Genetic Associations and Differential mRNA Expression Levels of Host Genes Suggest a Viral Trigger for Endemic Pemphigus Foliaceus

The long search for the environmental trigger of the endemic pemphigus foliaceus (EPF, fogo selvagem) has not yet resulted in any tangible findings. Here, we searched for genetic asso ciations and the differential expression of host genes involved in early viral infections and innate antiviral defense. Genetic variants could alter the structure, expression sites, or levels of the gene products, impacting their functions. By analyzing 3063 variants of 166 candidate genes in 227 EPF patients and 194 controls, we found 12 variants within 11 genes associated with differential suscepti bility (p < 0.005) to EPF. The products of genes TRIM5, TPCN2, EIF4E, EIF4E3, NUP37, NUP50, NUP88, TPR, USP15, IRF8, and JAK1 are involved in different mechanisms of viral control, for example, the regulation of viral entry into the host cell or recognition of viral nucleic acids and proteins. Only two of nine variants were also associated in an independent German cohort of sporadic PF (75 patients, 150 controls), aligning with our hypothesis that antiviral host genes play a major role in EPF due to a specific virus–human interaction in the endemic region. Moreover, CCL5, P4HB, and APOBEC3G mRNA levels were increased (p < 0.001) in CD4+ T lymphocytes of EPF patients. Because there is limited or no evidence that these genes are involved in autoimmunity, their crucial role in antiviral responses and the associations that we observed support the hypothesis of a viral trigger for EPF, presumably a still unnoticed flavivirus. This work opens new frontiers in searching for the trigger of EPF, with the potential to advance translational research that aims for disease prevention and treatment

O impacto funcional de uma variante genética em sítio de ligação a microRNAs em MDM4, um gene associado ao câncer de mama

Orientadora: Profa Dra Daniela Fiori GradiaCoorientadora: Profa Dra Karin Braun PradoDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa : Curitiba, 27/03/2020Inclui referências: p. 113-125Resumo: O câncer de mama é o segundo tipo de câncer de maior incidência no mundo, sendo o de maior mortalidade entre as mulheres. O controle da progressão tumoral é dado, entre outros fatores, pela ação de genes supressores tumorais, entre eles o gene TP53, essencial na resposta aos danos ao DNA. Dessa forma, mudanças nos níveis de p53 e de seus reguladores podem afetar a função como supressor de tumor e predispor ao desenvolvimento de câncer. As proteínas MDM2 e MDM4 participam dessa regulação, pois inibem a função de transativação de p53 bloqueando o sítio de ligação de fatores basais de transcrição, além de participar da via de poliubiquitinação marcando a proteína p53 para posterior degradação por proteossomo. As proteínas MDM2 e MDM4 podem ser reguladas ao nível de mRNA, por meio de regulação póstranscricional mediada por miRNA. A interação entre miRNA e mRNA ocorre pelo pareamento de bases de sequências complementares entre a região seed do miRNA e a região 3' não traduzida (3' UTR) do mRNA alvo. Polimorfismos de nucleotídeo único (SNPs) situados nestes sítios de interação podem alterar a afinidade de ligação entre miRNAs e mRNA alvo. As análises computacionais de predição in silico são capazes de fornecer uma lista de genes alvos candidatos de regulação por miRNAs, levando em consideração a ocorrência de SNPs dentro dos sítios alvos de miRNA. Este trabalho parte da integração de dados de bioinformática e de regulação gênica relevantes ao câncer de mama previamente realizadas, que resultou na identificação de miRNAs que podem estar envolvidos no câncer de mama de maneira dependente de polimorfismos no sítio alvo. Foram selecionados como candidatos para a validação funcional, o SNP (rs4245739) A>C do gene MDM4 e os miRNAs miR-887-3p e miR-191- 5p, preditos para se ligar ao mRNA na presença do alelo C. O ensaio de gene repórter luciferase foi utilizado com o objetivo de validar experimentalmente a predição in silico. A sequência 3'UTR do gene MDM4 contendo o SNP rs4245739 foi clonada à jusante do gene da firefly luciferase, presente no vetor pmirGLO. A linhagem celular MDA-MB-231 foi utilizada para a co-transfecção dos vetores contendo os insertos A e C do SNP rs4245739 e o pre-miR-887-3p ou os miRNAs mimics miR-887-3p e miR-191-5p. A partir do ensaio luciferase foi possível observar a ligação específica dos miRNAs 887-3p e 191-5p ao vetor contendo a variante C e que as concentrações de mimic transfectado, de 50nM e 100 nM, não mostraram diferenças significativas na interação miRNA-mRNA. Células transfectadas apenas com os mimics foram usadas para avaliação dos níveis de mRNA do gene MDM4 por RT-qPCR. Apesar de observarmos uma diminuição na expressão do RNA de MDM4, esta não foi estatisticamente significativa, sugerindo que outros mecanismos de regulação, como o bloqueio de tradução do mRNA mediado por miRNAs seja o modelo mais relevante Desta forma, foi demonstrado que a variante genética rs4245739 no sítio de ligação para os miRNAs 887-3p 191-5p é relevante do ponto de vista funcional, uma vez que modula a afinidade de ligação destes de maneira diferencial, ou seja, dependente da presença das variantes A ou C.Abstract: Breast cancer is the second most prevalent cancer in the world, with the highest mortality among women. Tumor progression is controlled among other factors due to tumor suppressor genes including the TP53 gene, essential in response to DNA damage. Thus, changes in levels of p53 and their regulators may affect tumor suppressor function and predispose to cancer development. MDM2 and MDM4 proteins participate in this regulation as they inhibit the transactivation function of p53 by blocking the basal transcription factors binding site as well as participating in the polyubiquitination pathway by marking the p53 protein for further proteasome degradation. MDM2 and MDM4 proteins can be regulated at the mRNA levels by miRNA-mediated post-transcriptional regulation. The interaction between miRNA and mRNA occurs by base pairing between the miRNA seed region and target mRNA 3' untranslated region (3' UTR). Single nucleotide polymorphisms (SNPs) within these interaction sites may alter the binding affinity between miRNAs and mRNA targets. In silico prediction, computational analysis provides a list of target genes candidates to miRNA regulation considering the occurrence of SNPs within the target sites. This study is based on previously performed bioinformatics integration of gene regulation data relevant to breast cancer, which resulted in the identification of miRNAs that may be involved in breast cancer in polymorphisms-depending manner. MDM4 SNP (rs4245739) A>C and miR-887-3p and miR-191-5p predicted to bind to mRNA in the presence of the C allele, were selected for functional validation. The luciferase reporter gene assay was used to experimentally validate in silico prediction. MDM4 3'UTR sequence containing the rs4245739 SNP was cloned downstream of the firefly luciferase gene in the pmirGLO vector. MDA-MB-231 breast cancer cell line was used for the co-transfection of vectors containing SNP rs4245739 A and C inserts and the pre-miR-887 or the miRNA mimics miR-887- 3p and miR-191-5p. From the luciferase assay was possible to observe the specific binding of the mimics 887-3p and 191-5p to the vector containing the C allele and the concentrations of transfected mimics - 50nM and 100nM - did not show significant differences in the miRNA-mRNA interaction. The transfected cells with mimics were used to assesses MDM4 mRNA levels by RT-qPCR. Although we observed a decrease in mRNA level from MDM4, this was not statistically significant, suggesting that other regulatory mechanisms, such as miRNA-mediated blocking of mRNA translation are the most relevant model. Thus, it has been shown that the genetic variant rs4245739 at the binding site for the miRNAs 887-3p and 191-5p is functionally relevant, since it modulates their binding affinity in a differential way, i.e., dependent on the presence of variants A or C

ATLANTIC BIRD TRAITS: a data set of bird morphological traits from the Atlantic forests of South America

Scientists have long been trying to understand why the Neotropical region holds the highest diversity of birds on Earth. Recently, there has been increased interest in morphological variation between and within species, and in how climate, topography, and anthropogenic pressures may explain and affect phenotypic variation. Because morphological data are not always available for many species at the local or regional scale, we are limited in our understanding of intra- and interspecies spatial morphological variation. Here, we present the ATLANTIC BIRD TRAITS, a data set that includes measurements of up to 44 morphological traits in 67,197 bird records from 2,790 populations distributed throughout the Atlantic forests of South America. This data set comprises information, compiled over two centuries (1820–2018), for 711 bird species, which represent 80% of all known bird diversity in the Atlantic Forest. Among the most commonly reported traits are sex (n = 65,717), age (n = 63,852), body mass (n = 58,768), flight molt presence (n = 44,941), molt presence (n = 44,847), body molt presence (n = 44,606), tail length (n = 43,005), reproductive stage (n = 42,588), bill length (n = 37,409), body length (n = 28,394), right wing length (n = 21,950), tarsus length (n = 20,342), and wing length (n = 18,071). The most frequently recorded species are Chiroxiphia caudata (n = 1,837), Turdus albicollis (n = 1,658), Trichothraupis melanops (n = 1,468), Turdus leucomelas (n = 1,436), and Basileuterus culicivorus (n = 1,384). The species recorded in the greatest number of sampling localities are Basileuterus culicivorus (n = 243), Trichothraupis melanops (n = 242), Chiroxiphia caudata (n = 210), Platyrinchus mystaceus (n = 208), and Turdus rufiventris (n = 191). ATLANTIC BIRD TRAITS (ABT) is the most comprehensive data set on measurements of bird morphological traits found in a biodiversity hotspot; it provides data for basic and applied research at multiple scales, from individual to community, and from the local to the macroecological perspectives. No copyright or proprietary restrictions are associated with the use of this data set. Please cite this data paper when the data are used in publications or teaching and educational activities. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

ATLANTIC BIRD TRAITS

Scientists have long been trying to understand why the Neotropical region holds the highest diversity of birds on Earth. Recently, there has been increased interest in morphological variation between and within species, and in how climate, topography, and anthropogenic pressures may explain and affect phenotypic variation. Because morphological data are not always available for many species at the local or regional scale, we are limited in our understanding of intra- and interspecies spatial morphological variation. Here, we present the ATLANTIC BIRD TRAITS, a data set that includes measurements of up to 44 morphological traits in 67,197 bird records from 2,790 populations distributed throughout the Atlantic forests of South America. This data set comprises information, compiled over two centuries (1820–2018), for 711 bird species, which represent 80% of all known bird diversity in the Atlantic Forest. Among the most commonly reported traits are sex (n = 65,717), age (n = 63,852), body mass (n = 58,768), flight molt presence (n = 44,941), molt presence (n = 44,847), body molt presence (n = 44,606), tail length (n = 43,005), reproductive stage (n = 42,588), bill length (n = 37,409), body length (n = 28,394), right wing length (n = 21,950), tarsus length (n = 20,342), and wing length (n = 18,071). The most frequently recorded species are Chiroxiphia caudata (n = 1,837), Turdus albicollis (n = 1,658), Trichothraupis melanops (n = 1,468), Turdus leucomelas (n = 1,436), and Basileuterus culicivorus (n = 1,384). The species recorded in the greatest number of sampling localities are Basileuterus culicivorus (n = 243), Trichothraupis melanops (n = 242), Chiroxiphia caudata (n = 210), Platyrinchus mystaceus (n = 208), and Turdus rufiventris (n = 191). ATLANTIC BIRD TRAITS (ABT) is the most comprehensive data set on measurements of bird morphological traits found in a biodiversity hotspot; it provides data for basic and applied research at multiple scales, from individual to community, and from the local to the macroecological perspectives. No copyright or proprietary restrictions are associated with the use of this data set. Please cite this data paper when the data are used in publications or teaching and educational activities. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ