UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA VETERINÁRIA CÂMPUS ARAÇATUBA MARIA FERNANDA DA SILVA LOPES Substâncias obesogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional Araçatuba 2021 MARIA FERNANDA DA SILVA LOPES Substâncias obesogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional Araçatuba 2021 Dissertação apresentada à Faculdade de Medicina Veterinária de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, como parte dos requisitos para a obtenção do título de Mestre em Ciência Animal (Área de Medicina Veterinária Preventiva e Produção Animal). Orientadora: Pesquisadora Flávia Lombardi Lopes L864s Lopes, Maria Fernanda da Silva Substâncias obesogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional / Maria Fernanda da Silva Lopes. -- Araçatuba, 2021 47 p. Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Faculdade de Medicina Veterinária, Araçatuba Orientadora: Flávia Lombardi Lopes 1. Epigenética. 2. Substâncias obesogênicas. 3. Camundongo. 4. LncRNA. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Medicina Veterinária, Araçatuba. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Araçatuba CERTIFICADO DE APROVAÇÃO Título: AUTORA: MARIA FERNANDA DA SILVA LOPES ORIENTADORA: FLÁVIA LOMBARDI LOPES             Aprovada como parte das exigências para obtenção do Título de Mestra em CIÊNCIA ANIMAL, área: Medicina Veterinária Preventiva e Produção Animal pela Comissão Examinadora: Pesquisadora FLÁVIA LOMBARDI LOPES (Participaçao Virtual) Departamento de Produção e Saúde Animal / Faculdade de Medicina Veterinária de Araçatuba - Unesp Profa. Dra. CRISTINA ANTONIALI SILVA (Participaçao Virtual) Departamento. de Ciências Básicas / Faculdade de Odontologia de Araçatuba-UNESP Prof. Dr. YURI TANI UTSUNOMIYA (Participaçao Virtual) Programa de Pós-Graduação em Ciência Animal da Faculdade de Medicina Veterinária - Câmpus de Araçatuba/UNESP Araçatuba, 14 de janeiro de 2021. Faculdade de Medicina Veterinária - Câmpus de Araçatuba - Rua Clóvis Pestana, 793, 16050680, Araçatuba - São Paulo http://www.fmva.unesp.br/#!/pos-graduacao/mestrado-doutorado/CNPJ: 48031918003905. Stamp FreeText Substâncias obsogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional. UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Araçatuba CERTIFICADO DE APROVAÇÃO Título: Substâncias obsogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional AUTORA: MARIA FERNANDA DA SILVA LOPES ORIENTADORA: FLÁVIA LOMBARDI LOPES             Aprovada como parte das exigências para obtenção do Título de Mestra em CIÊNCIA ANIMAL, área: Medicina Veterinária Preventiva e Produção Animal pela Comissão Examinadora: Pesquisadora FLÁVIA LOMBARDI LOPES (Participaçao Virtual) Departamento de Produção e Saúde Animal / Faculdade de Medicina Veterinária de Araçatuba - Unesp Profa. Dra. CRISTINA ANTONIALI SILVA (Participaçao Virtual) Departamento. de Ciências Básicas / Faculdade de Odontologia de Araçatuba-UNESP Prof. Dr. YURI TANI UTSUNOMIYA (Participaçao Virtual) Programa de Pós-Graduação em Ciência Animal da Faculdade de Medicina Veterinária - Câmpus de Araçatuba/UNESP Araçatuba, 14 de janeiro de 2021. Faculdade de Medicina Veterinária - Câmpus de Araçatuba - Rua Clóvis Pestana, 793, 16050680, Araçatuba - São Paulo http://www.fmva.unesp.br/#!/pos-graduacao/mestrado-doutorado/CNPJ: 48031918003905. UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Araçatuba CERTIFICADO DE APROVAÇÃO Título:Título: Substâncias obsogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional AUTORA: MARIA FERNANDA DA SILVA LOPES ORIENTADORA: FLÁVIA LOMBARDI LOPES             Aprovada como parte das exigências para obtenção do Título de Mestra em CIÊNCIA ANIMAL, área: Medicina Veterinária Preventiva e Produção Animal pela Comissão Examinadora: Pesquisadora FLÁVIA LOMBARDI LOPES (Participaçao Virtual) Departamento de Produção e Saúde Animal / Faculdade de Medicina Veterinária de Araçatuba - Unesp Profa. Dra. CRISTINA ANTONIALI SILVA (Participaçao Virtual) Departamento. de Ciências Básicas / Faculdade de Odontologia de Araçatuba-UNESP Prof. Dr. YURI TANI UTSUNOMIYA (Participaçao Virtual) Programa de Pós-Graduação em Ciência Animal da Faculdade de Medicina Veterinária - Câmpus de Araçatuba/UNESP Araçatuba, 14 de janeiro de 2021. Faculdade de Medicina Veterinária - Câmpus de Araçatuba - Rua Clóvis Pestana, 793, 16050680, Araçatuba - São Paulo http://www.fmva.unesp.br/#!/pos-graduacao/mestrado-doutorado/CNPJ: 48031918003905. 7 À minha família, com muito amor e carinho, por sua intensa dedicação e apoio durante a elaboração deste trabalho. 8 AGRADECIMENTOS Agradeço à minha família, em especial aos meus pais, à minha irmã e ao meu namorado Lucas, que sempre acreditaram em mim e me deram todo o apoio e incentivo nos momentos em que mais precisei, principalmente nos mais difíceis. Sou imensamente grata por tudo que fizeram por mim e por aceitarem compartilhar esta jornada ao meu lado. Amo vocês, serão sempre a minha base de tudo! Às minhas amigas que a ciência me presenteou: Amanda Furlan, Beatriz Trigo, Juliana Felix, Jéssica Troiano, Mariana Cordeiro, Natália Scaramelle e Nayra Herrera. Obrigada pelo companheirismo, pelas conversas, conselhos, alegrias e tristezas compartilhadas durante essa caminhada. À minha orientadora, Profa. Dra. Flávia Lombardi Lopes, que me deu a oportunidade de fazer parte da equipe do seu laboratório de Epigenômica, confiando inteiramente a mim a condução deste trabalho. Palavras não são suficientes para descrever minha imensa gratidão. À UNESP – Universidade Estadual Paulista “Júlio de Mesquita Filho”, em especial ao Programa de Pós-Graduação em Ciência Animal da Faculdade de Medicina Veterinária de Araçatuba (UNESP/FMVA), pela oportunidade de realização do mestrado. A todos os professores e colaboradores do Departamento de Produção e Saúde Animal. À minha banca de qualificação, Prof. Dr. Yuri Tani Utsunomyia e a Profa. Dra. Joselaine de Oliveira, que me servem de exemplo profissional pela dedicação e comprometimento, obrigado pelas considerações dadas ao meu trabalho. À Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, pela concessão da bolsa de mestrado. 9 “O que é escrito sem esforço, em geral, é lido sem prazer”. (Samuel Johnson) 10 LOPES, M.F.S. Substâncias obesogênicas alteram a expressão de RNAs longos não-codificadores de maneira transgeracional. 2021. 47 f. Dissertação (Mestrado) – Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Araçatuba, 2021. RESUMO A epidemia da obesidade é considerada uma crise mundial de Saúde Pública, tendo como fatores contribuintes o aumento da ingestão calórica, estilos de vida sedentários e/ou predisposições genéticas. Ainda que o balanço energético positivo seja uma das causas mais significativas da obesidade, pesquisas recentes relacionam a epidemia da doença à exposição precoce a produtos químicos desreguladores endócrinos (DEs) (do inglês, Endocrine-Disrupting Chemicals - EDCs). A Sociedade Endócrina define DEs como “substâncias químicas exógenas, ou misturas de substâncias químicas, que interferem em qualquer aspecto da ação hormonal”. Substâncias obesogênicas compõem um subconjunto de DEs que estimulam inadequadamente a adipogênese e o armazenamento de gordura direta ou indiretamente, aumentando o número e/ou o tamanho das células adiposas, ou interferindo na regulação hormonal do metabolismo, apetite e saciedade. A exposição a essas substâncias químicas durante o período intrauterino e/ou de lactação pode influenciar fortemente a predisposição tardia à obesidade. Os RNAs longos não-codificadores (lncRNAs) são caracterizados por desempenhar relevantes papéis regulatórios em diversos processos biológicos. Considerando a importância da nutrição materna para a regulação dos padrões epigenéticos da prole, o presente estudo visa elucidar os efeitos da exposição ancestral a substâncias obesogênicas na expressão de lncRNAs no tecido adiposo gonadal de camundongos. Os dados de RNA-Seq de 8 amostras de tecido adiposo gonadal de camundongos F4 adultos expostos ancestralmente a dimetilsulfóxido (DMSO) (0,1%) como grupo controle, ou ao DE TBT (50 nM) durante o desenvolvimento uterino e lactação, foram obtidos de estudo previamente realizado (NCBI - GEO Datasets - GSE105051). Após alinhamento e contagem das reads, foi realizada análise de expressão diferencial, e identificamos 74 lncRNAs diferencialmente expressos (p<0.05), visto que 22 apresentaram expressão aumentada no grupo TBT e 52 estavam mais expressos no grupo DMSO em relação ao TBT. As análises dos termos de ontologia gênica (do inglês Gene Ontology - GO) revelaram o enriquecimento dos processos relacionados ao metabolismo da glicose e 11 à atividade do receptor ativado por Wnt. Sendo assim, nossos resultados sugerem que a regulação dos lncRNAs e de seus genes parceiros podem contribuir para o controle do processo de adipogênese. Palavras-chave: Epigenética. Substâncias obesogênicas. Camundongo. LncRNA. 12 LOPES, M. F. S. Obesogenic substances alter the expression of long non-coding RNAs in a transgerational manner. 2021. 47 f. Dissertação (Mestrado) – Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Araçatuba, 2021. ABSTRACT The obesity epidemic is considered a global public health crisis. Increased caloric intake, sedentary lifestyles and/or genetic predisposition are all factors contributing to the epidemic. Although the positive energy balance is one of the most significant causes of obesity, recent research has linked early exposure to Endocrine-Disrupting Chemicals (EDCs) to the disease. The Endocrine Society defines EDCs as "exogenous chemicals, or mixtures of chemicals, that interfere with any aspect of hormonal action". Obesogenic substances make up a subset of EDCs that inadequately stimulate adipogenesis and fat storage directly or indirectly, increasing the number and/or size of fat cells, or interfering with the hormonal regulation of metabolism, appetite and satiety. Exposure to these chemicals during intrauterine and/or lactation periods can strongly influence the late predisposition to obesity. Long noncoding RNAs (lncRNAs) are characterized by playing important regulatory roles in several biological processes. Considering the importance of maternal nutrition to the regulation of epigenetic patterns in the offspring, the present study aimed at investigating the effects of ancestral exposure to obesogenic substances on the expression of lncRNAs in the gonadal adipose tissue of mice. RNA-seq data from 8 gonadal adipose tissue samples from adult F4 mice ancestrally exposed to dimethyl sulfoxide (DMSO) (0.1%) as a control group, or to EDC TBT (50 nM) during uterine development and lactation, were obtained from a previously conducted study (NCBI - GEO Datasets - GSE105051). After reading alignment and counting, differential expression analysis was performed, and we identified 74 differentially expressed lncRNAs were identified (p <0.05), as 22 showed increased expression in the TBT group and 52 were more expressed in the TBT group. The analysis of the GO terms revealed an enrichment for glucose metabolism and the activity of the receptor activated by Wnt. Therefore, our results suggest that the regulation of lncRNAs and their partner genes may contribute to the control of the adipogenesis process. Keywords: Epigenetics. Obesogenic substances. Mouse. LncRNA. 13 SUMÁRIO 1 INTRODUÇÃO GERAL ......................................................................................... 14 1.1 Objetivos ............................................................................................................ 16 1.1.1 Objetivo Geral ................................................................................................. 16 1.1.2 Objetivos Específicos ...................................................................................... 16 2 CAPÍTULO 1 - CORRELATION ANALYSIS OF LNCRNA AND MRNA IN MICE ANCESTRALLY EXPOSED TO OBESOGENIC SUBSTANCES INDENTIFIES POTENTIAL ADIPOGENESIS REGULATORY GENES ........................................... 17 2.1 Resumo.............................................................................................................. 18 2.2 Abstract .............................................................................................................. 19 2.3 Introduction ........................................................................................................ 20 2.4 Methods ............................................................................................................. 21 2.4.1 RNA-seq datasets ........................................................................................... 22 2.4.2 Bioinformatic identification of lncRNAs in RNA-seq datasets .......................... 22 2.4.3 Prediction analysis of lncRNAs-mRNAs with cis action ................................... 23 2.4.4 Functional Enrichment Analysis ...................................................................... 23 2.5 Results ............................................................................................................... 24 2.6 Discussion .......................................................................................................... 28 2.7 References ......................................................................................................... 30 APÊNDICE A – Referências da Introdução Geral .................................................... 34 ANEXO A – Normas da Revista Molecular and Cellular Endocrinology ................... 37 14 1 INTRODUÇÃO GERAL A epidemia da obesidade é considerada uma crise mundial de Saúde Pública, tendo como fatores contribuintes o aumento da ingestão calórica, estilos de vida sedentários e/ou predisposições genéticas. Ainda que o balanço energético positivo seja uma das causas mais significativas da obesidade, pesquisas recentes relacionam a exposição precoce a produtos químicos desreguladores endócrinos (DEs) (do inglês, Endocrine-Disrupting Chemicals - EDCs) à doença. A Sociedade Endócrina (The Endocrine Society) e a Organização Mundial da Saúde definem DEs como “substâncias químicas exógenas, ou misturas de substâncias químicas, que alteram funções do sistema endócrino e, por consequência, causam efeitos adversos em um organismo, em sua prole ou em populações” (1). Substâncias obesogênicas compõem um subconjunto de DEs que estimulam inadequadamente a adipogênese e o armazenamento de gordura direta ou indiretamente, aumentando o número e/ou o tamanho das células adiposas, ou interferindo na regulação hormonal do metabolismo, apetite e saciedade. O tecido adiposo é, particularmente, um reservatório significativo dos DEs e sua presença nos tecidos corporais refletem não apenas ao contato recente, mas também a exposições anteriores a essas substâncias químicas (2,3). Efeitos transgeracionais foram observados com alguns tipos de DEs, como: Bisfenol A, também conhecido como BPA - composto químico que está presente na composição do plásticos policarbonato; Dicloro-difenil-tricloroetano ou DDT, pesticida utilizado para controlar pestes agrícolas; Dibutilftalato (DBP) – plastificante utilizado como aditivo em adesivos e tintas de impressão; Trifenilestanho (TPT) e Tributilestanho (TBT) - compostos orgânicos de estanho, comumente utilizados em tintas anti-incrustante de navios (4). O TBT apresenta baixa solubilidade em água e perfil lipofílico, podendo causar efeitos no sistema endócrino e cardiovascular. Estudos recentes mostram que a exposição pré-natal a obesogênicos ambientais pode produzir efeitos permanentes nos animais expostos (F0) e, transgeracionalmente, em seus descendentes. A exposição a essas substâncias químicas durante o período intrauterino e/ou de lactação pode influenciar fortemente a predisposição tardia à obesidade (1). Estudos epidemiológicos sobre a nutrição materna durante diversas fases da gestação revelam que nesta fase da vida as fêmeas podem induzir transformações 15 permanentes na estrutura, na fisiologia e/ou no metabolismo da prole (5). A nutrição materna pode ocasionar alterações epigenéticas no genoma fetal (6) como, por exemplo, no perfil de metilação do DNA, podendo levar a mudanças fenotípicas permanentes na prole (7). Fetal programming ou programação fetal é o efeito do ambiente uterino, através de mecanismos epigenéticos, no desenvolvimento fetal, sendo capaz de resultar em alterações permanentes na fisiologia e metabolismo dos descendentes (8). Em epigenética, as principais modificações incluem a metilação do DNA, modificações químicas na cauda das histonas e a ação dos RNAs não-codificadores (ncRNAs) (9). O termo epigenética foi usado por Conrad Waddington na década de 1940, para elucidar as interações de genes com o meio ambiente e as possíveis alternativas que cada célula pode desempenhar durante o desenvolvimento (10). A mesma é definida como um processo capaz de regular a expressão gênica sem alteração no código genético, um exemplo clássico são as células de um mesmo organismo que, apesar de geneticamente idênticas, são capazes de se distinguir fenotipicamente, dependendo de sua localização e/ou função, mediante o controle da expressão gênica (11). A condição vital para o desenvolvimento de um novo organismo é a programação epigenética dos gametas e embriões em início de desenvolvimento. Esta programação engloba a integração dos três processos epigenéticos, interferindo na transcrição de genes (12). Tais eventos epigenéticos equilibram a expressão gênica através do controle da transcrição e/ou tradução, e são altamente dependentes das condições uterinas no momento do desenvolvimento de embriões ou gametas da prole. Aproximadamente 62% do DNA funcional é transcrito em ncRNAs que apresentam amplas funções, como: regulação da expressão gênica, regulação da síntese de proteínas, inativação de cromossomos, modulação da cromatina, dentre outras (13– 15). Os ncRNAs são classificados de acordo com o tamanho: ncRNAs longos (acima de 200 nucleotídeos) ou ncRNAs pequenos (abaixo de 200 nucleotídeos) (13,14,16,17). Análises recentes sobre os RNAs longos não-codificadores (lncRNAs) apontam seu importante papel na regulação transcricional, pós-transcricional e epigenética da expressão gênica, podendo assim, silenciar ou ativar genes e loci cromossômicos 16 específicos (18). A inativação do cromossomo X e o imprinting gênomico são importantes exemplos da ação de lncRNAs (19–21). Considerando a importância da nutrição materna para regulação dos padrões epigenéticos da prole e os diversos papéis que os lncRNAs desempenham, o presente estudo visa elucidar os efeitos da exposição ancestral a substâncias obesogênicas na expressão de lncRNAs no tecido adiposo gonadal de camundongos. 1.1 Objetivos 1.1.1 Objetivo Geral Analisar os efeitos da exposição ancestral a substâncias obesogênicas na expressão de RNAs longos não-codificadores no tecido adiposo gonadal de camundongos descendentes F4 (efeito transgeracional). 1.1.2 Objetivos Específicos I. Caracterizar, in silico, os lncRNAs diferencialmente expressos no tecido adiposo gonadal de camundongos, transgeracionalmente expostos a substâncias obesogênicas; II. Analisar o perfil de co-expressão de lncRNAs–mRNA diferencialmente expressos. 17 2 CAPÍTULO 1 - CORRELATION ANALYSIS OF LNCRNA AND MRNA IN MICE ANCESTRALLY EXPOSED TO OBESOGENIC SUBSTANCES IDENTIFIES POTENTIAL ADIPOGENESIS REGULATORY GENES Maria Fernanda da Silva Lopesa, Juliana de Souza Felixa, Natália Francisco Scaramelea, Mariana Cordeiro Almeidaa, Jéssica Antonini Troianoa, Flávia Regina Florêncio de Athaydea, Flávia Lombardi Lopesa aSão Paulo State University (Unesp), School of Veterinary Medicine, Araçatuba, Brazil. Corresponding author: Flávia Lombardi Lopes, São Paulo State University (Unesp), School of Veterinary Medicine, Araçatuba. R. Clóvis Pestana, 793 - Dona Amelia, 16050-680, Araçatuba, SP, Brasil. E-mail: flavia.lopes@unesp.br. Phone: +55(18) 3636-0032. 18 2.1 Resumo A epidemia de obesidade é considerada uma crise global de saúde pública, com aumento da ingestão calórica, sedentarismo e / ou predisposições genéticas como fatores contribuintes. Embora o balanço energético positivo seja uma das causas mais significativas da obesidade, pesquisas recentes relacionaram a exposição precoce a Produtos Químicos Desreguladores Endócrinos (EDCs) à doença. Além de suas ações no perfil hormonal, os EDCs podem induzir alterações de longo prazo na expressão gênica, possivelmente devido a alterações nos padrões epigenéticos. Os longos RNAs não codificantes (lncRNAs) são um mediador epigenético que desempenha importantes papéis regulatórios em diversos processos biológicos. Considerando a importância da nutrição materna na regulação do padrão epigenético da prole, o presente estudo tem como objetivo elucidar os efeitos da exposição ancestral a substâncias obesogênicas sobre a expressão de lncRNAs no tecido adiposo gonadal de camundongos. Neste estudo, exploramos a expressão diferencial de lncRNAs em amostras de tecido adiposo branco gonadal de camundongos F4 adultos expostos ancestralmente a EDC TBT durante o desenvolvimento uterino e a lactação, analisando dados de RNA-seq do conjunto de dados público GSE105051. Um total de 74 lncRNAs foram expressos diferencialmente, 22 foram regulados positivamente e 52 regulados negativamente no grupo exposto ao TBT quando comparado aos controles (DMSO). Seguindo o gene parceiro do mRNA para a predição dos lncRNAs de ação cis, a análise dos termos do GO revelou o enriquecimento dos processos relacionados ao metabolismo da glicose e à atividade do receptor ativado por Wnt. Em conclusão, nossos resultados indicam que a regulação de lncRNAs e seus genes-parceiros no tecido adiposo gonadal branco de camundongos ancestralmente expostos ao EDC TBT pode estar diretamente relacionada ao controle do processo de adipogênese, sugerindo que esta regulação pode ser hereditária epigeneticamente. Palavras-chave: Epigenética. Substâncias obesogênicas. Camundongo. LncRNA. 19 2.2 Abstract The obesity epidemic is considered a global public health crisis, with an increase in caloric intake, sedentary lifestyles and/or genetic predispositions as contributing factors. Although the positive energy balance is one of the most significant causes of obesity, recent research has linked early exposure to Endocrine-Disrupting Chemicals (EDCs) to the disease. In addition to their actions on the hormonal profile, EDCs can induce long-term changes in gene expression, possibly due to changes in epigenetic patterns. The long non-coding RNAs (lncRNAs) is an epigenetic mediator that play important regulatory roles in several biological processes. Considering the importance of maternal nutrition for regulating epigenetic patterning in the offspring, the present study aims to elucidate the effects of ancestral exposure to obesogenic substances on the expression of lncRNAs in the gonadal adipose tissue of mice. In this study, we explored the differential expression of lncRNAs in gonadal white adipose tissue samples from adult F4 mice ancestrally exposed to EDC TBT during uterine development and lactation, by analyzing RNA-seq data the public dataset GSE105051. A total of 74 lncRNAs were diferentially expressed, 22 were up-regulated and 52 were down-regulated in the group exposed to TBT when compared to controls (DMSO). Following mRNA gene-partner to cis-acting lncRNAs prediction, analysis of GO terms unveiled enrichment of processes related to glucose metabolism and Wnt-activated receptor activity. In conclusion, our results indicate that regulation of lncRNAs and their gene-partners in the white gonadal adipose tissue of mice ancestrally exposed to EDC TBT may be directly related to the control of the adipogenesis process, suggesting that this regulation may be epigenetically inherited. Keywords: Epigenetics. Obesogenic substances. Mouse. LncRNA. 20 2.3 Introduction Exposure to environmental factors during embryonic development has been linked to the risk of diseases such as obesity and type 2 diabetes mellitus later in life. The obesity epidemic is considered a global public health crisis, having as contributing factors increased caloric intake, sedentary lifestyles and/or genetic predispositions. Although the positive energy balance is one of the most significant causes of obesity, recent research has linked early exposure to endocrine disrupting chemicals (EDCs) to the disease (Chamorro-Garcia et al., 2017). EDCs are a group of different chemical compounds that interfere with the production, release, transport, metabolism, action or elimination of endogenous hormones responsible for maintaining homeostasis and regulating developmental processes (Shahnazaryan et al., 2019). Obesogenic substances comprise a subset of EDCs, which can generate an increase in the accumulation of lipids, inadequately stimulating adipogenesis, hypertrophy or hyperplasia of adipocytes or interfering with the hormonal regulation of metabolism, appetite and satiety (Gupta et al., 2020). There is a growing range of evidence showing that exposure to these chemicals during intrauterine development or lactation can strongly influence offspring's late predisposition to obesity (Street et al., 2018). In addition to their actions on the hormonal profile, EDCs can induce long-term changes in gene expression, possibly due to changes in epigenetic patterns (Alavian- Ghavanini and Rüegg, 2018). Shoucri et al. (2017) demonstrated that the obesogenic effects of prenatal tributyltin (TBT) exposure are propagated transgenerationally to unexposed generations, presumably through epigenetic changes. In fact, transgenerational effects have been observed with some types of EDCs, such as bisphenol A (BPA), dichlorodiphenyltrichloroethane (DDT), dibutyl phthalate (DBP), triphenyl tin (TPT) and TBT. Obesogens were defined as chemical substances that lead to an increase in the accumulation of white adipose tissue (WAT). Egusquiza and Blumberg (2020) reported that TBT induces obesity by promoting the differentiation of adipocytes in the body while stimulating the activity of the RXR-PPARγ complex. Interestingly, Chamorro-Garcia and his collaborators (2017) showed in their study that prenatal exposure to environmental obesogens, especially EDC TBT, can produce permanent effects on exposed animals (F0) and, transgenerationally, on their *Text according to the Guidelines for Authors of the journal Molecular and Cellular Endocrinology – Available in ANEXO A 21 descendants of the F4 generation, such as global changes in DNA methylation, altered expression of genes relevant to metabolism and obesity consistent with leptin resistance. A considerable body of studies within the past two decades have shown that maternal nutrition can cause changes in the fetal epigenome, i.e, the DNA methylation profile, post-translational histone modifications and regulation of and by non-coding RNAs (ncRNAs), which can lead to permanent phenotypic changes in the offspring reviewed by Greco et al. (2019). Epigenetic mechanisms play essential roles in the processes that determine adult phenotypes, through epigenetic programming. While research has shown that exposure to EDC during periods of development can disrupt epigenetic programming, it is not clear whether these changes are actually causing adverse outcomes (Jacobs et al., 2017). Geisler and Coller (2016) proposed that exposure to environmental toxins like bisphenol A, benzo(a)pyrene, tributyltin, and phthalates produces alterations in the expression of long noncoding RNAs (lncRNAs) that may be associated with a range of diseases. Long non-coding RNAs (lncRNAs) are ncRNAs composed of more than 200 nucleotides and play an important role in the transcriptional, post-transcriptional and epigenetic regulation of gene expression, thus being able to silence or activate specific genes or loci (Charles Richard and Eichhorn, 2018). LncRNAs are classified according to their location in the genome as: 1) sense or antisense, when they overlap one or more exons of coding genes, on the same strand or on the opposite, respectively; 2) bidirectional, when the transcription of lncRNA and a coding transcript on the opposite strand are located nearby; 3) intronic, when it is derived from the intronic region of a protein-coding gene; 4) intergenic, when it is located between coding genes (Kopp and Mendell, 2018). Considering the importance of maternal nutrition for regulating epigenetic patterning on the offspring (and transgenerationally on their descendents), the diverse roles that lncRNAs play on gene expression control, and the altered expression of genes relevant to metabolism (Chamorro-Garcia et al., 2017), the present study aimed at evaluating the effects of ancestral exposure to obesogenic substances on the expression of lncRNAs in the gonadal white adipose tissue (gWAT) of mice. 2.4 Methods 22 2.4.1 RNA-Seq data sets RNA-seq data were obtained from the Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under the bioproject PRJNA414476, with accession number GSE105051. This dataset was produced by Chamorro-Garcia et al. (2017) in simultaneous experiments using samples of gWAT from C57BL/6J male F4 mice (7 weeks of age) ancestrally exposed via drinking water to EDC TBT (50 nM) (n = 4) or to the vehicle dimethylsulfoxide (DMSO) (0.1%) as a control group (n = 4) (both dilute in 0.5% carboxymethyl cellulose in water for solubility) during pregnancy and lactation. In total, 8 samples were used, consisting of 4 samples from the TBT exposed group and 4 samples from the control group. 2.4.2 Bioinformatic identification of lncRNAs in RNA-Seq datasets Quality of the extracted RNA-seq readings was evaluated with FastQC on the Galaxy web plataform and we used the public server at www.usegalaxy.org to analyse data (Afgan et al., 2016). The data were then aligned to the latest mouse genome reference sequence (GRCm38.p6, as provided by GENCODE) using HISAT2 version 2.1.0+galaxy5 (Kim et al., 2015) with the Burrows-Wheeler Transformation (BWT) and the Ferragina-Manzini (FM) indexing algorithms. The resulting BAM file was then passed to FeatureCounts version 1.6.4+galaxy1 (Liao et al., 2014) to perform read counts using the GENCODE M25 (mouse) annotation as reference. Quality control of all steps was carried out with MultiQC. Next, DESeq2 (version 2.11.40.6 + galaxy1) was used to perform statistical analyses of differential expression of lncRNA between samples from the TBT exposed group and the control group. This tool estimates the average variance in the count data and tests the differential expression using a binomial distribution model as basis. By default, the DESeq2 tool in Galaxy uses the Wald test to identify genes that are differentially expressed between two sample classes (Love et al., 2014). Using the Biomart tool, the biotypes “processed_transcript”, “pseudogene”, “lincRNA”, “3prime_overlapping_ncrna”, “antisense”, “sense_intronic” and sense_overlapping” were classified as lncRNAs (http://www.ensembl.org/info/genome/genebuild/biotypes.html). Differentially expressed (DE) lncRNAs (p-value<0.05) were clustered with the heatmap3 package in R using “complete linkage” method and Euclidian distance as 23 parameters (https://www.rdocumentation.org/packages/heatmap3/versions/1.1.7/topics/heatmap 3). 2.4.3 Prediction analysis of lncRNAs-mRNAs with cis action The lncRNAs can act in the regulation of genes in cis or trans forms. The cis function refers to the action of lncRNAs in target genes neighboring its transcription site, whereas lncRNAs that act in trans exercise their function in different location to which they were transcribed. Differentially expressed lncRNAs (P-value <0.05) were selected for the prediction of the cis- and trans-target genes. We used the FEELnc tool (FlExible Extraction of Long non-coding RNAs) to predict nearby partner genes (cis- genes) of DE lncRNAs (within the window size of 10.000 - 100.000 nt). For each lncRNA-mRNA interaction, a best lncRNA:RNA-partner interaction was identified (isBest score = 1). Next, Pearson correlation coefficient method and Student`s t-test were used to analyze correlations between all DE mRNAs and DE lncRNAs. The lncRNA-mRNA interaction was considered significant when Pearson`s correlation |r| ≥ 0.8 and p<0.05 assuming that r is normally-distributed under the null hypothesis of true r = 0. 2.4.4 Functional Enrichment Analysis We used the g:GOSt (Function Profiling) tool within gProfiler (https://biit.cs.ut.ee/gprofiler/gost) for analysis of the Gene Ontology (GO) annotation of all significant lncRNAs and partner mRNA pairs (https://biit.cs.ut.ee/gprofiler/gost). All p-values were adjusted using the Benjamini-Hochberg (FDR) method (FDR- adjusted p-value<0.05). 24 Figure 1 - Workflow of lncRNA analysis and functional predictions. 2.5 Results Out of 1324 differentially expressed transcripts, 74 were considered to have a lncRNA biotype, according to the classification available on the Ensembl Biomart tool (Table 1). Table 1 – Biomart classification of biotype, counts and percentage of total transcripts. Biotype Counts % of total Antisense 20 1.51 bidirectional_promoter_lncRNA 4 0.30 lincRNA 29 2.19 processed_transcript 6 0.45 sense_intronic 1 0.08 25 TEC 14 1.06 IG_C_gene 6 0.45 IG_J_gene 1 0.08 IG_V_gene 23 1.74 miRNA 1 0.08 Mt_tRNA 2 0.15 processed_pseudogene 16 1.21 protein_coding 1194 90.18 pseudogene 1 0.08 snoRNA 2 0.15 transcribed_processed_pseudogene 1 0.08 transcribed_unprocessed_pseudogene 2 0.15 unprocessed_pseudogene 1 0.08 Total 1324 100 Total lncRNAs 74 5.59 Of these 74 DE lncRNAs in the contrast of the EDC TBT and DMSO control groups (filtered using p-value <0.05 for the Wald test for log2FoldChange difference between groups, after adjusting for the rate of false discoveries, 22 showed increased expression in the group exposed to the EDC TBT when compared to controls (DMSO). The other 52 lncRNAs were downregulated in the EDC TBT ancestrally exposed group (Figure 2). 26 Figure 2 – Heatmap of 74 differentially expressed lncRNAs between samples of gWAT ancestrally exposed to EDC TBT and samples of the control group (DMSO). Expression of lncRNA is represented according to the colour scale shown at the top, corresponding to the z-score. Red represents high expression, and green represents low expression. From the correlation matrix containing 11.685 interactions between lncRNA- mRNA and from the prediction analysis of the cis lncRNAs-mRNAs pairs performed on the FEELnc tool, we observed that, of a total of 12 cis-lncRNA-mRNAs considered as best (isBest=1), only 3 DE mRNAs (Figure 3) were found to have a significant correlation of expression with their lncRNA predicted cis-partner (Pearson`s correlation |r| ≥ 0.8 and p-value <0.05; Table 2). 27 Figure 3 - Cis-acting lncRNAs and their differentially expressed gene-partners between samples of gWAT ancestrally exposed to EDC TBT and samples of the control group (DMSO). Y-axis shows expression (log2 fold change) of DE lncRNAs and gene-partners. On the x-axis lncRNAs are shown in blue and gene-partners in orange. Table 2 – Top cis-acting Pearson’s correlation between lncRNAs-gene partners LncRNA mRNA Name Biotype Name P-value Correlation Gm26704 Bidirectional promoter lncRNA Fzd6 4.33 x 10-6 0.87 B130024G19Rik Processed transcript NR2F2 1.18 x 10-8 0.86 Gm38843 lincRNA HK2 1.29 x 10-8 0.93 GO analysis was performed to obtain information about the function of the identified target genes of all DE lncRNAs. In our analysis of enrichment of GO terms, we identified that the lncRNAs partner genes were significantly enriched for a total 384 GO terms, 27 for molecular function, 332 for biological processes and 25 for cellular 28 component. The top 10 ranked GO terms in molecular function of the EDC TBT group, are shown in Table 3. Table 3 – Top 10 GO terms enrichment analysis for Molecular Function related to glucose metabolism. GO - ID Term P-value GO:0004340 glucokinase activity 4.60 x 10-3 GO:0004396 hexokinase activity 4.60 x 10-3 GO:0008865 fructokinase activity 4.60 x 10-3 GO:0019158 mannokinase activity 4.60 x 10-3 GO:0001972 retinoic acid binding 8.40 x 10-3 GO:0042813 Wnt-activated receptor activity 8.40 x 10-3 GO:0005536 glucose binding 8.40 x 10-3 GO:0019200 carbohydrate kinase activity 1.01 x 10-2 GO:0004879 nuclear receptor activity 1.13 x 10-2 GO:0005501 retinoid binding 1.13 x 10-2 2.6 Discussion In the present study, we analyzed the expression profiles of lncRNAs in the gWAT of F4 mice ancestrally exposed to obesogenic substances. Based on the RNA- Seq data, we identified a total of 74 differentiatly expressed lncRNAs in the group ancestrally exposed to the EDC TBT, and analyzed the expression correlation with their respective partner genes, as lncRNAs are known to regulate neighboring protein- coding genes (Kopp and Mendell, 2018). Enrichment of GO terms revealed that the partner genes of our DE lncRNAs are primarily involved in glucose and lipid metabolism and in Wnt-activated receptor activity, all of which play an important role in regulating adipogenesis. The B130024G19Rik and Gm26704 lncRNAs, which interact with the gene- partners NR2F2 and Fzd6, respectivelywere downregulated in our study. The nuclear receptor of subfamily 2, group F, member 2 (NR2F2, also known as COUP-TFII, Chicken ovalbumin upstream promoter-transcription factor II) is expressed in adipose tissue, and may contribute to the pathogenesis of obesity, playing a role fundamental in adipogenesis and energy homeostasis (Polvani et al., 2020). Overexpression of 29 NR2F2 impairs adipogenesis by suppressing the expression of pro-adipogenic factors in adipocytes, whereas downregulation of NR2F2 promotes fibroblast differentiation into fat cells, and can act on development of chronic diseases ranging from obesity to metabolic syndrome (Li et al., 2010). Okamura (2009) and collaborators demonstrated that NR2F2 provides a direct link between the Wnt/beta-catenin signaling pathway and modulation of PPARγ. Wnt activates the expression of NR2F2, which in turn, prevents the expression of PPARγ, resulting in inhibition of adipogenesis. The gene-partner to Gm26704, Fzd6, belongs to the family of 10 Frizzled receptors, which play important roles in embryonic development, regulation of stem cells and tissue homeostasis, and its stimulation can activate the canonical Wnt/β-catenin and non-canonical Wnt/Ca 2+ pathways (Kozielewicz et al., 2020). Chamorro-Garcia et al. (2017), in the study that generated the data employed in our analysis, showed that ancestral exposure to TBT stimulated fat storage in adulthood. We propose that sustained inhibition of NR2F2 expression through lncRNA modulation is allowing for the differentiation of fibroblasts into adipocytes, thus contributing to the observed development of obesity. The lncRNA Gm38843, up regulated in the TBT group, is correlated with the HK2 gene partner. GO term analysis indicated that HK2, also up-regulated in the TBT group, is associated with glucose metabolism (GO:0019158 - mannokinase activity; GO:0008865 - fructokinase activity; GO:0004396 - hexokinase activity and GO:0004340 - glucokinase activity). Initial glucose metabolism is regulated by four main steps: I) glucose uptake; II) action of hexokinase (HK); III) action of phosphofrutokinase (PFK); and IV) lactate import and export (Tanner et al., 2018). HK2 is one of four hexokinase isoforms highly expressed in skeletal muscle and adipose tissue. It acts as a fundamental part of the glycolysis process, participating in the conversion of glucose to glucose-6-phosphate (G6P) (Rabbani and Thornalley, 2019). High concentration of glucose in stem derived adipose cells increases the accumulation of lipids and positively regulates the expression of the adipogenic factors PPAR and LPL (Kolodziej et al., 2019). Therefore, our results suggest that the positive regulation of HK2 expression in the gonadal white adipose tissue of mice ancestrally exposed to the obesogenic substance TBT may be directed at glucose homeostasis within the adipose tissue as a response to the observed lipid accumulation in these mice (Chamorro-Garcia et al., 2017). Our findings indicate that modulation of lncRNAs may play a fundamental role in the adipogenesis process. In fact, a recent review published by Squillaro et al. 30 (2020), focuses on the role of lncRNAs in the biology and metabolism of adipocytes. In this review, the authors discuss that lncRNAs such as SRA, ADINR and HOTAIR, are involved in the transcriptional silencing of genes involved in the differentiation of adipocytes in WAT. On the other hand, high expression of lncRNAs ASMER-1, ASMER-2 and Plnc1 in the adipose tissue of obese mice positively regulates the expression of adipogenic transcription factors, such as PPAR, which play a fundamental role during adipogenesis (Squillaro et al., 2020). Identification of target genes for cis-acting lncRNAs provides information that can help uncover the function of lncRNAs modulated during the adipogenesis process. Endocrine Disrupting Chemicals, e.g tributyltin (TBT), promote adipogenesis by altering the development of fat cells and/or by increasing energy storage in the adipose tissue (Papalou et al., 2019). 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SLAS Technol. 2018;23(6):493–506. 19. Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2012;25(18):1915–1927. 20. Bhat SA, Ahmad SM, Mumtaz PT, Malik AA, Dar MA, Urwat U, et al. Long non- coding RNAs: Mechanism of action and functional utility. Non-coding RNA Res. 36 2016;1(1):43–50. 21. Sun Z, Nair A, Chen X, Prodduturi N, Wang J, Kocher P. UClncR : Ultrafast and comprehensive long non-coding RNA detection from RNA-seq. Sci Rep. 2017;7(14196):1–10. AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 ANEXO A – Normas da Revista Molecular and Cellular Endocrinology TABLE OF CONTENTS . . • Description • Audience • Impact Factor • Abstracting and Indexing • Editorial Board • Guide for Authors p.1 p.1 p.1 p.2 p.2 p.4 DESCRIPTION . Molecular and Cellular Endocrinology was established in 1974 to meet the demand for integrated publication on all aspects related to the genetic and biochemical effects, synthesis and secretions of extracellular signals (hormones, neurotransmitters, etc.) and to the understanding of cellular regulatory mechanisms involved in hormonal control. The journal is fulfilling this aim by publishing full-length original research papers, rapid papers, reviews, invited Special Issues, and book reviews. The scope encompasses all subjects related to genetic, epigenetic, biochemical, and molecular aspects of endocrine research and cell regulation. These include: (1) mechanisms of action of extracellular signals (hormones, neurotransmitters, etc.), (2) interaction of these factors with receptors, (3) generation, action and role of intracellular signals such as cyclic nucleotides and calcium, (4) hormone-regulated gene expression, (5) impact of gene structure on endocrine functions, (6) structure and physicochemical properties of hormones, hormone receptors and other hormone-binding components, (7) synthesis, secretion, metabolism and inactivation of hormones, neurotransmitters, etc. (8) hormonal control of differentiation, (9) related control mechanisms in non-mammalian systems, (10) methodological and theoretical aspects related to hormonal control processes, (11) clinical and translational studies as far as they throw new light on basic research in this field, (12) control of intermediary metabolism at the cellular level, (13) ultrastructural aspects related to hormone secretion and action, (14) comparative aspects of endocrinology only if they elucidate novel hormonal mechanisms. AUDIENCE . Workers in endocrinology, biochemistry and pharmacology IMPACT FACTOR . 2019: 3.871 © Clarivate Analytics Journal Citation Reports 2020 ISSN: 0303-7207 http://www.elsevier.com/locate/mce AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 38 ABSTRACTING AND INDEXING . BIOSIS Citation Index Current Contents - Life Sciences Embase PubMed/Medline Informedicus Reference Update Scopus EDITORIAL BOARD . Editors C.M. Klinge, University of Louisville, Louisville, United States R. Laybutt, Garvan Institute of Medical Research, Sydney, Australia C.A. Stratakis, National Institute of Child Health and Human Development, Bethesda, Maryland, United States Special Issues and Reviews Editor V. Laudet, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan Editorial Board M. Bellingham, Glasgow, Scotland, United Kingdom D. Belsham, Toronto, Ontario, Canada D. Berryman, Athens, Ohio, United States W. Bourguet, Montpellier, France C. Carlberg, KUOPIO, Finland Y. Chen, Shanghai, China M.H. Choi, Seongbuk-gu, Korea, Republic of B.-C. Chung, Taipei, Taiwan F. Claessens, Leuven, Belgium C. Corbitt, Louisville, Kentucky, United States E. Estébanez-Perpiñá, Barcelona, Spain P. Fowler, Aberdeen, United Kingdom D. J. Good, Blacksburg, Virginia, United States D. Grammatopoulos, Warwick, United Kingdom B. Groner, Frankfurt am Main, Germany H.M. Hoffmann, East Lansing, Michigan, United States I.T. Huhtaniemi, London, United Kingdom J. C. Jonas, Bruxelles, Belgium E. Kassi, Athens, Greece S. Kimura, Bethesda, Maryland, United States T. Kino, Doha, Qatar J. Köhrle, Berlin, Germany N. Kotaja, Turku, Finland R. Kumar, Houston, Texas, United States D. Kurrasch, Calgary, Alberta, Canada E. Lalli, Paris, France E.W.F. Lam, London, United Kingdom M. Levine, Philadelphia, Pennsylvania, United States M. Maggiolini, Cosenza, Italy I.J. McEwan, Aberdeen, United Kingdom Y. Nakamura, Sendai, Japan R. Narayanan, Memphis, Tennessee, United States S. Nef, Geneva, Switzerland R. Nogueiras, Santiago de Compostela, Spain T.M. Penning, Philadelphia, Pennsylvania, United States M. Plateroti, Lyon, France M. Rae, Edinburgh, United Kingdom W.E. Rainey, Ann Arbor, Michigan, United States K.H. Storbeck, Stellenbosch, South Africa M. Theodoropoulou, Munich, Germany J. Toppari, Turku, Finland A. Ulloa-Aguirre A. Walker, Riverside, California, United States D.B. Wilson, Saint Louis, Missouri, United States A.S.T. Wong, Hong Kong, Hong Kong http://www.elsevier.com/locate/mce AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 39 9 GUIDE FOR AUTHORS . Your Paper Your Way We now differentiate between the requirements for new and revised submissions. You may choose to submit your manuscript as a single Word or PDF file to be used in the refereeing process. Only when your paper is at the revision stage, will you be requested to put your paper in to a 'correct format' for acceptance and provide the items required for the publication of your article. To find out more, please visit the Preparation section below. Types of article • Research Paper • Review Article Unsolicited reviews will be considered only in exceptional cases and should be preceded by a letter of enquiry from the prospective author, who should be a recognized expert in the field of the proposed article. Pre-submission enquiries may be sent to the Editorial Office mce@elsevier.com and will be evaluated by the Special Issues and Reviews Editor of Molecular and Cellular Endocrinology. Specifically, authors must provide the following in their review proposal: 1) both your own and any co-author(s) affiliation and full contact details; 2) an explanation of the current interest and significance to the broad readership of the journal, that is, compelling reasons why the review should be considered; 3) a 500-600 word summary which clearly outlines what will be discussed in the article, plus up to 20 key references that indicate the intended breadth of the proposed article (please note that references should include work published in the past 2-4 years). Only proposals that include this information will be considered. Submission checklist You can use this list to carry out a final check of your submission before you send it to the journal for review. Please check the relevant section in this Guide for Authors for more details. Ensure that the following items are present: One author has been designated as the corresponding author with contact details: • E-mail address • Full postal address All necessary files have been uploaded: Manuscript: • Include keywords • All figures (include relevant captions) • All tables (including titles, description, footnotes) • Ensure all figure and table citations in the text match the files provided • Indicate clearly if color should be used for any figures in print Graphical Abstracts / Highlights files (where applicable) Supplemental files (where applicable) Further considerations • Manuscript has been 'spell checked' and 'grammar checked' • All references mentioned in the Reference List are cited in the text, and vice versa • Permission has been obtained for use of copyrighted material from other sources (including the Internet) • A competing interests statement is provided, even if the authors have no competing interests to declare • Journal policies detailed in this guide have been reviewed • Referee suggestions and contact details provided, based on journal requirements For further information, visit our Support Center. BEFORE YOU BEGIN Ethics in publishing Please see our information pages on Ethics in publishing and Ethical guidelines for journal publication. http://www.elsevier.com/locate/mce mailto:mce@elsevier.com https://service.elsevier.com/app/home/supporthub/publishing/ https://www.elsevier.com/about/policies/publishing-ethics https://www.elsevier.com/authors/journal-authors/policies-and-ethics AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 39 Studies in humans and animals If the work involves the use of human subjects, the author should ensure that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The manuscript should be in line with the Recommendations for the Conduct, Reporting, Editing and Publication of Scholarly Work in Medical Journals and aim for the inclusion of representative human populations (sex, age and ethnicity) as per those recommendations. The terms sex and gender should be used correctly. Authors should include a statement in the manuscript that informed consent was obtained for experimentation with human subjects. The privacy rights of human subjects must always be observed. All animal experiments should comply with the ARRIVE guidelines and should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and the authors should clearly indicate in the manuscript that such guidelines have been followed. The sex of animals must be indicated, and where appropriate, the influence (or association) of sex on the results of the study. Declaration of interest All authors must disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work. Examples of potential competing interests include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Authors must disclose any interests in two places: 1. A summary declaration of interest statement in the title page file (if double-blind) or the manuscript file (if single-blind). If there are no interests to declare then please state this: 'Declarations of interest: none'. This summary statement will be ultimately published if the article is accepted. 2. Detailed disclosures as part of a separate Declaration of Interest form, which forms part of the journal's official records. It is important for potential interests to be declared in both places and that the information matches. More information. Submission declaration and verification Submission of an article implies that the work described has not been published previously (except in the form of an abstract, a published lecture or academic thesis, see 'Multiple, redundant or concurrent publication' for more information), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright- holder. To verify originality, your article may be checked by the originality detection service Crossref Similarity Check. Preprints Please note that preprints can be shared anywhere at any time, in line with Elsevier's sharing policy. Sharing your preprints e.g. on a preprint server will not count as prior publication (see 'Multiple, redundant or concurrent publication' for more information). Use of inclusive language Inclusive language acknowledges diversity, conveys respect to all people, is sensitive to differences, and promotes equal opportunities. Content should make no assumptions about the beliefs or commitments of any reader; contain nothing which might imply that one individual is superior to another on the grounds of age, gender, race, ethnicity, culture, sexual orientation, disability or health condition; and use inclusive language throughout. Authors should ensure that writing is free from bias, stereotypes, slang, reference to dominant culture and/or cultural assumptions. We advise to seek gender neutrality by using plural nouns ("clinicians, patients/clients") as default/wherever possible to avoid using "he, she," or "he/she." We recommend avoiding the use of descriptors that refer to personal attributes such as age, gender, race, ethnicity, culture, sexual orientation, disability or health condition unless they are relevant and valid. These guidelines are meant as a point of reference to help identify appropriate language but are by no means exhaustive or definitive. Author contributions For transparency, we encourage authors to submit an author statement file outlining their individual contributions to the paper using the relevant CRediT roles: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; 40 http://www.elsevier.com/locate/mce https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/ http://www.icmje.org/recommendations/ http://www.icmje.org/recommendations/ https://www.who.int/gender-equity-rights/understanding/gender-definition/en/ https://www.nc3rs.org.uk/arrive-guidelines https://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm https://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm https://service.elsevier.com/app/answers/detail/a_id/286/supporthub/publishing/ https://www.elsevier.com/authors/journal-authors/policies-and-ethics https://www.elsevier.com/authors/journal-authors/policies-and-ethics https://www.elsevier.com/editors/perk/plagiarism-complaints/plagiarism-detection https://www.elsevier.com/editors/perk/plagiarism-complaints/plagiarism-detection https://www.elsevier.com/about/policies/sharing/preprint https://www.elsevier.com/about/policies/sharing https://www.elsevier.com/authors/journal-authors/policies-and-ethics https://www.elsevier.com/authors/journal-authors/policies-and-ethics AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 40 Software; Supervision; Validation; Visualization; Roles/Writing - original draft; Writing - review & editing. Authorship statements should be formatted with the names of authors first and CRediT role(s) following. More details and an example Changes to authorship Authors are expected to consider carefully the list and order of authors before submitting their manuscript and provide the definitive list of authors at the time of the original submission. Any addition, deletion or rearrangement of author names in the authorship list should be made only before the manuscript has been accepted and only if approved by the journal Editor. To request such a change, the Editor must receive the following from the corresponding author: (a) the reason for the change in author list and (b) written confirmation (e-mail, letter) from all authors that they agree with the addition, removal or rearrangement. In the case of addition or removal of authors, this includes confirmation from the author being added or removed. Only in exceptional circumstances will the Editor consider the addition, deletion or rearrangement of authors after the manuscript has been accepted. While the Editor considers the request, publication of the manuscript will be suspended. If the manuscript has already been published in an online issue, any requests approved by the Editor will result in a corrigendum. Article transfer service This journal is part of our Article Transfer Service. This means that if the Editor feels your article is more suitable in one of our other participating journals, then you may be asked to consider transferring the article to one of those. If you agree, your article will be transferred automatically on your behalf with no need to reformat. Please note that your article will be reviewed again by the new journal. More information. Copyright Upon acceptance of an article, authors will be asked to complete a 'Journal Publishing Agreement' (see more information on this). An e-mail will be sent to the corresponding author confirming receipt of the manuscript together with a 'Journal Publishing Agreement' form or a link to the online version of this agreement. Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their institutions. Permission of the Publisher is required for resale or distribution outside the institution and for all other derivative works, including compilations and translations. If excerpts from other copyrighted works are included, the author(s) must obtain written permission from the copyright owners and credit the source(s) in the article. Elsevier has preprinted forms for use by authors in these cases. For gold open access articles: Upon acceptance of an article, authors will be asked to complete an 'Exclusive License Agreement' (more information). Permitted third party reuse of gold open access articles is determined by the author's choice of user license. Author rights As an author you (or your employer or institution) have certain rights to reuse your work. More information. Elsevier supports responsible sharing Find out how you can share your research published in Elsevier journals. Role of the funding source You are requested to identify who provided financial support for the conduct of the research and/or preparation of the article and to briefly describe the role of the sponsor(s), if any, in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. If the funding source(s) had no such involvement then this should be stated. Open access Please visit our Open Access page for more information. 41 http://www.elsevier.com/locate/mce https://www.elsevier.com/authors/journal-authors/policies-and-ethics/credit-author-statement https://www.elsevier.com/authors/article-transfer-service https://www.elsevier.com/about/policies/copyright https://www.elsevier.com/about/policies/copyright/permissions https://www.elsevier.com/__data/assets/word_doc/0007/98656/Permission-Request-Form.docx https://www.elsevier.com/about/policies/copyright https://www.elsevier.com/about/policies/open-access-licenses https://www.elsevier.com/about/policies/copyright https://www.elsevier.com/about/policies/copyright https://www.elsevier.com/authors/journal-authors/submit-your-paper/sharing-and-promoting-your-article https://www.elsevier.com/journals/molecular-and-cellular-endocrinology/0303-7207/open-access-options AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 41 Elsevier Researcher Academy Researcher Academy is a free e-learning platform designed to support early and mid-career researchers throughout their research journey. The "Learn" environment at Researcher Academy offers several interactive modules, webinars, downloadable guides and resources to guide you through the process of writing for research and going through peer review. Feel free to use these free resources to improve your submission and navigate the publication process with ease. Language (usage and editing services) Please write your text in good English (American or British usage is accepted, but not a mixture of these). Authors who feel their English language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English may wish to use the English Language Editing service available from Elsevier's Author Services. Submission Our online submission system guides you stepwise through the process of entering your article details and uploading your files. The system converts your article files to a single PDF file used in the peer-review process. Editable files (e.g., Word, LaTeX) are required to typeset your article for final publication. All correspondence, including notification of the Editor's decision and requests for revision, is sent by e-mail. Referees Please submit the names and institutional e-mail addresses of several potential referees. For more details, visit our Support site. Note that the editor retains the sole right to decide whether or not the suggested reviewers are used. PREPARATION NEW SUBMISSIONS Submission to this journal proceeds totally online and you will be guided stepwise through the creation and uploading of your files. The system automatically converts your files to a single PDF file, which is used in the peer-review process. As part of the Your Paper Your Way service, you may choose to submit your manuscript as a single file to be used in the refereeing process. This can be a PDF file or a Word document, in any format or lay- out that can be used by referees to evaluate your manuscript. It should contain high enough quality figures for refereeing. If you prefer to do so, you may still provide all or some of the source files at the initial submission. Please note that individual figure files larger than 10 MB must be uploaded separately. References There are no strict requirements on reference formatting at submission. References can be in any style or format as long as the style is consistent. Where applicable, author(s) name(s), journal title/ book title, chapter title/article title, year of publication, volume number/book chapter and the article number or pagination must be present. Use of DOI is highly encouraged. The reference style used by the journal will be applied to the accepted article by Elsevier at the proof stage. Note that missing data will be highlighted at proof stage for the author to correct. Formatting requirements There are no strict formatting requirements but all manuscripts must contain the essential elements needed to convey your manuscript, for example Abstract, Keywords, Introduction, Materials and Methods, Results, Conclusions, Artwork and Tables with Captions. If your article includes any Videos and/or other Supplementary material, this should be included in your initial submission for peer review purposes. Divide the article into clearly defined sections. Figures and tables embedded in text Please ensure the figures and the tables included in the single file are placed next to the relevant text in the manuscript, rather than at the bottom or the top of the file. The corresponding caption should be placed directly below the figure or table. Peer review This journal operates a single blind review process. All contributions will be initially assessed by the editor for suitability for the journal. Papers deemed suitable are then typically sent to a minimum of two independent expert reviewers to assess the scientific quality of the paper. The Editor is responsible for the final decision regarding acceptance or rejection of articles. The Editor's decision is final. More information on types of peer review. 42 http://www.elsevier.com/locate/mce https://researcheracademy.elsevier.com/ https://webshop.elsevier.com/language-editing-services/language-editing/ https://webshop.elsevier.com/language-editing-services/language-editing/ https://service.elsevier.com/app/answers/detail/a_id/8238/kw/8238/p/10523/supporthub/publishing/ https://www.elsevier.com/reviewers/what-is-peer-review https://www.elsevier.com/reviewers/what-is-peer-review AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 42 REVISED SUBMISSIONS Use of word processing software Regardless of the file format of the original submission, at revision you must provide us with an editable file of the entire article. Keep the layout of the text as simple as possible. Most formatting codes will be removed and replaced on processing the article. The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also the Guide to Publishing with Elsevier). See also the section on Electronic artwork. To avoid unnecessary errors you are strongly advised to use the 'spell-check' and 'grammar-check' functions of your word processor. Article structure Subdivision - numbered sections Divide your article into clearly defined and numbered sections. Subsections should be numbered 1.1 (then 1.1.1, 1.1.2, ...), 1.2, etc. (the abstract is not included in section numbering). Use this numbering also for internal cross-referencing: do not just refer to 'the text'. Any subsection may be given a brief heading. Each heading should appear on its own separate line. Introduction State the objectives of the work and provide an adequate background, avoiding a detailed literature survey or a summary of the results. Material and methods Provide sufficient details to allow the work to be reproduced by an independent researcher. Methods that are already published should be summarized, and indicated by a reference. If quoting directly from a previously published method, use quotation marks and also cite the source. Any modifications to existing methods should also be described. Theory/calculation A Theory section should extend, not repeat, the background to the article already dealt with in the Introduction and lay the foundation for further work. In contrast, a Calculation section represents a practical development from a theoretical basis. Results Results should be clear and concise. Discussion This should explore the significance of the results of the work, not repeat them. A combined Results and Discussion section is often appropriate. Avoid extensive citations and discussion of published literature. Conclusions The main conclusions of the study may be presented in a short Conclusions section, which may stand alone or form a subsection of a Discussion or Results and Discussion section. Appendices If there is more than one appendix, they should be identified as A, B, etc. Formulae and equations in appendices should be given separate numbering: Eq. (A.1), Eq. (A.2), etc.; in a subsequent appendix, Eq. (B.1) and so on. Similarly for tables and figures: Table A.1; Fig. A.1, etc. Essential title page information • Title. Concise and informative. Titles are often used in information-retrieval systems. Avoid abbreviations and formulae where possible. • Author names and affiliations. Please clearly indicate the given name(s) and family name(s) of each author and check that all names are accurately spelled. You can add your name between parentheses in your own script behind the English transliteration. Present the authors' affiliation addresses (where the actual work was done) below the names. Indicate all affiliations with a lower- case superscript letter immediately after the author's name and in front of the appropriate address. Provide the full postal address of each affiliation, including the country name and, if available, the e-mail address of each author. • Corresponding author. Clearly indicate who will handle correspondence at all stages of refereeing and publication, also post-publication. This responsibility includes answering any future queries about Methodology and Materials. Ensure that the e-mail address is given and that contact details are kept up to date by the corresponding author. 43 http://www.elsevier.com/locate/mce https://www.elsevier.com/authors/journal-authors/submit-your-paper https://www.elsevier.com/authors/journal-authors/submit-your-paper AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 43 • Present/permanent address. If an author has moved since the work described in the article was done, or was visiting at the time, a 'Present address' (or 'Permanent address') may be indicated as a footnote to that author's name. The address at which the author actually did the work must be retained as the main, affiliation address. Superscript Arabic numerals are used for such footnotes. Highlights Highlights are mandatory for this journal as they help increase the discoverability of your article via search engines. They consist of a short collection of bullet points that capture the novel results of your research as well as new methods that were used during the study (if any). Please have a look at the examples here: example Highlights. Highlights should be submitted in a separate editable file in the online submission system. Please use 'Highlights' in the file name and include 3 to 5 bullet points (maximum 85 characters, including spaces, per bullet point). Abstract A concise and factual abstract is required. The abstract should state briefly the purpose of the research, the principal results and major conclusions. An abstract is often presented separately from the article, so it must be able to stand alone. For this reason, References should be avoided, but if essential, then cite the author(s) and year(s). Also, non-standard or uncommon abbreviations should be avoided, but if essential they must be defined at their first mention in the abstract itself. The abstract should be no more than 150 words. Graphical abstract Although a graphical abstract is optional, its use is encouraged as it draws more attention to the online article. The graphical abstract should summarize the contents of the article in a concise, pictorial form designed to capture the attention of a wide readership. Graphical abstracts should be submitted as a separate file in the online submission system. Image size: Please provide an image with a minimum of 531 × 1328 pixels (h × w) or proportionally more. The image should be readable at a size of 5 × 13 cm using a regular screen resolution of 96 dpi. Preferred file types: TIFF, EPS, PDF or MS Office files. You can view Example Graphical Abstracts on our information site. Authors can make use of Elsevier's Illustration Services to ensure the best presentation of their images and in accordance with all technical requirements. Keywords Immediately after the abstract, provide a maximum of 6 keywords, using British spelling and avoiding general and plural terms and multiple concepts (avoid, for example, 'and', 'of'). Be sparing with abbreviations: only abbreviations firmly established in the field may be eligible. These keywords will be used for indexing purposes. Abbreviations Define abbreviations that are not standard in this field in a footnote to be placed on the first page of the article. Such abbreviations that are unavoidable in the abstract must be defined at their first mention there, as well as in the footnote. Ensure consistency of abbreviations throughout the article. Acknowledgements Collate acknowledgements in a separate section at the end of the article before the references and do not, therefore, include them on the title page, as a footnote to the title or otherwise. List here those individuals who provided help during the research (e.g., providing language help, writing assistance or proof reading the article, etc.). Formatting of funding sources List funding sources in this standard way to facilitate compliance to funder's requirements: Funding: This work was supported by the National Institutes of Health [grant numbers xxxx, yyyy]; the Bill & Melinda Gates Foundation, Seattle, WA [grant number zzzz]; and the United States Institutes of Peace [grant number aaaa]. It is not necessary to include detailed descriptions on the program or type of grants and awards. When funding is from a block grant or other resources available to a university, college, or other research institution, submit the name of the institute or organization that provided the funding. If no funding has been provided for the research, please include the following sentence: 44 http://www.elsevier.com/locate/mce https://www.elsevier.com/authors/journal-authors/highlights https://www.elsevier.com/authors/journal-authors/graphical-abstract https://webshop.elsevier.com/illustration-services/ AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 44 This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Units Follow internationally accepted rules and conventions: use the international system of units (SI). If other units are mentioned, please give their equivalent in SI. Math formulae Please submit math equations as editable text and not as images. Present simple formulae in line with normal text where possible and use the solidus (/) instead of a horizontal line for small fractional terms, e.g., X/Y. In principle, variables are to be presented in italics. Powers of e are often more conveniently denoted by exp. Number consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text). Footnotes Footnotes should be used sparingly. Number them consecutively throughout the article. Many word processors build footnotes into the text, and this feature may be used. Should this not be the case, indicate the position of footnotes in the text and present the footnotes themselves separately at the end of the article. Artwork Image Manipulation Whilst it is accepted that authors sometimes need to manipulate images for clarity, manipulation for purposes of deception or fraud will be seen as scientific ethical abuse and will be dealt with accordingly. For graphical images, this journal is applying the following policy: no specific feature within an image may be enhanced, obscured, moved, removed, or introduced. Adjustments of brightness, contrast, or color balance are acceptable if and as long as they do not obscure or eliminate any information present in the original. Nonlinear adjustments (e.g. changes to gamma settings) must be disclosed in the figure legend. Electronic artwork General points • Make sure you use uniform lettering and sizing of your original artwork. • Preferred fonts: Arial (or Helvetica), Times New Roman (or Times), Symbol, Courier. • Number the illustrations according to their sequence in the text. • Use a logical naming convention for your artwork files. • Indicate per figure if it is a single, 1.5 or 2-column fittingimage. • For Word submissions only, you may still provide figures and their captions, and tables within a single file at the revision stage. • Please note that individual figure files larger than 10 MB must be provided in separate source files. A detailed guide on electronic artwork is available. You are urged to visit this site; some excerpts from the detailed information are given here. Formats Regardless of the application used, when your electronic artwork is finalized, please 'save as' or convert the images to one of the following formats (note the resolution requirements for line drawings, halftones, and line/halftone combinations given below): EPS (or PDF): Vector drawings. Embed the font or save the text as 'graphics'. TIFF (or JPG): Color or grayscale photographs (halftones): always use a minimum of 300 dpi. TIFF (or JPG): Bitmapped line drawings: use a minimum of 1000 dpi. TIFF (or JPG): Combinations bitmapped line/half-tone (color or grayscale): a minimum of 500 dpi is required. Please do not: • Supply files that are optimized for screen use (e.g., GIF, BMP, PICT, WPG); the resolution is too low. • Supply files that are too low in resolution. • Submit graphics that are disproportionately large for the content. Color artwork Please make sure that artwork files are in an acceptable format (TIFF (or JPEG), EPS (or PDF) or MS Office files) and with the correct resolution. If, together with your accepted article, you submit usable color figures then Elsevier will ensure, at no additional charge, that these figures will appear in color online (e.g., ScienceDirect and other sites) in addition to color reproduction in print. Further information on the preparation of electronic artwork. 45 http://www.elsevier.com/locate/mce https://www.elsevier.com/authors/author-schemas/artwork-and-media-instructions https://www.elsevier.com/authors/author-schemas/artwork-and-media-instructions https://www.elsevier.com/authors/author-schemas/artwork-and-media-instructions AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 45 Illustration services Elsevier's Author Services offers Illustration Services to authors preparing to submit a manuscript but concerned about the quality of the images accompanying their article. Elsevier's expert illustrators can produce scientific, technical and medical-style images, as well as a full range of charts, tables and graphs. Image 'polishing' is also available, where our illustrators take your image(s) and improve them to a professional standard. Please visit the website to find out more. Figure captions Ensure that each illustration has a caption. A caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used. Tables Please submit tables as editable text and not as images. Tables can be placed either next to the relevant text in the article, or on separate page(s) at the end. Number tables consecutively in accordance with their appearance in the text and place any table notes below the table body. Be sparing in the use of tables and ensure that the data presented in them do not duplicate results described elsewhere in the article. Please avoid using vertical rules and shading in table cells. References Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in the text. If these references are included in the reference list they should follow the standard reference style of the journal and should include a substitution of the publication date with either 'Unpublished results' or 'Personal communication'. Citation of a reference as 'in press' implies that the item has been accepted for publication. Reference links Increased discoverability of research and high quality peer review are ensured by online links to the sources cited. In order to allow us to create links to abstracting and indexing services, such as Scopus, CrossRef and PubMed, please ensure that data provided in the references are correct. Please note that incorrect surnames, journal/book titles, publication year and pagination may prevent link creation. When copying references, please be careful as they may already contain errors. Use of the DOI is highly encouraged. A DOI is guaranteed never to change, so you can use it as a permanent link to any electronic article. An example of a citation using DOI for an article not yet in an issue is: VanDecar J.C., Russo R.M., James D.E., Ambeh W.B., Franke M. (2003). Aseismic continuation of the Lesser Antilles slab beneath northeastern Venezuela. Journal of Geophysical Research, https://doi.org/10.1029/2001JB000884. Please note the format of such citations should be in the same style as all other references in the paper. Web references As a minimum, the full URL should be given and the date when the reference was last accessed. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different heading if desired, or can be included in the reference list. Data references This journal encourages you to cite underlying or relevant datasets in your manuscript by citing them in your text and including a data reference in your Reference List. Data references should include the following elements: author name(s), dataset title, data repository, version (where available), year, and global persistent identifier. Add [dataset] immediately before the reference so we can properly identify it as a data reference. The [dataset] identifier will not appear in your published article. References in a special issue Please ensure that the words 'this issue' are added to any references in the list (and any citations in the text) to other articles in the same Special Issue. Reference management software Most Elsevier journals have their reference template available in many of the most popular reference management software products. These include all products that support Citation Style Language styles, such as Mendeley. Using citation plug-ins from these products, authors only need to select 46 http://www.elsevier.com/locate/mce https://webshop.elsevier.com/illustration-services/ https://citationstyles.org/ https://citationstyles.org/ https://www.mendeley.com/reference-management/reference-manager/ AUTHOR INFORMATION PACK 12 Aug 2020 www.elsevier.com/locate/mce 37 46 the appropriate journal template when preparing their article, after which citations and bibliographies will be automatically formatted in the journal's style. If no template is yet available for this journal, please follow the format of the sample references and citations as shown in this Guide. If you use reference management software, please ensure that you remove all field codes before submitting the electronic manuscript. More information on how to remove field codes from different reference management software. Users of Mendeley Desktop can easily install the reference style for this journal by clicking the following link: http://open.mendeley.com/use-citation-style/molecular-and-cellular-endocrinology When preparing your manuscript, you will then be able to select this style using the Mendeley plug- ins for Microsoft Word or LibreOffice. Reference formatting There are no strict requirements on reference formatting at submission. References can be in any style or format as long as the style is consistent. Where applicable, author(s) name(s), journal title/ book title, chapter title/article title, year of publication, volume number/book chapter and the article number or pagination must be present. Use of DOI is highly encouraged. The reference style used by the journal will be applied to the accepted article by Elsevier at the proof stage. Note that missing data will be highlighted at proof stage for the author to correct. If you do wish to format the references yourself they should be arranged according to the following examples: Reference style Text: All citations in the text should refer to: 1. Single author: the author's name (without initials, unless there is ambiguity) and the year of publication; 2. Two authors: both authors' names and the year of publication; 3. Three or more authors: first author's name followed by 'et al.' and the year of publication. Citations may be made directly (or parenthetically). Groups of references can be listed either first alphabetically, then chronologically, or vice versa. Examples: 'as demonstrated (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999)…. Or, as demonstrated (Jones, 1999; Allan, 2000)… Kramer et al. (2010) have recently shown …' List: References should be arranged first alphabetically and then further sorted chronologically if necessary. More than one reference from the same author(s) in the same year must be identified by the letters 'a', 'b', 'c', etc., placed after the year of publication. Examples: Reference to a journal publication: Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2010. The art of writing a scientific article. J. Sci. Commun. 163, 51–59. https://doi.org/10.1016/j.Sc.2010.00372. Reference to a journal publication with an article number: Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2018. The art of writing a scientific article. Heliyon. 19, e00205. https://doi.org/10.1016/j.heliyon.2018.e00205. Reference to a book: Strunk Jr., W., White, E.B., 2000. The Elements of Style, fourth ed. Longman, New York. Reference to a chapter in an edited book: Mettam, G.R., Adams, L.B., 2009. How to prepare an electronic version of your article, in: Jones, B.S., Smith , R.Z. (Eds.), Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281–304. Reference to a website: Cancer Research UK, 1975. Cancer statistics reports for the UK. http://www.cancerresearchuk.org/ aboutcancer/statistics/cancerstatsreport/ (accessed 13 March 2003). Reference to a dataset: [dataset] Oguro, M., Imahiro, S., Saito, S., Nakashizuka, T., 2015. Mortality data for Japanese oak wilt disease and surrounding forest compositions. Mendeley Data, v1. https://doi.org/10.17632/ xwj98nb39r.1. Journal abbreviations source Journal names should be abbreviated according to the List of Title Word Abbreviations. Video Elsevier accepts video material and animation sequences to support and enhance your scientific r