analyses related to linear motifs in the four genes showed also a clear differentiation between FOXPs in mammals and non-mammals. We predicted for the first time the role of IDRs and SLIMs in the FOXP gene family associated with possible adaptive novelties within Tetrapoda. For instance, we found gain and loss of important phosphorylation sites in the Homo sapiens FOXP2 IDR regions, with possible implication for the evolution of human speech

Phase 2 study of buparlisib (BKM120), a pan-class I PI3K inhibitor, in patients with metastatic triple-negative breast cancer

Treatment options for triple-negative breast cancer remain limited. Activation of the PI3K pathway via loss of PTEN and/or INPP4B is common. Buparlisib is an orally bioavailable, pan-class I PI3K inhibitor. We evaluated the safety and efficacy of buparlisib in patients with metastatic triple-negative breast cancer. This was a single-arm phase 2 study enrolling patients with triple-negative metastatic breast cancer. Patients were treated with buparlisib at a starting dose of 100 mg daily. The primary endpoint was clinical benefit, defined as confirmed complete response (CR), partial response (PR), or stable disease (SD) for ≥ 4 months, per RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. A subset of patients underwent pre- and on-treatment tumor tissue biopsies for correlative studies. Fifty patients were enrolled. Median number of cycles was 2 (range 1-10). The clinical benefit rate was 12% (6 patients, all SD ≥ 4 months). Median PFS was 1.8 months (95% confidence interval [CI] 1.6-2.3). Median OS was 11.2 months (95% CI 6.2-25). The most frequent adverse events were fatigue (58% all grades, 8% grade 3), nausea (34% all grades, none grade 3), hyperglycemia (34% all grades, 4% grade 3), and anorexia (30% all grades, 2% grade 3). Eighteen percent of patients experienced depression (12% grade 1, 6% grade 2) and anxiety (10% grade 1, 8% grade 2). Alterations in PIK3CA / AKT1 / PTEN were present in 6/27 patients with available targeted DNA sequencing (MSK-IMPACT), 3 of whom achieved SD as best overall response though none with clinical benefit ≥ 4 months. Of five patients with paired baseline and on-treatment biopsies, reverse phase protein arrays (RPPA) analysis demonstrated reduction of S6 phosphorylation in 2 of 3 patients who achieved SD, and in none of the patients with progressive disease. Buparlisib was associated with prolonged SD in a very small subset of patients with triple-negative breast cancer; however, no confirmed objective responses were observed. Downmodulation of key nodes in the PI3K pathway was observed in patients who achieved SD. PI3K pathway inhibition alone may be insufficient as a therapeutic strategy for triple-negative breast cancer. Registered on 13 February 2013; . Registered on 27 June 2012

Role of BAFF in pulmonary autoantibody responses induced by chronic cigarette smoke exposure in mice

Emerging evidence suggests that autoimmune processes are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. We observed that BAFF levels were elevated in smokers and mice exposed to cigarette smoke. In mice, BAFF expression was rapidly induced in the lungs following 4 days of cigarette smoke exposure and remained elevated following 8 and 24 weeks of exposure. Alveolar macrophages were the major source of BAFF Blockade of BAFF using a BAFF receptor-Fc (BAFFR-Fc) construct prevented pulmonary ANA and TLT formation when delivered concurrent with cigarette smoke exposure. Under these conditions, no impact on lung inflammation was observed. However, administration of BAFFR-Fc following smoking cessation markedly reduced the number of TLTs and ANA levels and, of note, reduced pulmonary neutrophilia. Altogether, this study shows for the first time a central role of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggests that BAFF blockade following smoking cessation could have beneficial effects on persistent inflammatory processes.In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. Data presented show that BAFF plays a central role in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggest a therapeutic potential for BAFF blockade in limiting autoimmune processes associated with smoking

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Evolução dos genes da rede OXT - AVP - PRL: Aspectos moleculares, fisiológicos e comportamentais em mamíferos

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos.The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades

Oxytocin and arginine vasopressin systems in the domestication process

Domestication is of unquestionable importance to the technological revolution that has given rise to modern human societies. In this study, we analyzed the DNA and protein sequences of six genes of the oxytocin and arginine vasopressin systems (OXT-OXTR; AVP-AVPR1a, AVPR1b and AVPR2) in 40 placental mammals. These systems play an important role in the control of physiology and behavior. According to our analyses, neutrality does not explain the pattern of molecular evolution found in some of these genes. We observed specific sites under positive selection in AVPR1b ( = 1.429, p = 0.001) and AVPR2 ( = 1.49, p = 0.001), suggesting that they could be involved in behavior and physiological changes, including those related to the domestication process. Furthermore, AVPR1a, which plays a role in social behavior, is under relaxed selective constraint in domesticated species. These results provide new insights into the nature of the domestication process and its impact on the OXT-AVP system