DOI: 10.1016/j.cmet.2023.09.012

Resource: (Cell Signaling Technology Cat# 2794, RRID:AB_2183099)

Curator: @scibot

SciCrunch record: RRID:AB_2183099

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Este texto, que liguei, quer às aulas do Professor António Moreira, quer à sua bibliografia própria, quer à de outros autores que nos foi fornecida, levanta-me uma reflexão que desdobro em três tópicos que se vão entrosando: 1. Em primeiro lugar, trata-se de um estudo que realiza um exame crítico em torno de condições efetivas que garantam e-atividades produtivas. 2. Em segundo lugar, é um estudo que explana as condições de uma e-atividade em ecossistema digital com base numa posição "construtivista". 3. Em terceiro lugar, interroga o leitor relativamente às condições institucionais, que são objetivamente "políticas" (lato sensu), que consigam pôr em execução e-atividades "verdadeiras". Vejamos: 1. Este primeiro tópico aborda temas de vária ordem. Para o sucesso de e-atividades em ecossistema digital são necessárias, aparentemente, condições concretizáveis e materiais. O educador tem de ser, mais que um indivíduo, um designer de ensino. Igualmente se abordam números e mesmo idade dos participantes. E enumeram-se dados relativos aos benefícios principais que as e-atividades devidamente compreendidas produzem (no plano pedagógico, académico, social, etc.). Nestas condições assume importância o "5-Model Stage" de Salmon, que desenha uma escala que vai da motivação ao desenvolvimento de uma criadora interatividade, passando pela socialização, pela troca de informação, pela construção do conhecimento. Este tópico do estudo parece-me pretender delimitar fronteiras "reais". 2. O segundo coloca em destaque o núcleo duro do que que realmente nos desafia e é para mim entusiasmante: como desenhar e efetivar um ecossistema digital que exprima, em simultâneo, a sua autenticidade através da expressão afetiva, da coesão grupal, da inter-comunicação incondicional e incondicionada? Parece-me claro que a esta ideia de fundo subjaz a natureza do pressuposto de fundo: o construtivismo. Devo dizer que, para mim, esta é a questão principal. Por uma razão: o construtivismo é uma posição que nem sempre é bem compreendida porque assume particularidades muito vincadas em função dos domínios do conhecimento (no âmbito das ciências sociais e humanas) a que se aplica. Refiro uma história real: há tempos, assisti a um colóquio sobre transdisciplinaridade onde um reconhecido tecnocientista fez uma conterência de abertura muito interessante, que para mim, contudo, mais não foi do que o reconhecimento da importância do cruzamento de ideias em torno de uma área aplicada da tecnociência (embora com importância social). Quando lhe falei de epistemologias de fundo que ajuízam sobre a necessidade de se dessacralizar a tecnociência para a libertar das suas amarras sociais, políticas e mesmo civilizacionais, ocorrendo-me citar o "anarquista" Paul Feyerabend, a reação desse conceituado cientista foi exclamar em tom de desabafo: é um construtivista! E pronto, assunto encerrado. Com este exemplo quero apontar que o construtivismo é mal compreendido em muitos setores e incompreendido em muitos outros. Ora, o ponto de partida construtivista pretende, neste tema que aqui nos ocupa, ajustar e-atividades com e-moderação e todo um conjunto de métodos e trabalho que potencie o trabalho colaborativo e não simplesmente cooperativo. Trata-se, assim, de um paradigma que aponta para a uma verdadeira interação que produza auto-consciência, promova inteligência emocional, participação, que, com presença social, cognitiva, educativa, harmonize e favoreça uma organização complexiva que é um ecossistema digital. Esta constituição de um ecossistema digital tem algumas dificuldades de monta em conseguir exibir a sua indiscutível importância. Talvez a principal pareça ser aquilo que vou designar, em termos genéricos, a "espessura" do mundo. Em termos de debate sobre a validade de modelos verdadeiros de interação creio bem que se deve sempre colocar em tensão dois pólos: um deles será por mim denominado com uma frase do conhecido pensador Jürgen Habermas: o homem não pode não aprender; com esta afirmação este Autor quis designar algo muito relevante para o nosso tema: a comunicação aberta tem sempre viabilidade, apesar das suas inevitáveis distorções, porque no fundo o acordo racional e presencial entre iguais é o "telos" de qualquer linguagem. Esta tese, que não resume a sua obra gigantesca, exige alguns pré-requisitos difíceis que aqui não vou abordar, mas aponta para uma "presença" onde o "outro" é co-constitutivo de quem o escuta e vice-versa. Julgo que pode ser um contributo para uma compreensão da idealidade real, passe o paradoxo, de um ecossistema digital. Mas não chega. Será necessária a tensão proveniente de um outro pólo: aquele que estabelece com os objetos e as situações do/no mundo uma troca lúdica, cobrindo a sua multiplicidade. E aqui apelo para um autor como John Dewey, conhecido pedagogo, mas também politólogo e filósofo das relações internacionais (de onde parto para o conhecer). Este Autor, como outros (entre os quais G. H. Mead), segue, não por uma construção apriorística da ação, mas da praxis que mantemos com os outros e os objetos que nos cercam, que permite diversas interpretações, flexibilidade perante possibilidades conjugadas. A ação e a construção de possibilidades e intencionalidades estão, deste modo, abertas a construções e reconstruções contínuas, e só quando a intencionalidade aberta ao mundo embate na realidade tudo se inunda com a auto-consciência que tal choque produz. A solução de problemas é indissociável de um ideal de interação prévio que ganha força quando se redireciona face ao imprevisto ou, em última instância, ao impossível. Será caso para dizer: se impossível, mais necessário! É claro que um ecossistema digital, onde se fundem sujeitos e objetos em interação que possui modelos que se vão afinando com a realidade, pode abrir outras possibilidades teóricas. Não posso ignorar que há toda uma corrente estrutural-funcionalista, cujo grande autor é sem dúvida Niklas Luhmann, que parte do princípio de que organização, decisão, ação e intercomunicação, constituem sistemas autopoiéticos que vivem e se reproduzem por si e para si, não passando os membros desse sistema de "sinapses" que asseguram comunicação entre "neurotransmissores". Esta tese que foi desenvolvida por Luhmann, como uma espécie de "outra pós-modernidade", não colhe, quanto a mim, legitimidade neste nosso caso, porque um ecossistema digital vive, não de acasos, ou de uma direção naturalista férrea, mas, antes, vive de uma interação imperfeita que, porque se auto-compreende como tal, se volta para a verdadeira interação enquanto horizonte de possibilidades situadas na nossa história comum. 3. A última parte deste estudo é, quanto a mim, a mais difícil de entender. Porque, no fundo, toca o papel das instituições. Estas fazem parte de uma estrutura montada que, em meu parecer, constitui a parte mais afastada da "instituição imaginária" de qualquer sociedade, para usar uma expressão de Castoriadis. Todo o sistema histórico parte do seu ato fundador, mas o facto é que o défice de dinâmica social (ideologias, modos de produção, circunstâncias básicas da vida humana como a escassez de bens, etc.) vai amortecendo os seus ideais, as suas promessas, as suas capacidades criadores, avultando a racionalidade funcionalista. Que é a atual. Assim, que fazer no plano institucional quando é aqui que se digladiam conceções de gestão funcional de recursos escassos? Sou cético neste tópico. para mim, um verdadeiro construtivismo terá de apostar na crítica reconstrutiva e na crítica desta crítica, em movimento incessante entre horizontes, possibilidades, deceções (a que o artigo alude, de resto, e que para mim radicam, hoje, na crise da motivação), e fixações de renovados faróis utópico-regulativos. A utopia, como nos diz a sua etimologia, é apenas o que está "fora do lugar", do "ainda não ser". Logo, creio bem que o entusiasmo endógeno a um ecossistema digital poderá ser uma boa bússola para caminhar na "meia-noite da História" em que de algum modo vivemos rumo a uma madrugada mais promissora. Desculpem meu desabafo po(i)ético. Cumprimentos. aluno 2302821. Silvério Cunha.

Os benefícios em usar as e-atividades parecem-me inúmeros, quer em ambientes totalmente virtuais quer em ambientes híbridos. Dos benefícios abordados neste artigo destaco em relação os benefícios académicos o impacto positivo no desempenho académico e nos resultados dos alunos e que podem promover uma melhor compreensão do material do curso e uma maior retenção de conhecimentos. Claro que para que tal aconteça as atividades devem ser bem estruturadas e fáceis de usar, com instruções claras e de compreensão fácil por parte dos alunos o que constituem benefícios do design. Os benefícios sociais e para os estudantes também me parecem extremamente relevantes uma vez que penso que as e-atividades promovem a colaboração e a interação entre os alunos através dos fóruns de discussão, projetos em grupo, feedback de colegas e construção de comunidades o que permite um maior envolvimento e maior motivação para os estudantes. Nos benefícios pedagógicos destaco a promoção da aprendizagem ativa e a incorporação de estratégias de ensino eficazes.

Por vezes, quando nos é proposta a transição para o digital centramos a nossa atenção nos aspetos tecnológicos. No entanto, o fundamental é que a aprendizagem ocorra, é aí que deve estar o foco. Este deve estar sempre na pedagogia e nunca na tecnologia; esta é apenas um meio que vai possibilitar/facilitar o processo de ensino-aprendizagem.

Catarina Paulos

O tópico “E-atividades” tem-me desafiado a (re)pensar a importância que sempre dei à planificação e tenho como certo que a planificação em ambientes de aprendizagem virtuais a elevam a uma categoria ainda maior no que à sua importância diz respeito. Conceber uma E-atividade não se pode limitar à ideia (apenas) de ela existir nem tão pouco conceber que a mesma não seja acompanhada de uma reflexão profunda quer no respeitante aos seus objetivos, quer à sua execução. Embora esta ideia pareça ser um pouco redutora (por tão óbvia) a verdade é que considero que este será um dos maiores desafios que se colocará ao professor (mediador) aquando da conceção de uma E-atividade.

Olá Colegas, Efetivamente todos os educadores/professores sentem a necessidade de se adaptarem às novas formas de ensino aprendizagem em ecossistemas de aprendizagem em rede onde se usam novos ambientes utilizando o que alguns designam de novas ferramentas digitais, mas que devem ser consideradas mais do que isso quando criam ambientes de relacionamento continuum e onde é possível sentir proximidade e afetividade com os colegas e relação contínua e interativa com os materiais.

Isabel Valente Unidade Curricular: Docência Digital em Rede , 2023 01

Não só a sobreposição mas também o cuidado que devemos ter ao desenhar e-atividades de forma a não sobrecarregar os formandos/alunos com demasiados ambientes. Embora as e-atividades que foram até agora utilizadas neste cursos mas pareçam exequíveis e complementares, dando ao formando (falo por experiencia) que se circula num ambiente muito interligado.

Existe um outro software para ana´lise de conteudo tb interessante: o Maxqda https://www.maxqda.com/

Blogs serão demasiado datados, não? Presentemente existem outras vias (como o anteriormente denominado twitter) que atraem mais os leitores. Ou estou enganada?

Creio que de facto o Hypotesis é uma excelente E-atividade para a leitura de textos que sejam distribuídos durante a formação porque permite ao formando opinar sobre aquilo que está a ler (a ideia aqui transcrita mais abaixo de integrating online students responses and probing) e a formador/docente aferir sobre as leituras que o formando faz.

A utilização de uma E-atividade, bem definida e motivadora permite potenciar o desenvolvimento das competências dos estudantes, fomentando a capacidade de construção autónoma do conhecimento. Uma e-atividade promove uma aprendizagem ativa, através de trabalho colaborativo estimulando a procura do conhecimento e a sua aplicabilidade.

Helena E Correia

Ao realizar uma pesquisa sobre o mesmo tema, encontrei duas outras referencias bibliográficas: um artigo de Garrison, et al. (1999) para o ensino superior e o artigo de Anderson et al. (2001).

Anderson, T., Rourke, L., Garrison, D. R., & Archer, W. (2001). Assessing teaching presence in a computer conferencing context. Journal of asynchronous learning networks, 5(2), 1-17. Garrison, D. R., Anderson, T., & Archer, W. (1999). Critical inquiry in a text-based environment: Computer conferencing in higher education. The Internet and Higher Education, 2(2-3), 87-105.

Margarida Saraiva | Docência Digital em Rede

Em relação ao impacto das atividades eletrónicas no envolvimento e motivação dos alunos em ambientes de aprendizagem online, o artigo sugere que as essas atividades podem ser altamente envolventes e interativas, e podem proporcionar aos alunos uma oportunidade clara e estruturada de participar e interagir de forma colaborativa. Além disso, as atividades eletrónicas podem ser adaptáveis entre cursos e disciplinas e podem fornecer uma estrutura confiável e prática para o design de aprendizagem online. De um modo geral, o artigo sugere que as atividades eletrónicas podem ser uma ferramenta útil para promover o envolvimento e a motivação dos alunos em ambientes de aprendizagem online, particularmente quando concebidas tendo em consideração cuidadosamente as necessidades e preferências dos alunos. Porém, na minha opinião, todos os intervenientes devem estar preparados para desenvolver essas atividades eletrónicas, sobretudo, no ensino superior, em que professores e alunos estão em patamares diferentes, quer relativamente ao envolvimento, quer à motivação, para o uso desses ambientes de aprendizagem online.

Margarida Saraiva | Docência Digital em Rede

Considero muito relevante o facto das e-atividades estarem teoricamente muito bem fundamentadas, pelo facto de assentarem em pedagogias socio-construtivistas. Com efeito, as teorias sócio-construtivistas, de autores como Jean Piaget e Vygotsky entre outros, defendem o princípio de que o conhecimento é uma construção social, que o conhecimento se constrói essencialmente a partir da interação entre o indivíduo e o meio. As atividades realizadas em ambientes digitais de aprendizagem deverão, neste sentido, ser organizadas para apelarem à participação dos estudantes, para promoverem a interação/relação entre os mesmos e com todos os que habitam um determinado ecossistema digital para o qual a e-atividade foi pensada. José Ramalho

As e-ativities devem ser estruturadas de forma a envolver os estudantes, estimular a interação e promover a realização de objetivos de aprendizagem específicos. Elas podem assumir várias formas, como discussões online, questionários, projetos colaborativos ou apresentações multimédia, de entre outras. As e-ativities devem fomentar o pensamento crítico, a resolução de problemas e a aquisição de conhecimento num ambiente online (ou em contextos híbridos). Susana Amante - IPViseu

Os resultados da investigação são interessantes e reforçam, entre outros aspetos, o carácter e importância da sociabilidade no seio do grupo. Se aceitarmos que a educação é uma questão de coração (Dom Bosco) ficamos mais conscientes que a tarefa do professor vai muito além das fronteiras metodológicas e será, com certeza, o seguro de um processo de ensino/aprendizagem, inserido num determinado contexto e conjunto de objetivos. Para o desenho das e-atividades considerar, a partir dos objetivos, os recursos necessários, as atividades a desenvolver propriamente ditas, a duração das mesmas, o processo de avaliação, não esquecendo o feedback que apoiará o aprendente durante todo o processo, reforçando-se que é imperativo as instruções de realização serem claras e objetivas. É igualmente importante, do lado do docente, a planificação de todo o processo através de um documento que funcione como guia orientador.

@ Microcredencial em Educação a Distância e Digital 2023

Como em qualquer ambiente, se não dominamos as ferramentas não podemos fazer bom uso delas. O meu B&D pode ser ótimo mas se não tiver conhecimentos para o usar não há solução.

As e-atividdaes mais adequadas são as que convidam à construção ativa do conhecimento a partir da interação com os outros (perspetiva sócio-construtivista, de aprendizagem colaborativa); as que recorrem à experimentação na resolução de problemas, estimulam a reflexão e o pensamento crítico.

A pedagogia da Presencialidade é, a meu ver, a essência da transição de um ensino remoto para uma educação digital em rede. O ensino à distância digital só fará sentido quando se garante esta presencialidade! A forma como se desenha e "executa" a e-atividade será determinante na promoção desta presença social, onde a expressão afetiva parece ter um papel muito importante.

Acredito que a docência digital em rede ao desafiar os docentes a aprenderem mais sobre modelos pedagógicos, processos de comunicação e interação, ao refletirem mais sobre o seu papel e capacidades de docência, vai contribuir em muito para a melhoria dos processos de ensino-aprendizagem no geral. Esta é uma oportunidade de mudança da pedagogia no Ensino Superior, que exige que os docentes recebam apoio e formação mas também motivação e tempo, tal como referido nesta parte do artigo.

Prueba

SCHOUERI, Luís E. Direito tributário. Editora Saraiva, 2023.:

A tributação segundo a capacidade contributiva pode assumir duas feições: a absoluta e a relativa. Enquanto do ponto de vista relativo (subjetivo) a capacidade contributiva se aplica a todos os tributos, no sentido absoluto (objetivo), ela é um critério a ser empregado para distinguir quem será contribuinte.

Este ponto nem sempre fica claro entre os que escrevem sobre a capacidade contributiva, e, por isso mesmo, é muito comum que alguns neguem a existência do Princípio e outros o apresentem como muito fluido: possivelmente, não estão falando sobre o mesmo fenômeno. A capacidade contributiva pode ser: (i) um limite ou critério para a graduação da tributação; ou (ii) um parâmetro para a distinção entre situações tributáveis e não tributáveis. No primeiro caso, falar­-se­-á em capacidade contributiva relativa ou subjetiva; no último, em capacidade contributiva absoluta ou objetiva. Esta será “a existência de uma riqueza apta a ser tributada (capacidade contributiva como pressuposto de tributação)”, enquanto no sentido subjetivo, será “a parcela dessa riqueza que será objeto da tributação em face de condições individuais (capacidade contributiva como critério de graduação e limite do tributo)”.

Quando encarada a capacidade contributiva do ponto de vista subjetivo, querem­-se conhecer as condições pessoais do contribuinte, i.e., se ele pode, ou não, suportar a carga tributária. A questão se resume a saber se existe um ponto, abaixo ou acima do qual descabe a incidência de um tributo, ou, ainda, até onde pode atingir a tributação; no primeiro caso, estar­-se­-á cogitando do mínimo de subsistência; ultrapassado o limite, versar­-se­-á sobre o confisco. Trata­-se da aptidão econômica, i.e., a capacidade de ser contribuinte.

É, neste sentido, algo além da mera capacidade econômica, já que a capacidade contributiva compreende aquela parcela da riqueza de que o contribuinte pode dispor para voltar­-se à coletividade. “Allí donde no existe tal capacidad, no pode existir el impuesto. Podrá haberse establecido en la Ley. Pero no llegará a ser una realidad social, porque no podrá ser pagado y suportado por quienes han de pagarlo y soportarlo. El impuesto que grava a quien carece de aptitud, de capacidad económica, es utópico. Es un impuesto que nace para no vivir, para fracassar, para morir, en suma.

A capacidade contributiva relativa pressupõe a existência de uma riqueza, mas não qualquer uma, senão aquela que gera um saldo (disponível). Assim, não basta, para aferir a existência de capacidade contributiva, investigar os rendimentos de uma pessoa. O exemplo, hoje clássico, é daquela pessoa que recebe alugueres razoáveis, mas que, por ter saúde precária, vê­-se obrigada a manter enfermeiros durante todo seu tratamento, além de altos custos de medicamentos. Terá ela, talvez, capacidade econômica; capacidade contributiva, entretanto, não cabe cogitar.

Nesta acepção relativa, parece que o Princípio da Capacidade Contributiva deve espraiar­-se por todas as categorias tributárias: não tendo o contribuinte o mínimo para sua sobrevivência, não pode ele ser constrangido a contribuir para as despesas públicas, ainda que ele as tenha causado (o serviço público é, sempre, de interesse público, ainda que dirigido a alguém). No caso de tributo com efeito de confisco, o próprio constituinte tratou de estender a proteção a qualquer espécie tributária, como se verá mais adiante. Mínimo existencial e confisco oferecem as balizas da capacidade contributiva, no sentido subjetivo, que “começa além do mínimo necessário à existência humana digna e termina aquém do limite destruidor da propriedade”869. Assim, no sentido subjetivo, o Princípio da Capacidade Contributiva não se limita aos impostos. Foi o que entendeu o Supremo Tribunal Federal no julgamento do RE 1.018.911-RR, em que se reconheceu a aplicabilidade da capacidade contributiva às taxas, no sentido de barreira à tributação se o contribuinte não tiver riqueza disponível (este julgado será retomado quando se tratar da base de cálculo de taxas, no Capítulo XII).

Por tais características, parece acertado afirmar que a capacidade contributiva, em sua feição relativa, tem feições de princípio jurídico, i.e., mandamento de otimização: deve o legislador, na medida do possível (ou ao máximo possível), buscar alcançar a capacidade contributiva; a base de cálculo do tributo deve ser medida que atinja, do melhor modo possível, aquela capacidade. Dentre duas bases de cálculo, o Princípio exigirá que se busque a mais exata; a alíquota do tributo não pode ser tão alta a ponto de a tributação ultrapassar a capacidade contributiva manifestada.

Outro é o raciocínio quando se toma a capacidade contributiva sob o ponto de vista objetivo; o que se quer é, apenas, que a situação que distinguirá os contribuintes (i.e., a situação que dirá que alguém deve pagar um tributo, ou, ainda mais claramente: a hipótese tributária) seja algo que, objetivamente, indique que quem nela se enquadra tem condições de suportar os gastos comuns.

A capacidade contributiva absoluta compreende o “momento que concerne à delimitação da base imponível, ou seja, a escolha de quais elementos aferidores da economia individual formam a fonte do tributo.

Neste sentido objetivo, absoluto, não se indaga se um determinado contribuinte pode, ou não, pagar o tributo; ao contrário, o legislador, em sua função generalizante, visando a concretizar a igualdade, dirá que quem está naquela situação deve poder pagar tributo.

A capacidade contributiva objetiva é verdadeira regra do ordenamento, já que proíbe que o legislador preveja hipóteses tributárias que não revelem, objetivamente, capacidade contributiva.

Assim, ser proprietário de um imóvel indica, objetivamente, ter capacidade contributiva; do mesmo modo, possuir um automóvel ou auferir renda. São todas situações que indicam, objetivamente, capacidade contributiva. São, melhor dizendo, signos presuntivos de riqueza.

Claro que é possível que alguém seja proprietário de um automóvel mas não tenha capacidade contributiva. Por exemplo, se um mendigo encontra um bilhete de uma rifa e vem a ganhar um automóvel, não tem ele, por isso, capacidade contributiva; não obstante, se ele quiser continuar proprietário do automóvel, deverá ele pagar o imposto correspondente. É o ônus que ele tem por ostentar o veículo. Não querendo pagar o imposto, cabe­-lhe vender o veículo a outrem, que, tornando-se proprietário do automóvel, pagará aquele imposto.

Nesse sentido (objetivo), ter­-se­-á por acertado o art. 145, § 1º, da Constituição Federal, quando faz referência apenas aos impostos. Com efeito, embora, em princípio, idêntico raciocínio pudesse ser estendido às taxas – e em outros países se aceite que as taxas se dobrem ao Princípio da Capacidade Contributiva também em seu sentido objetivo – deve­-se repisar que o constituinte brasileiro, por meio do § 2º do art. 145, vedou o emprego de base de cálculo própria de impostos às taxas. Ora, base de cálculo “própria” de impostos é aquela que se vale da capacidade contributiva objetiva, pois é índice de riqueza.

Ao vedar o emprego de semelhante base de cálculo para as taxas, vedou o constituinte que considerações de capacidade contributiva objetiva se estendessem àquela espécie tributária, onde descabem considerações sobre solidariedade. Quanto às contribuições especiais, o tema já foi explorado acima, com idêntica conclusão: o Princípio da Capacidade Contributiva é o reflexo, na matéria tributária, do princípio da solidariedade e por tal razão é critério para discriminação entre contribuintes que se igualam em outros critérios.

Assim, enquanto nos impostos a capacidade contributiva aparece imediatamente como critério de discriminação exigido pelos Princípios da Igualdade e da Solidariedade, no caso das contribuições especiais, o primeiro critério de discriminação está na referibilidade (i.e.: pertencer, ou não, ao grupo afetado); dentro do grupo, será a capacidade contributiva que permitirá que se efetuem diferenciações entre contribuintes.

• RE 588322
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 16/06/2010
• Publicação: 03/09/2010

Recurso Extraordinário 1. Repercussão geral reconhecida. 2. Alegação de inconstitucionalidade da taxa de renovação de localização e de funcionamento do Município de Porto Velho. 3. Suposta violação ao artigo 145, inciso II, da Constituição, ao fundamento de não existir comprovação do efetivo exercício do poder de polícia. 4. O texto constitucional diferencia as taxas decorrentes do exercício do poder de polícia daquelas de utilização de serviços específicos e divisíveis, facultando apenas a estas a prestação potencial do serviço público. 5. A regularidade do exercício do poder de polícia é imprescindível para a cobrança da taxa de localização e fiscalização. 6. À luz da jurisprudência deste Supremo Tribunal Federal, a existência do órgão administrativo não é condição para o reconhecimento da constitucionalidade da cobrança da taxa de localização e fiscalização, mas constitui um dos elementos admitidos para se inferir o efetivo exercício do poder de polícia, exigido constitucionalmente. Precedentes. 7. O Tribunal de Justiça de Rondônia assentou que o Município de Porto Velho, que criou a taxa objeto do litígio, é dotado de aparato fiscal necessário ao exercício do poder de polícia. 8. Configurada a existência de instrumentos necessários e do efetivo exercício do poder de polícia. 9. É constitucional taxa de renovação de funcionamento e localização municipal, desde que efetivo o exercício do poder de polícia, demonstrado pela existência de órgão e estrutura competentes para o respectivo exercício, tal como verificado na espécie quanto ao Município de Porto Velho/RO 10. Recurso extraordinário ao qual se nega provimento.

Tema - 217 - Comprovação do poder de polícia para cobrança de taxa de localização e funcionamento.

Tese - É constitucional taxa de renovação de funcionamento e localização municipal, desde que efetivo o exercício do poder de polícia, demonstrado pela existência de órgão e estrutura competentes para o respectivo exercício.

• A regulamentação é a atribuição conferida ao Presidente da República para editar decretos regulamentando a fiel execução das leis, prevista no art. 84, IV, CF. Trata-se de uma função política distanciada da neutralidade imposta às agências reguladoras. Na regulamentação, o Chefe do Poder executivo edita normas secundárias, complementares às leis, com o objetivo de lhes dar execução.

• A função regulatória, por outro lado, está prevista no art. 174, CF e diz respeito à elaboração de normas técnicas (não políticas), para normatização de um setor econômico em sentido amplo (serviços públicos e econômico em sentido estrito). Na função regulatória, busca-se que agentes neutros (imparciais) editem normas eminentemente técnicas ponderando custos e benefícios para o setor envolvido.

• ADI 1642
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EROS GRAU
• Julgamento: 03/04/2008
• Publicação: 19/09/2008

Ementa AÇÃO DIRETA DE INCONSTITUCIONALIDADE. ALÍNEA "d" DO INCISO XXIII DO ARTIGO 62 DA CONSTITUIÇÃO DO ESTADO DE MINAS GERAIS. APROVAÇÃO DO PROVIMENTO, PELO EXECUTIVO, DOS CARGOS DE PRESIDENTE DAS ENTIDADES DA ADMINISTRAÇÃO PÚBLICA INDIRETA ESTADUAL PELA ASSEMBLÉIA LEGISLATIVA. ALEGAÇÃO DE VIOLAÇÃO DO DISPOSTO NO ARTIGO 173, DA CONSTITUIÇÃO DO BRASIL. DISTINÇÃO ENTRE EMPRESAS ESTATAIS PRESTADORAS DE SERVIÇO PÚBLICO E EMPRESAS ESTATAIS QUE DESENVOLVEM ATIVIDADE ECONÔMICA EM SENTIDO ESTRITO. REGIME JURÍDICO ESTRUTURAL E REGIME JURÍDICO FUNCIONAL DAS EMPRESAS ESTATAIS. INCONSTITUCIONALIDADE PARCIAL. INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO. 1. Esta Corte em oportunidades anteriores definiu que a aprovação, pelo Legislativo, da indicação dos Presidentes das entidades da Administração Pública Indireta restringe-se às autarquias e fundações públicas, dela excluídas as sociedades de economia mista e as empresas públicas. Precedentes. 2. As sociedades de economia mista e as empresas públicas que explorem atividade econômica em sentido estrito estão sujeitas, nos termos do disposto no § 1º do artigo 173 da Constituição do Brasil, ao regime jurídico próprio das empresas privadas. 3. Distinção entre empresas estatais que prestam serviço público e empresas estatais que empreendem atividade econômica em sentido estrito 4. O § 1º do artigo 173 da Constituição do Brasil não se aplica às empresas públicas, sociedades de economia mista e entidades (estatais) que prestam serviço público. 5. A intromissão do Poder Legislativo no processo de provimento das diretorias das empresas estatais colide com o princípio da harmonia e interdependência entre os poderes. A escolha dos dirigentes dessas empresas é matéria inserida no âmbito do regime estrutural de cada uma delas. 6. Pedido julgado parcialmente procedente para dar interpretação conforme à Constituição à alínea "d" do inciso XXIII do artigo 62 da Constituição do Estado de Minas Gerais, para restringir sua aplicação às autarquias e fundações públicas, dela excluídas as empresas estatais, todas elas.

Di Pietro:

Quanto à maneira como concorrem para satisfazer ao interesse geral, os serviços podem ser: uti singuli e uti universi.

• Serviços uti singuli são aqueles que têm por finalidade a satisfação individual e direta das necessidades dos cidadãos. Pelo conceito restrito de serviço público adotado por Celso Antônio Bandeira de Mello, só esta categoria constitui serviço público: prestação de utilidade ou comodidade fruível diretamente pela comunidade. Entram nessa categoria determinados serviços comerciais e industriais do Estado (energia elétrica, luz, gás, transportes) e de serviços sociais (ensino, saúde, assistência e previdência social).

• Os serviços uti universi são prestados à coletividade, mas usufruídos apenas indiretamente pelos indivíduos. É o caso dos serviços de defesa do país contra o inimigo externo, dos serviços diplomáticos, dos trabalhos de pesquisa científica, de iluminação pública, de saneamento. Quanto a este último, o STF, pela Súmula nº 670 (atual Súmula Vinculante nº 41), consagrou o entendimento de que “o serviço de iluminação pública não pode ser remunerado mediante taxa”, exatamente por não ser usufruído uti singuli e não se enquadrar no conceito contido no artigo 145, II, da Constituição.

"A doutrina preconiza que a descentralização pode ser feita mediante outorga (descentralização por serviço) ou delegação (descentralização por colaboração) de serviços.

Na outorga, é transferida a titularidade e a execução do serviço público, a pessoa Jurídica diversa do Estado, ao passo que, na delegação, apenas a execução é transferida, permanecendo com o Estado a titularidade do serviço.

Para a doutrina majoritária, a outorga é conferida, somente, para pessoas jurídicas de direito público, como as autarquias ou fundações públicas de direito público, as quais se tornam titulares do serviço a elas transferido, executando essas atividades por sua conta e risco, sem, contudo, excluir o controle dos entes federativos." (CARVALHO, Matheus. Manual de direito administrativo. 4. ed. Salvador: Juspodivm, 2017, p. 163)

• ADI 3394
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EROS GRAU
• Julgamento: 02/04/2007
• Publicação: 15/08/2008

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. ARTIGOS 1º, 2º E 3º DA LEI N. 50, DE 25 DE MAIO DE 2.004, DO ESTADO DO AMAZONAS. TESTE DE MATERNIDADE E PATERNIDADE. REALIZAÇÃO GRATUITA. EFETIVAÇÃO DO DIREITO À ASSISTÊNCIA JUDICIÁRIA. LEI DE INICIATIVA PARLAMENTAR QUE CRIA DESPESA PARA O ESTADO-MEMBRO. ALEGAÇÃO DE INCONSTITUCIONALIDADE FORMAL NÃO ACOLHIDA. CONCESSÃO DEFINITIVA DO BENEFÍCIO DA ASSISTÊNCIA JUDICÁRIA GRATUITA. QUESTÃO DE ÍNDOLE PROCESSUAL. INCONSTITUCIONALIDADE DO INCISO I DO ARTIGO 2º. SUCUMBÊNCIA NA AÇÃO INVESTIGATÓRIA. PERDA DO BENEFÍCIO DA ASSISTÊNCIA JUDICIÁRIA GRATUITA. INCONSTITUCIONALIDADE DO INCISO III DO ARTIGO 2º. FIXAÇÃO DE PRAZO PARA CUMPRIMENTO DA DECISÃO JUDICIAL QUE DETERMINAR O RESSARCIMENTO DAS DESPESAS REALIZADAS PELO ESTADO-MEMBRO. INCONSTITUCIONALIDADE DO INCISO IV DO ARTIGO 2º. AFRONTA AO DISPOSTO NO ARTIGO 61, § 1º, INCISO II, ALÍNEA "E", E NO ARTIGO 5º, INCISO LXXIV, DA CONSTITUIÇÃO DO BRASIL .

1. Ao contrário do afirmado pelo requerente, a lei atacada não cria ou estrutura qualquer órgão da Administração Pública local. Não procede a alegação de que qualquer projeto de lei que crie despesa só poderá ser proposto pelo Chefe do Executivo. As hipóteses de limitação da iniciativa parlamentar estão previstas, em numerus clausus, no artigo 61 da Constituição do Brasil --- matérias relativas ao funcionamento da Administração Pública, notadamente no que se refere a servidores e órgãos do Poder Executivo. Precedentes.

2. Reconhecimento, pelas Turmas desta Corte, da obrigatoriedade do custeio do exame de DNA pelo Estado-membro, em favor de hipossuficientes.

3. O custeio do exame pericial da justiça gratuita viabiliza o efetivo exercício do direto à assistência judiciária, consagrado no artigo 5º, inciso LXXIV, da CB/88.

4. O disposto no inciso I consubstancia matéria de índole processual --- concessão definitiva do benefício à assistência judiaria gratuita --- tema a ser disciplinado pela União.

5. Inconstitucionalidade do inciso III do artigo 2º que estabelece a perda do direito à assistência judiciária gratuita do sucumbente na ação investigatória que tenha sido proposta pelo Ministério Público e que tenha como suporte o resultado positivo do exame de DNA. Violação do disposto no inciso LXXIV do artigo 5º da Constituição de 1.988.

6. Fixação de prazo para cumprimento da decisão judicial que determinar o ressarcimento das despesas realizadas pelo Estado-membro. Inconstitucionalidade do inciso IV do artigo 2º.

7. Ação direta julgada parcialmente procedente para declarar inconstitucionais os incisos I, III e IV, do artigo 2º, bem como a expressão "no prazo de sessenta dias a contar da sua publicação", constante do caput do artigo 3º da Lei n. 50/04 do Estado do Amazonas.

• ADI 5087
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 19/12/2019
• Publicação: 21/09/2020

ODS 16 - Paz, Justiça e Instituições Eficazes

CONSTITUCIONAL E ADMINISTRATIVO. EMENDA CONSTITUCIONAL 11/2013 DO ESTADO DO RIO GRANDE DO NORTE. IMPOSSIBILIDADE DE EMENDA PARLAMENTAR QUE ALTERE REGIME JURÍDICO DE SERVIDORES PÚBLICOS DA ADMINISTRAÇÃO PÚBLICA EM PROJETO DE EMENDA À CONSTITUIÇÃO ESTADUAL DE INICIATIVA PRIVATIVA DO PODER EXECUTIVO. INCONSTITUCIONALIDADE FORMAL RECONHECIDA. EC 41/2003. EFICÁCIA IMEDIATA DO TETO REMUNERATÓRIO. TEMAS 480 E 257 DA REPERCUSSÃO GERAL. INCONSTITUCIONALIDADE MATERIAL RECONHECIDA. PROCEDÊNCIA. 1. A jurisprudência desta CORTE assegura a possibilidade de os parlamentares apresentarem emendas a projetos de lei de iniciativa exclusiva de outro Poder, desde que delas não resulte “aumento de despesa pública, observada ainda a pertinência temática, a harmonia e a simetria à proposta inicial” (ADI 2.350, Rel. Min. MAURÍCIO CORRÊA, Tribunal Pleno, DJ de 30/4/2004). 2. Emenda parlamentar apresentada extrapolou o domínio temático da proposição original apresentada pelo Poder Executivo. A questão tratada na proposta original enviada à Assembleia local tinha como escopo adequar o teto remuneratório dos servidores públicos estaduais ao modelo estabelecido pela Constituição Federal, matéria essa que, conforme o art. 61, § 1º, II, a, da Constituição Federal, seria da iniciativa privativa da Chefe do Poder Executivo. 3. Possui eficácia imediata a redação do art. 37, XI, da Constituição Federal, inclusive para período anterior à promulgação da EC 41/2003. Entendimento firmado em sede de repercussão geral. Temas 480 e 257. 4. Medida Cautelar confirmada e Ação Direta de Inconstitucionalidade julgada procedente.

• As agências reguladoras independentes são autarquias de regime especial, caracterizadas por independência administrativa, ausência de subordinação hierárquica, mandato fixo, estabilidade de seus dirigentes e autonomia financeira.
• Esse regime especial foi concebido para lhes assegurar independência e isenção no desempenho de suas funções normativas, fiscalizatórias e sancionatórias. Justifica-se, desse modo, a previsão de normas funcionais mais rígidas tendo por finalidade a prevenção de potenciais conflitos de interesses que possam comprometer o interesse público subjacente às funções das agências. (...) “É constitucional norma legal que veda aos servidores titulares de cargo efetivo de agências reguladoras o exercício de outra atividade profissional, inclusive gestão operacional de empresa, ou de direção político-partidária”.

[ADI 6.033, rel. min. Roberto Barroso, j. 6-3-2023, P, DJE de 16-3-2023.]

• Para a desestatização de empresa estatal é suficiente a autorização prevista em lei que veicule programa de desestatização. (...) Autorização legislativa genérica é pautada em princípios e objetivos que devem ser observados nas diversas fases deliberativas do processo de desestatização. A atuação do Chefe do Poder Executivo vincula-se aos limites e condicionantes legais previstos.

[ADI 6.241, rel. min. Cármen Lúcia, j. 8-2-2021, P, DJE de 22-3-2021.]

• A lei específica autorizadora da criação das estatais é a ordinária, restringindo-se a exigência de lei complementar aos casos expressamente elencados na Constituição da República. No inc. XIX do art. 37 da Constituição, alterado pela Emenda Constitucional 19/1998, ao ser determinada a edição de lei complementar para a regulamentação das áreas de atuação, o poder constituinte derivado fez alusão tão somente às fundações.
• A interpretação gramatical deixa certo que a expressão ‘neste último caso’, no singular, refere-se ao antecedente ‘fundação’. A interpretação sistemática da Constituição também permite concluir não ser necessária a edição de lei complementar para a definição da atuação de empresas públicas ou sociedades de economia mista.

[ADI 4.895, voto da rel. min. Cármen Lúcia, j. 7-12-2020, P, DJE de 4-2-2021.]

• Resolução 790/2019 da ANP. (...) As Agências Reguladoras, criadas como autarquias especiais pelo Poder Legislativo (CF, art. 37, XIX), recebem da lei que as instituem uma delegação para exercer seu poder normativo de regulação, competindo ao Congresso Nacional a fixação das finalidades, dos objetivos básicos e da estrutura das Agências, bem como a fiscalização de suas atividades.
• As Agências Reguladoras não poderão, no exercício de seu poder normativo, inovar primariamente a ordem jurídica sem expressa delegação, tampouco regulamentar matéria para a qual inexista um prévio conceito genérico em sua lei instituidora (standards), ou criar ou aplicar sanções não previstas em lei, pois, assim como todos os Poderes, Instituições e órgãos do poder público, estão submetidas ao princípio da legalidade (CF, art. 37, caput).
• As normas técnicas veiculadas pela resolução impugnada inserem-se no espaço de conformação previsto pelo art. 8º, da Lei 9.478/1997, que atribui à ANP a implementação da política nacional de petróleo, gás natural e biocombustíveis com ênfase na proteção dos interesses dos consumidores quanto à qualidade dos produtos.
• A atribuição dos custos do monitoramento aos agentes regulados em questão revela tratamento isonômico quanto aos demais elos da cadeia de comercialização de combustíveis, sendo incapaz de violar os princípios da legalidade, da livre iniciativa, da liberdade de contratar e da proporcionalidade e razoabilidade.

[ADI 7.031, rel. min. Alexandre de Moraes, j. 8-8-2022, P, DJE de 16-8-2022.]

• Informativo nº 700
• 14 de junho de 2021.
• RECURSOS REPETITIVOS
• Processo: REsp 1.818.564-DF, Rel. Min. Moura Ribeiro, Segunda Seção, por unanimidade, julgado em 09/06/2021. (Tema 1025)

Ramo do Direito DIREITO CIVIL, DIREITO REGISTRAL, DIREITO URBANÍSTICO

Tema <br /> Imóvel particular desprovido de registro. Loteamento irregular. Usucapião. Possibilidade. Tema 1025.

DESTAQUE - É cabível a aquisição de imóveis particulares situados no Setor Tradicional de Planaltina/DF, por usucapião, ainda que pendente o processo de regularização urbanística.

INFORMAÇÕES DO INTEIRO TEOR - Tem-se, inicialmente, que a possibilidade de registro da sentença declaratória da usucapião não é pressuposto ao reconhecimento do direito material em testilha, o qual se funda, essencialmente, na posse ad usucapionem e no decurso do tempo.

• A propósito da questão da regularização fundiária, a doutrina esclarece que ela compreende três dimensões: (a) a dimensão urbanística, relacionada aos investimentos necessários para melhoria das condições de vida da população; (b) a dimensão jurídica, que diz respeito aos instrumentos que possibilitam a aquisição da propriedade nas áreas privadas e o reconhecimento da posse nas áreas públicas; e (c) a dimensão registrária, com o lançamento nas respectivas matrículas da aquisição destes direitos, a fim de atribuir eficácia para todos os efeitos da vida civil.

• Não há, portanto, como negar o direito à usucapião sob o pretexto de que o imóvel está inserido em loteamento irregular, porque o direito de propriedade declarado pela sentença (dimensão jurídica) não se confunde com a certificação e publicidade que emerge do registro (dimensão registrária) ou com a regularidade urbanística da ocupação levada a efeito (dimensão urbanística).

• O reconhecimento da usucapião não impede a implementação de políticas públicas de desenvolvimento urbano. Muito ao revés, constitui, em várias hipóteses, o primeiro passo para restabelecer a regularidade da urbanização.

• No mesmo sentido, o Pleno do STF, ao julgar o RE 422.349/RS, sob a relatoria do Ministro Dias Toffoli, fixou a tese de que preenchidos os requisitos do art. 183 da Constituição Federal, o reconhecimento do direito à usucapião especial urbana não pode ser obstado por legislação infraconstitucional que estabeleça módulos urbanos na respectiva área em que situado o imóvel (dimensão do lote).

• Admitindo-se que aquele não era o único imóvel da região com metragem inferior ao módulo mínimo legal, parece razoável sustentar que o STF, ao fim e ao cabo, reconheceu a possibilidade de usucapião de glebas inseridas em loteamentos não regularizados.

• Nesse contexto, é preciso ter em mente que Poder Público não faz favor nenhum quando promove a regularização de áreas ocupadas irregularmente. Muito pelo contrário, limita-se a desempenhar uma obrigação que lhe foi expressamente confiada pela CF. Admitindo-se que a regularização fundiária concorre para a segurança, saúde e bem estar da população e, bem assim, que esses são deveres essenciais do Estado, nada mais lógico do que concluir que a Administração Pública tem o dever de promover a regularização fundiária.

• Não parece acertado assumir como linha de princípio que que as ocupações irregulares do solo atentem, todas elas, contra o interesse público. Muito ao revés, o que atenta contra o interesse público é a inércia do Estado em promover e disciplinar a ocupação do solo.

• No caso, essa omissão estatal é mais do que flagrante. A ocupação da área está sedimentada há décadas e contou com a anuência implícita do Poder Público, que fingiu não ter visto nada, tolerou durante todos esses anos e ainda providenciou a instalação de vários serviços e equipamentos públicos, como pavimentação de ruas, iluminação pública, linhas de ônibus, praça pública, posto do DETRAN; etc. Não por outro motivo, a região é conhecida como Setor Tradicional de Planaltina, o que bem denota a idade do parcelamento do solo.

Atentar para o conceito jurídico indeterminado, isto é, o órgão deverá entender que a atividade econômica é capaz de causar significativo impacto ambiental.

Ademais, embora não haja significativa degradação ambiental em decorrência da atividade econômica, não estará o poluidor isento de outras medidas cautelares de avaliação do impacto ambiental.

Author Response

The following is the authors’ response to the original reviews.

We thank the reviewers for recognizing the importance of our work and for their insightful suggestions. A point-by-point response to their comments is listed underneath each reviewer’s section.

Reviewer #1 (Recommendations For The Authors):

Major comments

1) Have the authors optimized the expression level of dCas9? I cannot find this information in this paper or in their 2021 paper. It is important to avoid the toxicity phenomenon that occurs when using guide RNAs that share specific five base seed sequences (referred to as 'bad seeds').

Cui L., Vigouroux A., Rousset F., Varet H., Khanna V., Bikard D. A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat. Commun. 2018; 9:1912.

Rostain W., Grebert T., Vyhovskyi D., Thiel Pizarro P., Tshinsele-Van Bellingen G., Cui1 L., Bikard D. Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity. Nucleic Acids Research, 2023, gkad170.

2) One guide per gene is highly unusual given that different guides block the RNA polymerase with different efficiency. This was even shown by the Machner lab in the Legionella context in Figure 1c of Ellis et al. 2021 for sidM and vipD. Typically, genes need three guides minimum to ensure that the gene of interest is knocked down fully unless it is not possible as the gene is too small and/or it is difficult to find an NGG sequence. The authors have used one guide per effector, how can they be sure that each gene is knocked down? The Machner lab themselves in Figure 3c of Ellis et al. 2021 shows not all genes targeted using multiplex CRISPRi are equally efficiently knocked down. Please justify why only one guide per gene was chosen and add controls to validate the results. The authors themselves state that the strategy of one guide may be problematic. Lines 315-316 it reads... A possible explanation was the incomplete knockdown of a seemingly important process.

3) Given what the Machner lab observed about spacer location in Ellis et al. 2021 would it not make more sense to take one set of redundant effectors and make multiplex randomized CRISPRi with them in different locations? For Figure 1 at least.

4) Following infection, it seems that the bacteria were not plated onto antibiotic media, so it is not known how well the plasmid harboring guides is kept through infection.

Specific comments

A) The first results paragraph describes the set-up of 10-plex synthesized CRISPR arrays, where 10 effector encoding genes of specific gene families are selected. The rationale of the choice of these genes is not given. Please explain.

B) Please also add some biological data on what these genes code for, and what is their known or predicted function. It is not very informative and exciting to have tables of lpg numbers without any knowledge of what these genes code for and why they were selected, at least some.

C) Figure 1 A Why are only some of the MC arrays shown? Please, at least include in supplementary the others. Again one array in detail would be more informative, showing true knockdown of all genes by qPCR and ideally by western blot.

D) I am not convinced that the gene silencing efficiency qPCR comparison is done in the correct way. In my opinion, each of the genes to be knocked down should be tested against the expression of a control gene e.g. rpoS and then these results should be compared and not the results of empty plasmid or CRISPR array containing plasmid directly. L. pneumophila are very sensitive to growth conditions and inoculum, thus the two strains might not be completely at the same growth stage when being compared which can impact the results.

E) Figure 1 B As stated in general comment number 4, the authors do not appear to plate onto antibiotic so we don't know how well the plasmid harboring the guides is kept through infection. The sustained presence of the guide is particularly important for CRISPRi.

F) The authors found only a few growth phenotypes and mainly this was due to single genes and not combinations of genes. This might again be due to the fact that only one guide per gene was used. How do the authors know that all genes targeted were indeed knocked down?

G) Line 119 Alternatively, the genes were not 100% all knocked down, escaping the knockdown effect expected. Could authors take three genes with three guides each and look at impact instead of only one?

H) The authors then develop the randomized multiplexed arrays and chose to test 44 TME encoding genes. Line 141 Justify why these effectors were chosen in the text.

I) Unfortunately, the method is not clearly described, and many parts are complicated and the text needs to be re-read several times to be understood (lines 150 - 166). Please re-write to better explain to the reader. In line 156 the authors point to a supplementary note 1. This information should be in the main text.

J) What is the copy number of the CRISPR plasmid? Please add in the Material and Method section also the origin of this plasmid.

Figure 2

K) In the paper (line 154-160) and the extra notes, it states that authors attempt to size select CRISPR arrays. However, this is not apparent in Figure 2 schematic. Or are the authors stating that plasmids only containing one guide were selected out? However, line 312 would suggest not. Please clarify

L) A limiting factor in making multiplex guide CRISPR, as the authors are trying to establish in this study, is cloning of multiple guides. In the pre-determined CRISPR arrays in this study, the guides were synthesized. For the randomized multiplex CRISPR in this study, the authors adapt a Golden Gate cloning method to generate multiple sgRNAs in the Cas9 vector. A similar protocol was established in the below paper. Please add this reference.

Zuckermann, M.; Hlevnjak, M.; Yazdanparast, H.; Zapatka, M.; Jones, D.T.W.; Lichter, P.; Gronych, J. A novel cloning strategy for one-step assembly of multiplex CRISPR vectors. Sci. Rep. 2018

M) As the authors note, Zuckermann et al. similarly note that plex of 3 or 4 is most common and above 5 is rare. This thus appears to still be the limiting step of multiplex CRISPR technology. Please discuss

Figure 4

N) The idea of multiplexed CRISPRi seq to address the biological phenomenon of redundancy is an interesting one, however, I am missing the in-depth functional characterization and discussion of at least one of the redundant functions discovered. Please add.

Figure5/6

O) As noted above, the strength of the experiments is undermined by how CRISPRi is set up. Having an average multiplex of 2 or three and again only using one guide per gene weakens the study and the results obtained. Furthermore, as stated in general comment number 4, the authors do not appear to plate onto antibiotic so again, we don't know how well the plasmid harboring the guides is kept through infection. The sustained presence of the guide is particularly important for CRISPRi. Please add a validation that the guides are all present.

Response to Reviewer #1

We are grateful to the reviewers for their insightful comments and suggestions on how to further improve the manuscript.

Regarding the issue of ‘bad seed sequences’ (comment #1), we had previously evaluated the expression level of dcas9 (plotted in Figure 1b of the 2021 Communications Biol paper) and tuned our induction conditions accordingly (40 ng/mL as described in the Methods). Since all strains used in this study express dcas9 from the chromosome, not a plasmid, this eliminates the possibility of fluctuations in expression levels due to variabilities in plasmid copy numbers.

In the rare event that toxicity by any given guide occurs, we would expect that guide to already be underrepresented or missing in the input pool following 24+ hours of CRISPRi induction during axenic growth. Our data, now discussed in the manuscript (Lines 211-216 and Figure S2), revealed that this was not the case and that all guide-encoding spacers were present in roughly equal amounts (median of >5000 occurrences). As with any knockdown study, the creation of true chromosome deletions was performed throughout as to alleviate the issue of false positives.

Regarding comments #2, #3, and specific comments made under point F, G, and O, on the topic of using single vs. multiple guides, we agree that there are circumstances under which using more than one guide per target may be advantageous, for example when attempting to delete a gene from mammalian cells using conventional CRISPR engineering. In the study described here, this is not the case. In fact, we did create a second array library with alternative guides targeting the same group of genes at locations other than the “optimal location” identified in our 2021 paper and found that these “sub-optimal” guides were inefficient for identifying critical effectors as described in Supplemental Note S1 under the heading “Sub-optimal annealing sites” (Lines 919+). These data suggest that adding sub-optimal guides to the arrays of optimal guides might ‘poison’ the arrays and limit rather than enhance their ability to identify gene combinations.

Regarding comment #2, #3, and specific comments made under point C, F, and G, on the topic of confirming efficient gene knockdown for the identification of critical genes, we remind Reviewer 1 that we did confirm knockdown of 60 of the target genes of the 10-plex screen to be at least 2-fold, with an average fold repression of one order of magnitude or more (Figure 1A). While knockdown of every gene in every 10-plex construct would be an unprecedented ask of any published CRISPR screen, we believe that these 60 genes provide a large enough sampling of all guides to elucidate the range of knockdown to be expected by our CRISPRi platform. As with other knockdown technologies, such as RNAi, there is no expectation of accomplishing complete knockdown for any given target. Hence, the data in Figure 1A suggest that the lack of identifying critical genes using pre-determined 10-plex arrays was not due to a lack of knockdown efficiency, but rather the difficulty to accurately predict redundancy within a cohort of uncharacterized genes, accentuating the need for array randomization with MuRCiS.

On the topic of antibiotic use for plasmid selection (comments #4, E and O), we would like to clarify that the CRISPR plasmids were selected by thymidine prototrophy, not antibiotic resistance, and we apologize for not making this clearer. The laboratory strain Lp02 is a thymidine auxotroph (thyA-) L. pneumophila variant, and plasmid retention is routinely achieved by including the thymidine biosynthesis gene (thyA) on the plasmid backbone. Only with a plasmid bearing the thyA gene can L. pneumophila grow on CYE (thymidine-) plates. Our use of vectors bearing thyA and plating on CYE plates is described in the Methods section. Further, in Figure 7 of our 2021 paper, we show that CRISPR plasmids are efficiently retained by Lp02 for the duration of a 48-hour infection, resulting in efficient multi-gene knockdown even at the end of the intracellular growth experiment.

Regarding comments A and B, on publishing the biological data used to classify genes in gene families for 10-plex silencing, we do not consider it critical to provide additional information beyond the broad classification (e.g. kinases, phosphatases, etc) described in Table S1. Structural predictions constantly change due to continuously evolving databases. Our initial analyses were made in 2015 using HHPRED Hidden-Markov models and, in all likelihood, those predictions have been refined since then. Moreover, with the recent advent of Alphafold, anyone interested in learning more about select effectors from our list is advised to simply access the most recent functional predictions directly on the Alphafold webpage (https://alphafold.ebi.ac.uk/). We clarify how predictions were made on Lines 97-101.

Regarding specific comment D, on our method for qPCR normalization and comparison, we point Reviewer 1 to the Methods section (Lines 460+) where we describe that data obtained from each CRISPRi strain were in fact normalized to the levels of rpsL prior to comparing them to the normalized data from the strain with the empty control plasmid. This normalization to rpsL, a gene encoding a ribosomal protein, also corrects for growth differences between samples.

Regarding specific comment H, the justification for studying 44 transmembrane effector-encoding genes was driven by the fact that activities mediated by transmembrane proteins are difficult (though not impossible) to be replaced by cytosolic proteins, for example the transport of metabolites across the LCV membrane. And since transmembrane regions can be predicted with high confidence, we decided to probe this group of TMEs for synthetic lethality with the randomized CRISPRi approach as proof-of-concept. To make this clearer, we have added more detail to the text (Lines 151-155).

Regarding specific comment I, we have further simplified the description of the cloning technique to increase clarity (Lines 156+). The information listed under Supplemental Note S1, though informative, is not critical for the overall understanding of this highly technical section, and since the reviewer already considered this section to be difficult to follow, we would prefer to not further complicate the text by including these non-essential details.

Regarding the origin of the CRISPRi plasmid (specific comment J), we point Reviewer 1 to the reference (Hammer BK and Swanson MS (Mol Microbiol 1999)) listed in Table S10: Strains and Plasmids Used in this Study.

Regarding specific comment K and O, on the clarity of depicting the CRISPR array size selection process, we have updated the Figure 2 schematic. Reviewer 1 is correct in that despite our best efforts to exclude short CRISPR arrays, inevitably some 1-plex arrays remained in our input vector pool. Still, the average length of all arrays used in our pilot study exceeded three crRNA-encoding spacers. Further, having a population of 1- or 2-plex arrays in our libraries did allow us to pin-point the most critical effectors of a larger arrays within the same MuRCiS experiment (Table S5 and Table S7), a strength of MuRCiS as described in the discussion (Lines 378+).

Regarding specific comment L, we appreciate Reviewer 1’s suggestion of an additional reference and we have added it to the manuscript as reference #23 (Line 71). While this reference does use a Golden Gate strategy to build a multiplex array, that array was not randomized but had a predefined order. Hence, our assembly method is unique due to its randomization.

Regarding specific comment M, on array length cloning limitations, we agree with the conclusion of Zuckermann in Figure 1d of their article that longer inserts are generally harder to get into vector backbones. The challenge of cloning longer inserts is a common phenomenon of general biology and is not unique to cloning CRISPR arrays. We have altered the wording in our manuscript to better describe the intrinsic competition between short and long inserts during cloning (Lines 162-164).

Regarding specific comment N, we second Reviewer 1’s desire to learn more about the critical effector pairs discovered here. With that said, the goal of this manuscript is to report the development of a novel MuRCiS pipeline to identify these critical pairs. Biochemical and molecular investigations of the encoded protein pairs are on-going and will be the topic of a future manuscript.

Reviewer #2 (Recommendations For The Authors):

Specific points

1) The effector repertoire of L. pneumophila seems to have evolved in response to the plethora of potential protozoan hosts (PMID: 31988381). To further assess evolutionary aspects of the vast L. pneumophila effector arsenal, it would be interesting to test the single and double effector mutant strains (Fig. 5FG, Fig. 6EF) for growth in protozoa other than A. castellanii.

2) Most CRISPR arrays comprising genes encoding functionally similar proteins or encoding evolutionarily conserved proteins did not substantially affect intracellular growth of L. pneumophila (Fig. 1B). This rather surprising result should be further discussed.

3) l. 118/119: "Similar results ..., where none of the MC arrays ..." This statement should be phrased more precisely, since some CRISPR arrays did indeed have an effect on intracellular growth of L. pneumophila in U937 macrophages, while none affected intracellular growth in A. castellanii (Fig. 1B).

4) Typos:

• l. 852: ... (arbitrarily set to -100).

• l. 862: ... Legionella-containing vacuole (LCV).

• l. 895: ... and so we would recommend ...

Regarding point 1, we thank Reviewer 2 for the suggestion of testing effector mutants in different hosts. While the primary purpose of the current manuscript was to optimize the MuRCiS platform, future studies using this technology to investigate specific biological questions related to Legionella infection would certainly benefit from including more than one amoebaean species.

Regarding point 2, we agree that the lack of substantial growth defects seems surprising. Yet only two of the seven core effectors (found in all Legionella sp.), lpg2300 and mavN, individually attenuated Legionella intracellular growth when deleted (Burstein 2016 Nat Genetics; Isaac et al., 2015 PNAS). Thus, we hypothesize that the functions many effectors fulfil are of such importance for intracellular survival that that redundancy reaches beyond the boundary of conservation or like-function. We have added a statement emphasizing this at the end of the Figure 1 results section (Line 122-125).

Regarding points 3 and 4, we thank Reviewer 2 for catching these errors and have corrected where needed in the text.

-l. 852 (now Line 874): … (arbitrarily set to -100,000) is correct for Figure 6E.

from - https://hypothes.is/a/axloiHSPEe62H09PAJfBPw

RECURSO ESPECIAL - AÇÃO DE CANCELAMENTO DE GRAVAMES - PROCEDIMENTO ESPECIAL DE JURISDIÇÃO VOLUNTÁRIA - IMPENHORABILIDADE E INCOMUNICABILIDADE - DOAÇÃO - MORTE DO DOADOR - RESTRIÇÃO DO DIREITO DE PROPRIEDADE - INTERPRETAÇÃO DO CAPUT DO ARTIGO 1.911 DO CÓDIGO CIVIL DE 2002 . INSURGÊNCIA DA AUTORA. Quaestio Iuris: Cinge-se a controvérsia em definir a interpretação jurídica a ser dada ao caput do art. 1.911 do Código Civil de 2002 diante da nítida limitação ao pleno direito de propriedade, para definir se a aposição da cláusula de impenhorabilidade e/ou incomunicabilidade em ato de liberalidade importa automaticamente, ou não, na cláusula de inalienabilidade. 1. A exegese do caput do art. 1.911 do Código Civil de 2002 conduz ao entendimento de que: a) há possibilidade de imposição autônoma das cláusulas de inalienabilidade, impenhorabilidade e incomunicabilidade, a critério do doador/instituidor; b) uma vez aposto o gravame da inalienabilidade, pressupõe-se, ex vi lege, automaticamente, a impenhorabilidade e a incomunicabilidade; c) a inserção exclusiva da proibição de não penhorar e/ou não comunicar não gera a presunção do ônus da inalienabilidade; e d) a instituição autônoma da impenhorabilidade, por si só, não pressupõe a incomunicabilidade e vice-versa. 2. Caso concreto: deve ser acolhida a pretensão recursal veiculada no apelo extremo para, julgando procedente o pedido inicial, autorizar o cancelamento dos gravames, considerando que não há que se falar em inalienabilidade do imóvel gravado exclusivamente com as cláusulas de impenhorabilidade e incomunicabilidade. 3. Recurso especial provido. (REsp n. 1.155.547/MG, relator Ministro Marco Buzzi, Quarta Turma, julgado em 6/11/2018, DJe de 9/11/2018.)

CIVIL E PROCESSUAL CIVIL. EMBARGOS DE DIVERGÊNCIA EM AGRAVO EM RECURSO ESPECIAL. TELEFONIA FIXA. COBRANÇA INDEVIDA. AÇÃO DE REPETIÇÃO DE INDÉBITO DE TARIFAS. APLICAÇÃO DO PRAZO PRESCRICIONAL DECENAL DO CÓDIGO CIVIL (ART. 205 DO CÓDIGO CIVIL). CONHECIMENTO E PROVIMENTO. 1. Trata-se de embargos de divergência interpostos contra acórdão em que se discute o lapso prescricional cabível aos casos de repetição de indébito por cobrança indevida de valores referentes a serviços não contratados, promovida por empresa de telefonia. 2. A Primeira Seção, no julgamento do REsp 1.113.403/RJ, de relatoria do Min. Teori Albino Zavascki (DJe 15/9/2009), submetido ao regime dos recursos repetitivos do art. 543-C do Código de Processo Civil e da Resolução STJ 8/2008, firmou o entendimento de que, ante a ausência de disposição específica acerca do prazo prescricional aplicável à prática comercial indevida de cobrança excessiva, é de rigor a incidência das normas gerais relativas à prescrição insculpidas no Código Civil na ação de repetição de indébito de tarifas de água e esgoto. Assim, tem-se prazo vintenário, na forma estabelecida no art. 177 do Código Civil de 1916, ou decenal, de acordo com o previsto no art. 205 do Código Civil de 2002. Diante da mesma conjuntura, não há razões para adotar solução diversa nos casos de repetição de indébito dos serviços de telefonia. 3. A tese adotada no âmbito do acórdão recorrido, de que a pretensão de repetição de indébito por cobrança indevida de valores referentes a serviços não contratados, promovida por empresa de telefonia, configuraria enriquecimento sem causa e, portanto, estaria abrangida pelo prazo fixado no art. 206, § 3º, IV, do Código Civil, não parece ser a melhor. A pretensão de enriquecimento sem causa (ação in rem verso) possui como requisitos: enriquecimento de alguém; empobrecimento correspondente de outrem; relação de causalidade entre ambos; ausência de causa jurídica; inexistência de ação específica. Trata-se, portanto, de ação subsidiária que depende da inexistência de causa jurídica. A discussão acerca da cobrança indevida de valores constantes de relação contratual e eventual repetição de indébito não se enquadra na hipótese do art. 206, § 3º, IV, do Código Civil, seja porque a causa jurídica, em princípio, existe (relação contratual prévia em que se debate a legitimidade da cobrança), seja porque a ação de repetição de indébito é ação específica. Doutrina. 4. Embargos de divergência conhecidos e providos, de sorte a vingar a tese de que a repetição de indébito por cobrança indevida de valores referentes a serviços não contratados, promovida por empresa de telefonia, deve seguir a norma geral do lapso prescricional (10 anos - art. 205 do Código Civil), a exemplo do que decidido e sumulado (Súmula 412/STJ) no que diz respeito ao lapso prescricional para repetição de indébito de tarifas de água e esgoto. (EAREsp n. 750.497/RS, relator Ministro Og Fernandes, Corte Especial, julgado em 20/2/2019, DJe de 11/6/2019.)

Desapropriação judicial pelo particular

• Informativo nº 792
• 24 de outubro de 2023.
• TERCEIRA TURMA
• Processo: REsp 2.088.100-SP, Rel. Ministra Nancy Andrighi, Terceira Turma, por unanimidade, julgado em 17/10/2023.

Ramo do Direito DIREITO CIVIL

Paz, Justiça e Instituições EficazesTema <br /> Débito prescrito. Cobrança judicial e extrajudicial. Instituto de direito material. Plano da eficácia. Princípio da indiferença das vias. Prescrição que não atinge o direito subjetivo. Cobrança de dívida prescrita. Impossibilidade.

DESTAQUE - O reconhecimento da prescrição da pretensão impede tanto a cobrança judicial quanto a cobrança extrajudicial do débito.

INFORMAÇÕES DO INTEIRO TEOR - Para o deslinde da controvérsia, é necessário que se examine a atuação da prescrição no plano da eficácia, o que perpassa, inicialmente, pela distinção entre os conceitos de direito subjetivo e de pretensão, pois, somente esta é, propriamente, atingida pela prescrição.

• Segundo a doutrina, a pretensão é o poder de exigir um comportamento positivo ou negativo da outra parte da relação jurídica. Observa-se, desse modo, que, antes do advento da pretensão, já existe direito e dever, mas em situação estática. Isso porque a dinamicidade do direito subjetivo surge, tão somente, com o nascimento da pretensão, que pode ser ou não concomitante ao surgimento do próprio direito subjetivo. Somente a partir desse momento, o titular do direito poderá exigir do devedor que cumpra aquilo a que está obrigado.

• No que diz respeito ao seu modo de atuação, restou demonstrado que a prescrição não atingiria a ação, mas sim a pretensão, o que representou fundamental virada dogmática com reflexos não só na nomenclatura, mas, sobretudo, na essência do instituto. Na doutrina brasileira, era relativamente comum, antes do advento do Código Civil de 2002, e em alguns casos, até mesmo, depois de sua entrada em vigor -, se apontar como alvo da eficácia da prescrição a própria ação.

• No entanto, o art. 189 do Código Civil de 2002, que representou importante inovação legislativa em face do direito anterior, acolheu a novel construção doutrinária ao estabelecer, expressamente, que o alvo da prescrição é mesmo a pretensão, instituto de direito material. Dessa forma, a doutrina defende que "eventuais projeções ao direito de ação (em sentido processual) só se justificam de modo reflexo." Isso porque, sendo a pretensão e a ação em sentido material encobertas pela prescrição, o seu titular não pode se servir dos remédios processuais da ação em sentido processual.

• A doutrina adverte que "a consequência processual de não poder se servir da 'ação', no entanto, não tem o condão de explicar o instituto. Trata-se de um resultado decorrente de uma prévia eficácia que se sucedeu no direito material". Nessa esteira de intelecção, não se pode olvidar, ainda, que a "pretensão se submete ao princípio da indiferença das vias, isto é, pode ser exercida tanto judicial, quanto extrajudicialmente".

• Nesse sentido, ao cobrar extrajudicialmente o devedor, o credor está, efetivamente, exercendo sua pretensão, ainda que fora do processo. Se a pretensão é o poder de exigir o cumprimento da prestação, uma vez paralisada em razão da prescrição, não será mais possível exigir o referido comportamento do devedor, ou seja, não será mais possível cobrar a dívida. Logo, o reconhecimento da prescrição da pretensão impede tanto a cobrança judicial quanto a cobrança extrajudicial do débito.

• Não há, portanto, duas pretensões, uma veiculada por meio do processo e outra veiculada extrajudicialmente. Independentemente do instrumento utilizado, trata-se da mesma pretensão, haurida do direito material. É a pretensão e não o direito subjetivo que permite a exigência da dívida. Uma vez prescrita, resta impossibilitada a cobrança da prestação. Nessas situações, não há que se falar em pagamento indevido, nem sequer em enriquecimento sem causa, nos termos do art. 882 do Código Civil, uma vez que o direito subjetivo (crédito) continua a existir. O que não há, de fato, é a possibilidade de exigí-lo.

• ADI 4815
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Julgamento: 10/06/2015
• Publicação: 01/02/2016

ODS 16 - Paz, Justiça e Instituições Eficazes Ementa EMENTA: AÇÃO DIRETA DE INCONSTITUCIONALIDADE. ARTS. 20 E 21 DA LEI N. 10.406/2002 (CÓDIGO CIVIL). PRELIMINAR DE ILEGITIMIDADE ATIVA REJEITADA. REQUISITOS LEGAIS OBSERVADOS. MÉRITO: APARENTE CONFLITO ENTRE PRINCÍPIOS CONSTITUCIONAIS: LIBERDADE DE EXPRESSÃO, DE INFORMAÇÃO, ARTÍSTICA E CULTURAL, INDEPENDENTE DE CENSURA OU AUTORIZAÇÃO PRÉVIA (ART. 5º INCS. IV, IX, XIV; 220, §§ 1º E 2º) E INVIOLABILIDADE DA INTIMIDADE, VIDA PRIVADA, HONRA E IMAGEM DAS PESSOAS (ART. 5º, INC. X). ADOÇÃO DE CRITÉRIO DA PONDERAÇÃO PARA INTERPRETAÇÃO DE PRINCÍPIO CONSTITUCIONAL. PROIBIÇÃO DE CENSURA (ESTATAL OU PARTICULAR). GARANTIA CONSTITUCIONAL DE INDENIZAÇÃO E DE DIREITO DE RESPOSTA. AÇÃO DIRETA JULGADA PROCEDENTE PARA DAR INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO AOS ARTS. 20 E 21 DO CÓDIGO CIVIL, SEM REDUÇÃO DE TEXTO. 1. A Associação Nacional dos Editores de Livros - Anel congrega a classe dos editores, considerados, para fins estatutários, a pessoa natural ou jurídica à qual se atribui o direito de reprodução de obra literária, artística ou científica, podendo publicá-la e divulgá-la. A correlação entre o conteúdo da norma impugnada e os objetivos da Autora preenche o requisito de pertinência temática e a presença de seus associados em nove Estados da Federação comprova sua representação nacional, nos termos da jurisprudência deste Supremo Tribunal. Preliminar de ilegitimidade ativa rejeitada. 2. O objeto da presente ação restringe-se à interpretação dos arts. 20 e 21 do Código Civil relativas à divulgação de escritos, à transmissão da palavra, à produção, publicação, exposição ou utilização da imagem de pessoa biografada.

1. A Constituição do Brasil proíbe qualquer censura. O exercício do direito à liberdade de expressão não pode ser cerceada pelo Estado ou por particular.
2. O direito de informação, constitucionalmente garantido, contém a liberdade de informar, de se informar e de ser informado. O primeiro refere-se à formação da opinião pública, considerado cada qual dos cidadãos que pode receber livremente dados sobre assuntos de interesse da coletividade e sobre as pessoas cujas ações, público-estatais ou público-sociais, interferem em sua esfera do acervo do direito de saber, de aprender sobre temas relacionados a suas legítimas cogitações.
3. Biografia é história. A vida não se desenvolve apenas a partir da soleira da porta de casa.
4. Autorização prévia para biografia constitui censura prévia particular. O recolhimento de obras é censura judicial, a substituir a administrativa. O risco é próprio do viver. Erros corrigem-se segundo o direito, não se coartando liberdades conquistadas. A reparação de danos e o direito de resposta devem ser exercidos nos termos da lei.
5. A liberdade é constitucionalmente garantida, não se podendo anular por outra norma constitucional (inc. IV do art. 60), menos ainda por norma de hierarquia inferior (lei civil), ainda que sob o argumento de se estar a resguardar e proteger outro direito constitucionalmente assegurado, qual seja, o da inviolabilidade do direito à intimidade, à privacidade, à honra e à imagem.
6. Para a coexistência das normas constitucionais dos incs. IV, IX e X do art. 5º, há de se acolher o balanceamento de direitos, conjugando-se o direito às liberdades com a inviolabilidade da intimidade, da privacidade, da honra e da imagem da pessoa biografada e daqueles que pretendem elaborar as biografias.
7. Ação direta julgada procedente para dar interpretação conforme à Constituição aos arts. 20 e 21 do Código Civil, sem redução de texto, para, em consonância com os direitos fundamentais à liberdade de pensamento e de sua expressão, de criação artística, produção científica, declarar inexigível autorização de pessoa biografada relativamente a obras biográficas literárias ou audiovisuais, sendo também desnecessária autorização de pessoas retratadas como coadjuvantes (ou de seus familiares, em caso de pessoas falecidas ou ausentes).

• Informativo nº 734
• 2 de maio de 2022.
• TERCEIRA TURMA
• Processo: Processo sob segredo de justiça, Rel. Min. Marco Aurélio Bellizze, Terceira Turma, por unanimidade, julgado em 26/04/2022, DJe 29/04/2022.

Ramo do Direito DIREITO CIVIL

Paz, Justiça e Instituições EficazesTema <br /> Responsabilidade civil. Eficácia transubjetiva das obrigações. Envio de carta desabonadora a patrocinadora de jogador de futebol. Teoria do terceiro cúmplice. Danos morais. Ocorrência.

DESTAQUE - Terceiro ofensor também está sujeito à eficácia transubjetiva das obrigações, haja vista que seu comportamento não pode interferir indevidamente na relação, perturbando o normal desempenho da prestação pelas partes, sob pena de se responsabilizar pelos danos decorrentes de sua conduta.

INFORMAÇÕES DO INTEIRO TEOR - A responsabilidade civil, em face da sua relevância e da sua natureza dinâmica, tem alargado seu horizonte, sem se restringir a um rol preestabelecido de direitos tutelados, buscando a proteção das mais variadas órbitas da dignidade da pessoa humana.

• A própria evolução da sociedade e o surgimento de relações jurídicas cada vez mais complexas exigiram a expansão da responsabilidade civil, notadamente para que esta cumpra sua função precípua (a de possibilitar o equilíbrio e a harmonia social), não se esgotando nos atributos tradicionais da personalidade humana - honra, nome, imagem, intimidade e vida privada.

• Dessa forma, diante do reconhecimento e da ampliação de novas áreas de proteção à pessoa humana, resultantes da nova realidade social e da ascensão de novos interesses, surgem também novas hipóteses de violações de direitos, o que impõe sua salvaguarda pelo ordenamento jurídico.

• Diante dessas considerações, viu-se a necessidade de analisar o comportamento daquele terceiro que interfere ou induz o inadimplemento de um contrato sob o prisma de uma proteção extracontratual, do capitalismo ético, da função social do contrato e da proteção das estruturas de interesse da sociedade, tais como a honestidade e a tutela da confiança.

• Assim, a responsabilização de um terceiro, alheio à relação contratual, decorre da sua não funcionalização sob a perspectiva social da autonomia contratual, incorporando como razão prática a confiança e o desenvolvimento social na conduta daqueles que exercem sua liberdade.

• Atualmente, difunde-se a ideia de que os contratos são protegidos por deveres de confiança, os quais se estendem a terceiros em razão da cláusula de boa-fé objetiva, pois, da mesma forma que um terceiro está protegido de contratos que possam vir a lhe prejudicar, os contratantes também estão protegidos da conduta de terceiro que possa prejudicar o vínculo.

• Portanto, um terceiro ofendido e que estava exposto aos riscos de danos pessoais e patrimoniais em decorrência da execução do contrato teria direito a uma indenização não por ter violado algum dever de prestação oriunda da relação obrigacional, mas por ter suportado uma ofensa à sua integridade psicofísica ou econômica.

• Em contrapartida, o terceiro ofensor também está sujeito à eficácia transubjetiva das obrigações, pois, segunda a doutrina, "o comportamento do terceiro não pode manifestamente interferir, perturbando o normal desempenho da prestação pelas partes. Nesse último sentido, o terceiro não pode se associar a uma das partes para descumprir com a obrigação. Nesse caso, seria um terceiro-cúmplice no inadimplemento daquela prestação".

• Uma das hipóteses em que a conduta condenável do terceiro pode gerar sua responsabilização é a indução interferente ilícita, na qual o terceiro imiscui-se na relação contratual mediante informações ou conselhos com o intuito de estimular uma das partes a não cumprir seus deveres contratuais.

• Destaca-se que a simples emissão de opinião não configura ato ilícito, pois a todos é lícito exprimir sua convicção sobre eventuais riscos ou desvios, o que, contudo, não pode ser exercido de forma maliciosa, exagerada ou proferida em contrariedade à boa-fé objetiva.

• De outro lado, nota-se que esse entendimento tem sido aplicado para se reconhecer a responsabilidade do terceiro em relação a danos materiais causados aos contratantes em razão de seu comportamento, mas esse raciocínio é plenamente aplicável aos casos em que o proceder do terceiro configure danos extrapatrimoniais.

• Apesar de não haver consenso quanto à definição de dano moral, tem prevalecido o entendimento de que ele não está atrelado à dor, mágoa ou sofrimento da vítima, pois as consternações e as dores de cada um são sensações subjetivas e pessoais, e cada ser humano percebe os flagelos da vida de uma forma particular.

• Sendo assim, foi editado o Enunciado n. 444 do Conselho da Justiça Federal, emitido na V Jornada de Direito Civil, com a seguinte redação: "O dano moral indenizável não pressupõe necessariamente a verificação de sentimentos humanos desagradáveis como dor ou sofrimento."

• Modernamente se tem aproximado o dano moral do princípio da dignidade da pessoa humana, mas sem se limitar a ele, sob pena de verificar um conceito abstrato e desprovido de segurança jurídica. Assim, será indenizável aquela violação a um interesse existencial concretamente merecedor de tutela, vislumbrando-se o ser humano diante de toda a coletividade e por meio de suas relações interpessoais.

• Em face dessas premissas, constata-se que o caso concreto configura uma hipótese em que o terceiro ofensor causou lesão a um interesse existencial do atleta.

• Conforme o quadro fático delineado, ao enviar correspondência à patrocinadora do jogador de futebol, fez-se expressa menção a uma denúncia criminal oferecida perante a Justiça da Espanha, mas sem se limitar à mera reprodução dos fatos narrados na acusação criminal, tendo emitido juízo de valor sobre as circunstâncias e adjetivado a conduta do atleta como mentirosa, fraudulenta e desonesta.

• A conduta não pode ser caracterizada como exercício de sua liberdade de expressão, porquanto este direito constitucional compreende a autonomia de receber e transmitir informações ou ideias sem uma validação ou censura prévias por terceiros, mas encontra limites nos demais direitos fundamentais individuais.

• O Superior Tribunal de Justiça, à procura de solução que melhor concilie as situações de conflito entre a liberdade de expressão e os direitos da personalidade, estabeleceu, entre outros, os seguintes elementos de ponderação: a) o compromisso ético com a informação verossímil; b) a preservação dos chamados direitos da personalidade, entre os quais incluem-se os direitos à honra, à imagem, à privacidade e à intimidade; e c) a vedação de veiculação de informações com intuito de difamar, injuriar ou caluniar a pessoa (animus injuriandi vel diffamandi) - (cf. REsp n. 801.109/DF, Rel. Min. Raul Araújo, Quarta Turma, julgado em 12/6/2012, DJe 12/3/2013).

• Por conseguinte, não configura ato ilícito a notícia de fatos verossímeis mediante opiniões severas ou irônicas, sobretudo quando se tratar de figuras públicas, o que, todavia, não justifica o ataque pessoal à vítima.

• Não se descura, também, de que o vínculo contratual entre atleta e patrocinadora não se rompeu após a emissão da carta, o que, contudo, não afasta a pretensão indenizatória do autor, pois, caso o contrato viesse a ser rescindido em decorrência da conduta da recorrente, a indenização abrangeria não apenas aquela decorrente dos dano morais, mas também a relativa aos danos patrimoniais por ele suportados.

• Tanto é que o pleito autoral se limita à indenização por danos morais em razão da atuação difamatória de um terceiro com o objetivo de estimular uma das partes a não cumprir seus deveres contratuais, imiscuindo-se na relação contratual extrapolando os limites da licitude.

• Do mesmo modo, é desinfluente o teor da carta remetida pela patrocinadora ao atleta em virtude do recebimento da primeira missiva, pois, repita-se, o que interessa é a ofensa à personalidade e à honra do jogador perpetrada na primeira, mediante emissão de juízo de valor negativo sobre a sua conduta, ainda que não tenha havido rescisão contratual ou algum outro prejuízo material ao ofendido.

• Por fim, importante relembrar que o art. 187 do CC reconhece como ilícito, e consequentemente gerador do dever de indenizar, o exercício abusivo de um direito, isto é, mesmo que se considerasse que a conduta foi um ato de liberdade de expressão, foi exercido o direito de forma abusiva, interferindo indevidamente em uma relação jurídica da qual não fazia parte.

• Assim, não obstante o alto grau de subjetivismo que envolve a matéria, a indenização deve ser um desestímulo a futuras condutas ilícitas, sem, contudo, gerar o enriquecimento sem causa da vítima e, de outro lado, a ruína econômica do ofensor, devendo ser pautada nos princípios da proporcionalidade e da razoabilidade.

• Informativo nº 736
• 16 de maio de 2022.
• TERCEIRA TURMA
• Processo: REsp 1.836.016-PR, Rel. Min. Ricardo Villas Bôas Cueva, Rel. Acd. Min. Nancy Andrighi, Terceira Turma, por maioria, julgado em 10/05/2022.

Ramo do Direito DIREITO CIVIL

Paz, Justiça e Instituições EficazesTema <br /> Responsabilidade civil. Indenização por danos materiais e morais. Prescrição. Termo inicial. Ciência inequívoca dos efeitos do ato lesivo. Teoria da actio nata. Viés subjetivo. Critérios.

DESTAQUE - São critérios que indicam a tendência de adoção excepcional do viés subjetivo da teoria da actio nata: a) a submissão da pretensão a prazo prescricional curto; b) a constatação, na hipótese concreta, de que o credor tinha ou deveria ter ciência do nascimento da pretensão, o que deve ser apurado a partir da boa-fé objetiva e de standards de atuação do homem médio; c) o fato de se estar diante de responsabilidade civil por ato ilícito absoluto; e d) a expressa previsão legal a impor a aplicação do sistema subjetivo.

INFORMAÇÕES DO INTEIRO TEOR

• Cinge-se a controvérsia sobre o dies a quo do prazo prescricional da pretensão compensatória dos danos morais.

• Trata-se de indagação da mais alta relevância, pois, como menciona a doutrina, "o início do prazo da prescrição é um fator estruturante do próprio instituto: dele depende, depois, todo o desenvolvimento subsequente".

• A determinação do termo inicial dos prazos prescricionais demanda, inicialmente, a distinção entre os conceitos de direito subjetivo e de pretensão.

• Nesse contexto, importa consignar que a pretensão é o poder de exigir um comportamento positivo ou negativo da outra parte da relação jurídica. Trata-se, a rigor, do chamado grau de exigibilidade do direito, nascendo, portanto, tão logo este se torne exigível.

• Desse modo, pode-se observar que, antes do advento da pretensão, já existe direito e dever, mas em situação estática. Especificamente no âmbito das relações jurídicas obrigacionais, por exemplo, antes mesmo do nascimento da pretensão, já há crédito (direito) e débito (dever) e, portanto, credor e devedor.

• A dinamicidade surge, tão somente, com o nascimento da pretensão, que pode ser ou não concomitante ao surgimento do próprio direito subjetivo. Somente a partir desse momento, o titular do direito poderá exigir do devedor que cumpra aquilo a que está obrigado.

• Nota-se que "a pretensão seria algo a mais do que o direito subjetivo, que é categoria eficacial de cunho estático. Quem tem em mãos um direito subjetivo é titular de uma situação jurídica ativa que é estática por estar destituída, ainda que em princípio, de um poder de exigibilidade, de uma possibilidade de atuação sobre a esfera jurídica alheia para se exigir um cumprimento".

• Exemplificativamente, pode-se mencionar os direitos sob condição suspensiva ou sob termo, que se encontram desprovidos de pretensão até o implemento dessa mesma condição ou o advento do referido termo.

• Assim, visando o encobrimento da eficácia da pretensão, a prescrição, como consequência lógica, possui como termo inicial do transcurso de seu prazo o nascimento dessa posição jurídica (a pretensão).

• Em síntese, "o prazo começa a correr assim que o direito possa ser exercido e independentemente do conhecimento que, disso, tenha o possa ter o respectivo credor".

• A propósito, a doutrina bem assevera que a ideia-chave é mesmo a de exigibilidade, acentuando que não se pode falar em "inércia" quando o direito subjetivo ainda não pode se fazer valer, de modo que o prazo prescricional só tem início no dia em que o direito poderia ter sido exercitado: "É óbvio que se a vítima não pode exigir (por razões de direito ou de fato), não há inércia punível com o encobrimento da pretensão, pela prescrição. E, sem essa ausência de uma atividade que poderia ter sido levada a efeito, mas não o foi, não há prescrição, como reiteradamente afirma a jurisprudência e já observava, ainda na vigência do Código de 1916, Miguel Reale".

• Daí a tão propalada teoria da actio nata - haurida dos trabalhos de Friedrich Carl Freiherr von Savigny - segundo a qual os prazos prescricionais se iniciariam no exato momento do surgimento da pretensão. Trata-se de reminiscência do brocardo romano "actioni nondum natae non praescribitur".

• De fato, somente a partir do instante em que o titular do direito pode exigir a sua satisfação é que se revela lógico imputar-lhe eventual inércia em ver satisfeito o seu interesse.

• Nesse contexto, eventuais injustiças produzidas pela adoção da vertente objetiva são mitigadas ou temperadas pelas regras atinentes à suspensão, à interrupção e ao impedimento dos prazos prescricionais.

• Do ponto de vista do direito positivo, a concepção perfilhada pelo atual Código Civil, ao dispor, no art. 189 foi que "violado o direito, nasce para o titular a pretensão, a qual se extingue, pela prescrição".

• No âmbito jurisprudencial, o STJ passou a admitir que, em determinadas hipóteses, o início dos prazos prescricionais deveria ocorrer a partir da ciência do nascimento da pretensão por seu titular, no que ficou conhecido como o viés subjetivo da teoria da actio nata.

• Com efeito, pelo sistema subjetivo, o início do prazo prescricional "só se dá quando o credor tenha conhecimento dos elementos essenciais relativos ao seu direito".

• Conforme já consignado pela Terceira Turma do STJ, no entanto, "a aplicação da teoria da actio nata em sua vertente subjetiva é excepcional" (REsp 1.736.091/PE, Terceira Turma, julgado em 14/05/2019, DJe 16/05/2019).

• É a hipótese, por exemplo, da prescrição relativa à indenização em virtude de incapacidade permanente, em que a jurisprudência do STJ fixou-se no sentido de que o prazo prescricional começa a fluir apenas a partir do momento em que a vítima toma ciência inequívoca de sua invalidez e da extensão da incapacidade de que restou acometida. A propósito: REsp 673.576/RJ, Primeira Turma, julgado em 02/12/2004, DJ 21/03/2005.

• Nesse sentido, foi editada a Súmula 278/STJ, segundo a qual o termo inicial do prazo prescricional, na ação de indenização, é a data em que o segurado teve ciência inequívoca da incapacidade laboral.

• Nesse contexto, do exame das vertentes objetiva e subjetiva, infere-se que a primeira prestigia o valor segurança, ao passo que a segunda, o valor justiça, ambos igualmente caros ao Direito, motivo pelo qual é imperioso delinear critérios para se determinar em quais hipóteses a regra excepcional (viés subjetivo da teoria da actio nata) merece ser aplicada.

• A adoção do sistema subjetivo pode revelar-se adequada na medida em que o estabelecimento do termo inicial do prazo prescricional na data do nascimento da pretensão (= sistema objetivo) possui o inconveniente de impor ao credor o pesado ônus de identificar, em curto espaço de tempo, quem é o devedor e a extensão de sua pretensão, o que nem sempre se revela fácil ou possível.

• Os inconvenientes da vertente objetiva também se revelam naquelas hipóteses em que a experiência comum aponta notória dificuldade para o titular do direito tomar conhecimento do nascimento da sua pretensão, como ocorre nas hipóteses em que há alguma distância física entre o titular do direito e o objeto tutelado pelo sistema jurídico (p. ex. propriedades rurais longínquas) ou naquelas outras em que existe algum lapso temporal entre o ato ilícito (dano-evento) e a lesão (dano-prejuízo), como ocorre, por exemplo, nos casos de problemas de saúde cujos sintomas demoram a surgir.

• Nesse cenário, a doutrina aponta que a vertente objetiva da teoria da actio nata se coaduna com prazos prescricionais mais longos, sob pena de, em muitas hipóteses, conduzir a flagrantes injustiças. Por outro lado, o viés subjetivo da teoria da actio nata amolda-se melhor a prazos prescricionais curtos, na medida em que a exiguidade dos prazos é, em certa medida, compensada pela flexibilização permitida pela adoção de critérios subjetivos para a aferição do termo inicial.

• Não por outro motivo, a doutrina clássica já destacava essa característica, asseverando que a "doutrina da contagem do prazo da prescrição da data da ciência da violação deve ser limitada às prescrições de curto prazo".

• A título de exemplo, a doutrina, debruçando-se sobre o prazo prescricional trienal, aponta que "a solução objetiva pode se revelar injusta quando, no intervalo de três anos, a parte lesada não pode descobrir a existência do dano, a sua extensão e o responsável. A situação é particularmente delicada quando não há causas de interrupção ou de suspensão aplicáveis".

• Desse modo, é seguro afirmar que prazos prescricionais curtos tendem a atrair com maior intensidade a adoção do viés subjetivo da teoria da actio nata, equilibrando, assim, a exiguidade do tempo com a flexibilidade do termo inicial.

• Além disso, partindo-se de uma interpretação teleológica, deve-se consignar que não é condizente com a finalidade do instituto imputar eventual inércia ao titular de um direito sem que este saiba ou deva razoavelmente saber que é titular de uma pretensão exercitável.

• Em outras palavras, deve-se observar que, a rigor, a impossibilidade de conhecer, desde logo, o nascimento da pretensão é fator que faz protrair o dies a quo do prazo prescricional.

• Nessa esteira de intelecção, a doutrina leciona que "se assim não ocorresse, seria punido quem não ficou 'inerte', pois nem sempre a não-ação tem como causa a inércia. Esta é a ausência de atividade quando esta (atividade) teria sido possível e, portanto, exigível".

• Nesse sentido, a Terceira Turma do STJ já teve a oportunidade de ressaltar que, em determinadas hipóteses deve-se, de fato, adotar o viés subjetivo da teoria da actio nata, "sob pena de reputar iniciado o prazo prescricional quando o lesado nem sequer detinha a possibilidade de exercer sua pretensão, em claro descompasso com a finalidade do instituto da prescrição e com a boa-fé objetiva, princípio vetor do Código Civil" (AgInt no AREsp 876.731/DF, Rel. Ministro Marco Aurélio Bellizze, Terceira Turma, julgado em 15/09/2016, DJe 30/09/2016).

• Desse modo, pode-se afirmar que outro critério a ser adotado para se perquirir a possibilidade de aplicação excepcional da vertente subjetiva da teoria da actio nata consiste em verificar, em cada hipótese concreta, se, a partir do postulado normativo da razoabilidade, o credor tinha ou devia ter conhecimento do nascimento da pretensão, o que deve ser apurado de acordo com a boa-fé objetiva e com standards objetivos de atuação do homem médio, devendo-se afastar, desde logo, hipóteses de culpa grave que atente, de modo extraordinariamente elevado, contra o cuidado exigível no tráfego.

• A par destas considerações, deve-se adicionar à análise a clássica distinção entre responsabilidade civil por ato ilícito relativo e por ato ilícito absoluto.

• Como cediço, a partir do exame do conteúdo eficacial das relações jurídicas é possível subdividi-las em relações jurídicas de direito relativo e relações jurídicas de direito absoluto.

• As relações jurídicas de direito relativo são aquelas que possuem sujeito passivo determinado ou determinável, de tal modo que as posições jurídicas do sujeito ativo são direcionadas, exclusivamente, ao sujeito passivo, ao qual são impostas as correlatas posições jurídicas passivas. São exemplos dessa espécie de relação as relações jurídicas obrigacionais.

• Nesse diapasão, eventual violação das referidas posições jurídicas ensejará a denominada responsabilidade civil por ato ilícito relativo, comumente chamada de responsabilidade civil contratual. A título de exemplo pode-se mencionar as hipóteses de inadimplemento, mora, adimplemento ruim, etc.

• Por outro lado, as relações jurídicas de direito absoluto são aquelas que possuem sujeito passivo indeterminado - o chamado sujeito passivo total ou universal. São exemplos as relações jurídicas de direito real (p. ex. a relação jurídica de propriedade) e a relação jurídica de direitos da personalidade.

• Com efeito, nas relações jurídicas de direito absoluto, as posições jurídicas titularizadas pelo sujeito ativo são oponíveis erga omnis, isto é, não contra um sujeito determinado, mas sim contra o sujeito passivo total ou universal, a quem é imposto um dever geral de abstenção.

• A título de exemplo, pode-se mencionar que o titular de determinado direito da personalidade ou direito real, por exemplo, é titular de uma pretensão consubstanciada no poder de exigir que todos os demais indivíduos se abstenham de violar esse seu direito.

• A violação de posição jurídica ativa conteúdo de relação jurídica de direito absoluto dá ensejo à responsabilidade por ato ilícito absoluto, que representa, a rigor, desrespeito ao mencionado dever geral de abstenção e que, via de regra, recebe o epíteto de responsabilidade civil extracontratual.

• É a hipótese, por exemplo, da prática do ato ilícito previsto nos arts. 186 e 927, do CC/2002.

• Diante destas considerações, importa consignar que o viés subjetivo da teoria da actio nata encontra maior campo de aplicação na hipótese de responsabilidade civil por ato ilícito absoluto (= responsabilidade civil extracontratual), pois da própria natureza jurídica desta espécie de responsabilidade e, sobretudo, da presença do sujeito passivo universal, decorre uma maior dificuldade para o credor determinar o causador e a extensão do dano sofrido.

• Tratando-se de sujeito passivo total ou universal e, portanto, de violação de um dever geral de abstenção a todos imposto, é lógico e razoável concluir que o credor terá maior dificuldade para tomar conhecimento da lesão, da sua extensão e do agente que praticou o ato ilícito.

• Em âmbito jurisprudencial, a Terceira Turma do STJ, no julgamento do REsp 1.711.581/PR, ressaltou que, muito embora se admita a aplicação do viés subjetivo da teoria da actio nata em determinadas situações, esta "tem sido aplicada por esta Corte em casos de ilícitos extracontratuais nos quais a vítima não tem como conhecer a lesão a sua esfera jurídica no momento em que ocorrida", prestigiando o acesso à justiça.

• No mesmo sentido, também a Quarta Turma, no julgamento do REsp 1.354.348/RS, fixou o entendimento de que "na responsabilidade contratual, em regra, o termo inicial da contagem dos prazos de prescrição encontra-se na lesão ao direito, da qual decorre o nascimento da pretensão, que traz em seu bojo a possibilidade de exigência do direito subjetivo violado, nos termos do disposto no art. 189 do Código Civil, consagrando a tese da actio nata no ordenamento jurídico pátrio. Contudo, na responsabilidade extracontratual, a aludida regra assume viés mais humanizado e voltado aos interesses sociais, admitindo-se como marco inicial não mais o momento da ocorrência da violação do direito, mas a data do conhecimento do ato ou fato do qual decorre o direito de agir, sob pena de se punir a vítima por uma negligência que não houve, olvidando-se o fato de que a aparente inércia pode ter decorrido da absoluta falta de conhecimento do dano" (REsp 1.354.348/RS, Quarta Turma, julgado em 26/08/2014, DJe 16/09/2014).

• Assim, pode-se afirmar que, em regra, o viés subjetivo da teoria da actio nata possui maior afinidade com as hipóteses de responsabilidade civil por ato ilícito absoluto, estabelecendo-se como termo a quo do prazo prescricional a data do conhecimento, pelo titular, do nascimento da pretensão.

• Por fim, deve-se apontar como critério serviente a guiar o intérprete na determinação do dies a quo dos prazos prescricionais a própria escolha levada a efeito pelo direito positivo.

• De fato, em algumas hipóteses o próprio legislador, de maneira expressa, impõe a adoção da vertente subjetiva da teoria da actio nata.

• É o que se verifica, por exemplo, no art. 27 do Código de Defesa do Consumidor - ao estatuir que o prazo prescricional se inicia a partir do conhecimento do dano e de sua autoria pelo consumidor - e no art. 206, § 1º, II, "b", do Código Civil, ao estabelecer que, nos contratos de seguro em geral, o termo a quo do prazo prescricional é a "ciência do fato gerador da pretensão".

• Destarte, é seguro afirmar que o sistema subjetivo de determinação do dies a quo deve prevalecer sempre que, por razões de política legislativa, a legislação expressamente o adotar.

• Assim, ainda que de modo não exaustivo e não cumulativo, pode-se concluir que são critérios que indicam a tendência de adoção do viés subjetivo da teoria da actio nata: a) a submissão da pretensão a prazo prescricional curto; b) a constatação, na hipótese concreta, de que o credor tinha ou deveria ter ciência do nascimento da pretensão, o que deve ser apurado a partir da boa-fé objetiva e de standards de atuação do homem médio; c) o fato de se estar diante de responsabilidade civil por ato ilícito absoluto; e d) a expressa previsão legal a impor a aplicação do sistema subjetivo.

• No caso, importa consignar que se está diante de pretensões indenizatórias e compensatórias, ambas submetidas ao prazo prescricional trienal previsto no art. 206, § 3º, V, do CC/2002. Cuida-se, portanto, de pretensões submetidas a prazo prescricional curto.

• Além disso, tratando-se, na origem, de ação de reparação por danos materiais e morais em virtude da indevida utilização do nome do autor para figurar como falso ocupante de cargo em comissão, infere-se que a causa de pedir da presente demanda é, exata e precisamente, a configuração de responsabilidade civil por ato ilícito absoluto (responsabilidade civil extracontratual).

• Some-se a isso o fato de que, na espécie, a adoção do viés objetivo da teoria da actio nata, estabelecendo-se como termo inicial do prazo prescricional a data em que o autor foi exonerado, conduziria à flagrante injustiça em prejuízo do jurisdicionado que foi prejudicado por conduta de ex-deputado estadual que o nomeou como funcionário fantasma sem o seu conhecimento.

• Com efeito, não se revela condizente com o postulado da razoabilidade supor - tomando como critério de atuação a boa-fé objetiva e o comportamento esperado do homem médio - que o autor soubesse ou devesse saber que havia sido nomeado, sem o seu consentimento e clandestinamente, como "funcionário fantasma" na Assembleia Legislativa do Estado por meio da indevida utilização de seus dados pessoais.

• Tampouco se extrai do arcabouço fático qualquer indício de negligência grosseira ou atuação com culpa grave por parte do autor capaz de afastar a presunção de boa-fé que milita a seu favor quando alega o desconhecimento da existência do ato ilícito de que foi vítima.

• Ressalte-se que o simples fato de o sítio eletrônico do Google e de outros motores de buscas na internet estarem operando normalmente desde o alegado evento danoso, não significa, por si só, que seja razoável supor que todo cidadão deva, diariamente, efetuar esta espécie de pesquisa.

• Essa situação revela-se ainda mais grave quando se imagina, ao menos em tese, a possibilidade de utilização do nome e dos dados pessoais de cidadãos vulneráveis que não possuem qualquer acesso à rede mundial de computadores ou que residem em locais remotos e que, portanto, não teriam condições de evitar a consumação da prescrição em seu desfavor.

• Tais pessoas, nesse contexto, poderiam ser facilmente utilizadas para esquemas deste jaez e seriam prejudicadas pela prescrição, caso se adotasse o viés objetivo da teoria da actio nata.

Seção XI

Da Organização e da Fiscalização das Fundações

Art. 764. O juiz decidirá sobre a aprovação do estatuto das fundações e de suas alterações sempre que o requeira o interessado, quando:

• I - ela for negada previamente pelo Ministério Público ou por este forem exigidas modificações com as quais o interessado não concorde;
• II - o interessado discordar do estatuto elaborado pelo Ministério Público.
• § 1º O estatuto das fundações deve observar o disposto na Lei nº 10.406, de 10 de janeiro de 2002 (Código Civil) .
• § 2º Antes de suprir a aprovação, o juiz poderá mandar fazer no estatuto modificações a fim de adaptá-lo ao objetivo do instituidor.

Art. 765. Qualquer interessado ou o Ministério Público promoverá em juízo a extinção da fundação quando:

• I - se tornar ilícito o seu objeto;
• II - for impossível a sua manutenção;
• III - vencer o prazo de sua existência.

PROCESSUAL CIVIL E TRIBUTÁRIO. EXECUÇÃO FISCAL. DÍVIDAS TRIBUTÁRIAS DA MATRIZ. PENHORA, PELO SISTEMA BACEN-JUD, DE VALORES DEPOSITADOS EM NOME DAS FILIAIS. POSSIBILIDADE. ESTABELECIMENTO EMPRESARIAL COMO OBJETO DE DIREITOS E NÃO COMO SUJEITO DE DIREITOS. CNPJ PRÓPRIO DAS FILIAIS. IRRELEVÂNCIA NO QUE DIZ RESPEITO À UNIDADE PATRIMONIAL DA DEVEDORA. 1. No âmbito do direito privado, cujos princípios gerais, à luz do art. 109 do CTN, são informadores para a definição dos institutos de direito tributário, a filial é uma espécie de estabelecimento empresarial, fazendo parte do acervo patrimonial de uma única pessoa jurídica, partilhando dos mesmos sócios, contrato social e firma ou denominação da matriz. Nessa condição, consiste, conforme doutrina majoritária, em uma universalidade de fato, não ostentando personalidade jurídica própria, não sendo sujeito de direitos, tampouco uma pessoa distinta da sociedade empresária. Cuida-se de um instrumento de que se utiliza o empresário ou sócio para exercer suas atividades. 2. A discriminação do patrimônio da empresa, mediante a criação de filiais, não afasta a unidade patrimonial da pessoa jurídica, que, na condição de devedora, deve responder com todo o ativo do patrimônio social por suas dívidas, à luz de regra de direito processual prevista no art. 591 do Código de Processo Civil, segundo a qual "o devedor responde, para o cumprimento de suas obrigações, com todos os seus bens presentes e futuros, salvo as restrições estabelecidas em lei". 3. O princípio tributário da autonomia dos estabelecimentos, cujo conteúdo normativo preceitua que estes devem ser considerados, na forma da legislação específica de cada tributo, unidades autônomas e independentes nas relações jurídico-tributárias travadas com a Administração Fiscal, é um instituto de direito material, ligado à questão do nascimento da obrigação tributária de cada imposto especificamente considerado e não tem relação com a responsabilidade patrimonial dos devedores prevista em um regramento de direito processual, ou com os limites da responsabilidade dos bens da empresa e dos sócios definidos no direito empresarial. 4. A obrigação de que cada estabelecimento se inscreva com número próprio no CNPJ tem especial relevância para a atividade fiscalizatória da administração tributária, não afastando a unidade patrimonial da empresa, cabendo ressaltar que a inscrição da filial no CNPJ é derivada do CNPJ da matriz. 5. Nessa toada, limitar a satisfação do crédito público, notadamente do crédito tributário, a somente o patrimônio do estabelecimento que participou da situação caracterizada como fato gerador é adotar interpretação absurda e odiosa. Absurda porque não se concilia, por exemplo, com a cobrança dos créditos em uma situação de falência, onde todos os bens da pessoa jurídica (todos os estabelecimentos) são arrecadados para pagamento de todos os credores, ou com a possibilidade de responsabilidade contratual subsidiária dos sócios pelas obrigações da sociedade como um todo (v.g. arts. 1.023, 1.024, 1.039, 1.045, 1.052, 1.088 do CC/2002), ou com a administração de todos os estabelecimentos da sociedade pelos mesmos órgãos de deliberação, direção, gerência e fiscalização. Odiosa porque, por princípio, o credor privado não pode ter mais privilégios que o credor público, salvo exceções legalmente expressas e justificáveis. 6. Recurso especial conhecido e provido. Acórdão submetido ao regime do art. 543-C do CPC e da Resolução STJ n. 8/08. (REsp n. 1.355.812/RS, relator Ministro Mauro Campbell Marques, Primeira Seção, julgado em 22/5/2013, DJe de 31/5/2013.)

Tema nº 614: - Inexiste óbices à penhora, em face de dívidas tributárias da matriz, de valores depositados em nome das filiais.

• Informativo nº 777
• 6 de junho de 2023.
• TERCEIRA TURMA
• Processo: REsp 1.900.843-DF, Rel. Ministro Paulo de Tarso Sanseverino (in memorian), Rel. para acórdão Ministro Ricardo Villas Bôas Cueva, Terceira Turma, por maioria julgado em 23/5/2023, DJe 30/5/2023.

Ramo do Direito DIREITO CIVIL, DIREITO DO CONSUMIDOR

Paz, Justiça e Instituições EficazesTema <br /> Incidente de desconsideração da personalidade jurídica. Relação de consumo. Art. 28, § 5º, do Código de Defesa do Consumidor. Teoria Menor. Sócio. Atos de gestão. Prática. Comprovação.

DESTAQUE - A despeito de não se exigir prova de abuso ou fraude para aplicação da Teoria Menor da desconsideração da personalidade jurídica, não é possível a responsabilização pessoal de sócio que não desempenhe atos de gestão, ressalvada a prova de que contribuiu, ao menos culposamente, para a prática de atos de administração.

INFORMAÇÕES DO INTEIRO TEOR - De acordo com a pacífica jurisprudência desta Corte Superior, para fins de aplicação da denominada Teoria Menor da desconsideração da personalidade jurídica, não se exige prova da fraude ou do abuso de direito, tampouco é necessária a prova de confusão patrimonial, bastando que o consumidor demonstre o estado de insolvência do fornecedor ou o fato de a personalidade jurídica representar um obstáculo ao ressarcimento dos prejuízos causados. - Considerando que o § 5º do art. 28 do Código de Defesa do Consumidor - CDC, em virtude do mero inadimplemento e da ausência de bens suficientes à quitação do débito, admite, a princípio, a responsabilização pessoal do sócio, torna-se necessário investigar a atuação na condução dos negócios da empresa.

• A rigor, a considerar as origens históricas da disregard doctrine, não se poderia afirmar que a hipótese contemplada no § 5º do art. 28 do CDC trata do mesmo instituto, a despeito das expressões utilizadas pelo legislador, tendo em vista que a desconsideração propriamente dita está necessariamente associada à fraude e ao abuso de direito, com desvirtuamento da função social da pessoa jurídica, criada com personalidade distinta da de seus sócios.

• Como bem acentua a doutrina, o instituto da desconsideração da personalidade jurídica é frequentemente confundido com hipóteses em que se atribui aos sócios, por mera opção legislativa, a responsabilidade ordinária por dívidas da sociedade.

• No julgamento do REsp n. 1.766.093/SP, tratou-se da possibilidade da inclusão, no polo passivo de ação de rescisão contratual cumulada com pedido de restituição de valores pagos, já em fase de cumprimento de sentença, de membros do conselho fiscal de uma cooperativa habitacional, à luz do disposto no § 5º do art. 28 do CDC.

• Nesse julgado, tudo o que se disse a respeito das regras aplicáveis às sociedades cooperativas teve como único propósito fixar a premissa de que membros do conselho fiscal desse tipo de sociedade não praticam, em regra, atos de gestão, a exigir, por isso, a comprovação da presença de indícios de que estes contribuíram, ao menos culposamente, e com desvio de função, para a prática de atos de administração.

• Também destacou-se que, de acordo com a doutrina, ainda que seja possível considerar o § 5º do art. 28 do CDC como hipótese autônoma e independente daquelas previstas em seu caput, na linha do que já decidiu esta Corte Superior, a desconsideração da personalidade jurídica, mesmo em tal hipótese, somente pode atingir pessoas incumbidas da gestão da empresa.

• Assim, a denominada Teoria Menor da desconsideração da personalidade jurídica, de que trata o § 5º do art. 28 do CDC, a despeito de dispensar a prova de fraude, abuso de direito ou confusão patrimonial, não dá margem para admitir a responsabilização pessoal I) de quem não integra o quadro societário da empresa, ainda que nela atue como gestor, e II) de quem, embora ostentando a condição de sócio, não desempenha atos de gestão, independentemente de se tratar ou não de empresa constituída sob a forma de cooperativa.

• Vale lembrar que a desconsideração, mesmo sob a vertente da denominada Teoria Menor, é uma exceção à regra da autonomia patrimonial das pessoas jurídicas, "instrumento lícito de alocação e segregação de riscos, estabelecido pela lei com a finalidade de estimular empreendimentos, para a geração de empregos, tributo, renda e inovação em benefício de todos" (art. 49-A do Código Civil, incluído pela Lei nº 13.874/2019), a justificar, por isso, a interpretação mais restritiva do art. 28, § 5º, do CDC.

• Informativo nº 440
• Período: 21 a 25 de junho de 2010.
• TERCEIRA TURMA

DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA INVERSA. - Discute-se, no REsp, se a regra contida no art. 50 do CC/2002 autoriza a chamada desconsideração da personalidade jurídica inversa. Destacou a Min. Relatora, em princípio, que, a par de divergências doutrinárias, este Superior Tribunal sedimentou o entendimento de ser possível a desconstituição da personalidade jurídica dentro do processo de execução ou falimentar, independentemente de ação própria. Por outro lado, expõe que, da análise do art. 50 do CC/2002, depreende-se que o ordenamento jurídico pátrio adotou a chamada teoria maior da desconsideração, segundo a qual se exige, além da prova de insolvência, a demonstração ou de desvio de finalidade (teoria subjetiva da desconsideração) ou de confusão patrimonial (teoria objetiva da desconsideração).

• Também explica que a interpretação literal do referido artigo, de que esse preceito de lei somente serviria para atingir bens dos sócios em razão de dívidas da sociedade e não o inverso, não deve prevalecer. Anota, após essas considerações, que a desconsideração inversa da personalidade jurídica caracteriza-se pelo afastamento da autonomia patrimonial da sociedade, para, contrariamente do que ocorre na desconsideração da personalidade propriamente dita, atingir, então, o ente coletivo e seu patrimônio social, de modo a responsabilizar a pessoa jurídica por obrigações de seus sócios ou administradores.
• Assim, observa que o citado dispositivo, sob a ótica de uma interpretação teleológica, legitima a inferência de ser possível a teoria da desconsideração da personalidade jurídica em sua modalidade inversa, que encontra justificativa nos princípios éticos e jurídicos intrínsecos à própria disregard doctrine, que vedam o abuso de direito e a fraude contra credores. Dessa forma, a finalidade maior da disregard doctrine contida no preceito legal em comento é combater a utilização indevida do ente societário por seus sócios.
• Ressalta que, diante da desconsideração da personalidade jurídica inversa, com os efeitos sobre o patrimônio do ente societário, os sócios ou administradores possuem legitimidade para defesa de seus direitos mediante a interposição dos recursos tidos por cabíveis, sem ofensa ao contraditório, à ampla defesa e ao devido processo legal. No entanto, a Min. Relatora assinala que o juiz só poderá decidir por essa medida excepcional quando forem atendidos todos os pressupostos relacionados à fraude ou abuso de direito estabelecidos no art. 50 do CC/2002.
• No caso dos autos, tanto o juiz como o tribunal a quo entenderam haver confusão patrimonial e abuso de direito por parte do recorrente. Nesse contexto, a Turma negou provimento ao recurso. Precedentes citados: REsp 279.273-SP, DJ 29/3/2004; REsp 970.635-SP, DJe 1°/12/2009, e REsp 693.235-MT, DJe 30/11/2009. REsp 948.117-MS, Rel. Min. Nancy Andrighi, julgado em 22/6/2010.

• Informativo nº 533
• Período: 12 de fevereiro de 2014.
• TERCEIRA TURMA

DIREITO CIVIL. LEGITIMIDADE ATIVA PARA REQUERER DESCONSIDERAÇÃO. INVERSA DE PERSONALIDADE JURÍDICA.

• Se o sócio controlador de sociedade empresária transferir parte de seus bens à pessoa jurídica controlada com o intuito de fraudar partilha em dissolução de união estável, a companheira prejudicada, ainda que integre a sociedade empresária na condição de sócia minoritária, terá legitimidade para requerer a desconsideração inversa da personalidade jurídica de modo a resguardar sua meação.
• Inicialmente, ressalte-se que a Terceira Turma do STJ já decidiu pela possibilidade de desconsideração inversa da personalidade jurídica - que se caracteriza pelo afastamento da autonomia patrimonial da sociedade, para, contrariamente do que ocorre na desconsideração da personalidade jurídica propriamente dita, atingir o ente coletivo e seu patrimônio social, de modo a responsabilizar a pessoa jurídica por obrigações do sócio -, em razão de uma interpretação teleológica do art. 50 do CC/2002 (REsp 948.117-MS, DJe 3/8/2010).
• Quanto à legitimidade para atuar como parte no processo, por possuir, em regra, vinculação com o direito material, é conferida, na maioria das vezes, somente aos titulares da relação de direito material. Dessa forma, a legitimidade para requerer a desconsideração é atribuída, em regra, ao familiar que tenha sido lesado, titular do direito material perseguido, consoante a regra segundo a qual "Ninguém poderá pleitear, em nome próprio, direito alheio, salvo quando autorizado por lei" (art. 6º do CPC).
• Nota-se, nesse contexto, que a legitimidade para requerer a desconsideração inversa da personalidade jurídica da sociedade não decorre da condição de sócia, mas sim da condição de companheira do sócio controlador acusado de cometer abuso de direito com o intuito de fraudar a partilha. Além do mais, embora a companheira que se considera lesada também seja sócia, seria muito difícil a ela, quando não impossível, investigar os bens da empresa e garantir que eles não seriam indevidamente dissipados antes da conclusão da partilha, haja vista a condição de sócia minoritária. REsp 1.236.916-RS, Rel. Min. Nancy Andrighi, julgado em 22/10/2013.

• Informativo nº 554
• Período: 25 de fevereiro de 2015.
• SEGUNDA SEÇÃO

DIREITO CIVIL. LIMITES À APLICABILIDADE DO ART. 50 DO CC. - O encerramento das atividades da sociedade ou sua dissolução, ainda que irregulares, não são causas, por si sós, para a desconsideração da personalidade jurídica a que se refere o art. 50 do CC. Para a aplicação da teoria maior da desconsideração da personalidade social - adotada pelo CC -, exige-se o dolo das pessoas naturais que estão por trás da sociedade, desvirtuando-lhe os fins institucionais e servindo-se os sócios ou administradores desta para lesar credores ou terceiros. É a intenção ilícita e fraudulenta, portanto, que autoriza, nos termos da teoria adotada pelo CC, a aplicação do instituto em comento. - Especificamente em relação à hipótese a que se refere o art. 50 do CC, tratando-se de regra de exceção, de restrição ao princípio da autonomia patrimonial da pessoa jurídica, deve-se restringir a aplicação desse disposto legal a casos extremos, em que a pessoa jurídica tenha sido instrumento para fins fraudulentos, configurado mediante o desvio da finalidade institucional ou a confusão patrimonial. - Dessa forma, a ausência de intuito fraudulento afasta o cabimento da desconsideração da personalidade jurídica, ao menos quando se tem o CC como o microssistema legislativo norteador do instituto, a afastar a simples hipótese de encerramento ou dissolução irregular da sociedade como causa bastante para a aplicação do disregard doctrine. - Ressalte-se que não se quer dizer com isso que o encerramento da sociedade jamais será causa de desconsideração de sua personalidade, mas que somente o será quando sua dissolução ou inatividade irregulares tenham o fim de fraudar a lei, com o desvirtuamento da finalidade institucional ou confusão patrimonial. - Assim é que o enunciado 146, da III Jornada de Direito Civil, orienta o intérprete a adotar exegese restritiva no exame do artigo 50 do CC, haja vista que o instituto da desconsideração, embora não determine a despersonalização da sociedade - visto que aplicável a certo ou determinado negócio e que impõe apenas a ineficácia da pessoa jurídica frente ao lesado -, constitui restrição ao princípio da autonomia patrimonial. - Ademais, evidenciando a interpretação restritiva que se deve dar ao dispositivo em exame, a IV Jornada de Direito Civil firmou o enunciado 282, que expressamente afasta o encerramento irregular da pessoa jurídica como causa para desconsideração de sua personalidade: "O encerramento irregular das atividades da pessoa jurídica, por si só, não basta para caracterizar abuso da personalidade jurídica". Entendimento diverso conduziria, no limite, em termos práticos, ao fim da autonomia patrimonial da pessoa jurídica, ou seja, regresso histórico incompatível com a segurança jurídica e com o vigor da atividade econômica. Precedentes citados: AgRg no REsp 762.555-SC, Quarta Turma, DJe 25/10/2012; e AgRg no REsp 1.173.067/RS, Terceira Turma, DJe 19/6/2012. EREsp 1.306.553-SC, Rel. Min. Maria Isabel Gallotti, julgado em 10/12/2014, DJe 12/12/2014.

• Informativo nº 643
• 29 de março de 2019.
• PRIMEIRA TURMA
• Processo: REsp 1.775.269-PR, Rel. Min. Gurgel de Faria, por unanimidade, julgado em 21/02/2019, DJe 01/03/2019

Ramo do Direito DIREITO TRIBUTÁRIO, DIREITO PROCESSUAL CIVIL

Tema <br /> Execução fiscal. Redirecionamento a pessoa jurídica do mesmo grupo econômico. Certidão de Dívida Ativa. Não identificação. Hipóteses dos arts. 134 e 135 do CTN. Não enquadramento. Incidente de desconsideração da personalidade jurídica. Necessidade.

DESTAQUE - É necessária a instauração do incidente de desconsideração da personalidade da pessoa jurídica devedora para o redirecionamento de execução fiscal a pessoa jurídica que integra o mesmo grupo econômico, mas que não foi identificada no ato de lançamento (Certidão de Dívida Ativa) ou que não se enquadra nas hipóteses dos arts. 134 e 135 do CTN.

INFORMAÇÕES DO INTEIRO TEOR - Inicialmente cumpre salientar que em atenção à presunção relativa de legitimidade da Certidão da Dívida Ativa, o STJ pacificou entendimento segundo o qual a execução fiscal pode ser redirecionada ao corresponsável nela indicado, cabendo à parte então executada defender-se por meio dos embargos do devedor. - Caso o pedido de redirecionamento da execução fiscal mire pessoas jurídicas não elencadas na Certidão de Dívida Ativa, após a comprovação, pela Fazenda, da caracterização de hipótese legal de responsabilização dos terceiros indicados, o magistrado também pode decidir pela inclusão no polo passivo sem a instauração do incidente de desconsideração, pois a responsabilização de terceiros tratada no CTN não necessita da desconsideração da pessoa jurídica devedora. - Ademais, a atribuição de responsabilidade tributária aos sócios gerentes, nos termos do art. 135 do CTN, não depende mesmo do incidente de desconsideração da personalidade jurídica da sociedade empresária prevista no art. 133 do CPC/2015, pois a responsabilidade dos sócios, de fato, já lhes é atribuída pela própria lei, de forma pessoal e subjetiva (v.g.: AgInt no REsp 1.646.648/SP, Rel. Ministro Gurgel de Faria, Primeira Turma, DJe 28/11/2017), na hipótese de "atos praticados com excesso de poderes ou infração de lei, contrato social ou estatutos". - Igualmente, a responsabilidade subsidiária do art. 134, VII, do CTN autoriza o redirecionamento da execução fiscal aos sócios na hipótese de não ser possível exigir o crédito tributário da sociedade empresária liquidada (v.g.: REsp 1.591.419/DF, Rel. Ministro Gurgel de Faria, Primeira Turma, DJe 26/10/2016). - Daí porque o art. 4º, incisos V e VI, da Lei n. 6.830/1980 explicita a possibilidade de ajuizamento da execução fiscal contra o responsável legal por dívidas, tributárias ou não, das pessoas jurídicas de direito privado e contra os sucessores a qualquer título. - Porém, essa conclusão não é adequada quando a pretensão fazendária de redirecionamento mira pessoa jurídica integrante do mesmo grupo econômico a que pertence a sociedade empresária originalmente executada, que não está indicada na Certidão de Dívida Ativa e à qual não é atribuída a responsabilidade, na qualidade de terceiro (arts. 134 e 135 do CTN). - Às exceções da prévia previsão em lei sobre a responsabilidade de terceiros e do abuso de personalidade jurídica, o só fato de integrar grupo econômico não torna uma pessoa jurídica responsável pelos tributos inadimplidos pelas outras. De forma semelhante, o art. 124 do CTN dispõe que "são solidariamente obrigadas as pessoas que: I - tenham interesse comum na situação que constitua o fato gerador da obrigação principal; e II - as pessoas expressamente designadas por lei". - Esse dispositivo não serve à pretensão de redirecionamento, tendo em vista estar relacionado com a impossibilidade de arguição do benefício de ordem na solidariedade dos devedores identificados no ato de constituição do crédito tributário, o qual, vale registrar, não pode ser refeito no decorrer do processo executivo (v.g.: Súmula n. 392 do STJ: "A Fazenda Pública pode substituir a certidão de dívida ativa (CDA) até a prolação da sentença de embargos, quando se tratar de correção de erro material ou formal, vedada a modificação do sujeito passivo da execução"). - O art. 30, IX, da Lei n. 8.212/1991 ("as empresas que integram grupo econômico de qualquer natureza respondem entre si, solidariamente, pelas obrigações decorrentes desta lei") não permite o redirecionamento de execução fiscal a pessoa jurídica que não tenha participado da situação de ocorrência do fato gerador, ainda que integrante do grupo econômico. - A correta leitura desse dispositivo depende de sua conjugação com as regras do CTN, daí porque o fisco deve lançar o tributo com a indicação das pessoas jurídicas que estejam vinculadas ao fato gerador, não lhe sendo permitido, no curso do processo executivo, redirecionar a cobrança para pessoa jurídica estranha ao fato imponível, ainda que integrante do mesmo grupo econômico da devedora original. - Em conclusão, o redirecionamento de execução fiscal a pessoa jurídica que integra o mesmo grupo econômico da sociedade empresária originalmente executada, mas que não foi identificada no ato de lançamento (nome na CDA) ou que não se enquadra nas hipóteses dos arts. 134 e 135 do CTN, depende mesmo da comprovação do abuso de personalidade, caracterizado pelo desvio de finalidade ou confusão patrimonial, tal como consta do art. 50 do Código Civil, daí porque, nessa hipótese, é obrigatória a instauração do incidente de desconsideração da personalidade da pessoa jurídica devedora.

DOI: 10.1016/j.celrep.2023.113277

Resource: CVCL_YC51

Curator: @scibot

SciCrunch record: RRID:CVCL_YC51

What is this?

O fabricante de produtos industriais tem a obrigação de fornecer informações importantes sobre seus produtos por meio de impressos apropriados que acompanham o produto, para garantir a segurança e informação do consumidor. Isso faz parte das medidas de proteção ao consumidor previstas no CDC.

As sociedades coligadas são definidas pelos arts. 1.097 a 1.101 do Código Civil, confira:

Art. 1.097. Consideram-se coligadas as sociedades que, em suas relações de capital, são controladas, filiadas, ou de simples participação, na forma dos artigos seguintes.

Art. 1.098. É controlada:

• I - a sociedade de cujo capital outra sociedade possua a maioria dos votos nas deliberações dos quotistas ou da assembléia geral e o poder de eleger a maioria dos administradores;

• II - a sociedade cujo controle, referido no inciso antecedente, esteja em poder de outra, mediante ações ou quotas possuídas por sociedades ou sociedades por esta já controladas.

Art. 1.099. Diz-se coligada ou filiada a sociedade de cujo capital outra sociedade participa com dez por cento ou mais, do capital da outra, sem controlá-la.

Art. 1.100. É de simples participação a sociedade de cujo capital outra sociedade possua menos de dez por cento do capital com direito de voto.

Art. 1.101. Salvo disposição especial de lei, a sociedade não pode participar de outra, que seja sua sócia, por montante superior, segundo o balanço, ao das próprias reservas, excluída a reserva legal.

• Parágrafo único. Aprovado o balanço em que se verifique ter sido excedido esse limite, a sociedade não poderá exercer o direito de voto correspondente às ações ou quotas em excesso, as quais devem ser alienadas nos cento e oitenta dias seguintes àquela aprovação.

As sociedades consorciadas são aquelas definidas pelo art. 278 da Lei das S/A, vide:

Art. 278 [...] § 1º O consórcio não tem personalidade jurídica e as consorciadas somente se obrigam nas condições previstas no respectivo contrato, respondendo cada uma por suas obrigações, sem presunção de solidariedade.

A doutrina entende haver a derrogação, pelo § 3º, art. 28, CDC, da norma do referido art. 278, constituindo, nas relações de consumo, vínculo de solidariedade entre as empresas consorciadas em benefício do consumidor.

Nessa mesma linha, é o precedente firmado pelo STJ:

• Como regra geral, as sociedades consorciadas apenas se obrigam nas condições previstas no respectivo contrato, respondendo cada uma por suas obrigações, sem presunção de solidariedade, de acordo com o disposto no art. 278, § 1º, da Lei das Sociedades Anônimas (Lei 6.404/76).

• Essa regra, no entanto, não é absoluta, havendo no ordenamento jurídico diversas normas que preveem a solidariedade entre as sociedades consorciadas, notadamente quando está em jogo interesse que prepondera sobre a autonomia patrimonial das integrantes do consórcio.

• Na hipótese de responsabilidade derivada de relação de consumo, afasta-se a regra geral da ausência de solidariedade entre as consorciadas por força da disposição expressa contida no art. 28, § 3º, do CDC. Essa exceção em matéria consumerista justifica-se pela necessidade de se atribuir máxima proteção ao consumidor, mediante o alargamento da base patrimonial hábil a suportar a indenização.

• Não obstante, é certo que, por se tratar de exceção à regra geral, a previsão de solidariedade contida no art. 28, § 3º, do CDC deve ser interpretada restritivamente, de maneira a abarcar apenas as obrigações resultantes do objeto do consórcio, e não quaisquer obrigações assumidas pelas consorciadas em suas atividades empresariais.

• Ademais, a exceção em comento não alcança o próprio consórcio, que apenas responderá solidariamente com suas integrantes se houver previsão contratual nesse sentido” (STJ – REsp 1.635.637/RJ – Terceira Turma – Rel. Min. Nancy Andrighi – j. 18.09.2018 – DJe 21.09.2018).

this is just so much fluff... sure right idea sort of but where is the actual material funding and resources for LEDCs to "promote public and private research"

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Referee #1

Evidence, reproducibility and clarity

Pollitt et al. investigated the role of Llgl1 in maturation of the ventricular wall in zebrafish. They examined zebrafish heart morphology with microscopy analysis of fluorescent reporter lines crossed with their CRISPR/Cas9 llgl1 line and observed apically extruding CMs in llgl1 mutant embryos, implicating a role for llgl1 in ventricular wall integrity. Further, they examined apicobasal polarity in the ventricular wall through quantification of Crb2a distribution at the apical membrane surface, with enhanced Crb2a retention at CM junctions in llgl1 mutant embryos in comparison to WT at 72 hpf but similar levels at 80 hpf. Further analyses indicated a requirement for llgl1 for the temporal establishment of the apical laminin sheath, llgl1 is required for the timely dissemination of epicardial cells which deposit laminin to maintain the integrity of the ventricular wall. This work is written well and provides new information on heart development in zebrafish, and provides additional information on the role of the epicardium in supporting the integrity of the ventricular wall during trabeculation.

Major:

1. Although information is provided in the introduction and discussion on the role of the Llgl1 homolog in Drosophila and speculation on LLGL1 contributing to heart defects in SMS patients in the discussion, have Llgl1 homologs been examined in other vertebrate animal models during heart development or regeneration?
2. In Fig. 3I-O: The authors described the spatial dynamics of laminin in llgl1 mutants at 72 and 80m hpf. However, it is hard to say the schematic depicting of laminin for llg1 mutant in Fig. 3O reflect the real laminin staining signals in Fig. 3J' and 3L'.
3. It is mentioned that llgl1 CRISPR/Cas9 mutants are viable as adults on pg. 3 of the Results section. Have the authors examined heart morphology in these mutants in juvenile or adult fish?
4. In Fig. 4J-M', there is no Cav1 signals after wt1a MO but still laminin signals. Where these laminins come from?
5. As pan-epicardial transgenes like tcf21 reporters have been widely used, the authors should use such reporters to verify the expression of laminin gene expression in epicardial cells, and the efficacy and efficiency of depleting epicardial cells after wt1 MO injection.

Minor:

1. On page 3 of the manuscript, Fig. 1A should be included with Fig. 1B in the first sentence of paragraph 2 of the Results subsection "Llgl1 regulates ventricular wall integrity and trabeculation".
2. Fig. 2E: Is Fig. 2E from WT or llgl1 embryos? This information isn't indicated in the image panels or the figure legend. It might also be beneficial to include a similar representative image for the WT or llgl1 mutant embryo as this was used for quantification.
3. Fig. 3G: As the entirety of Fig. 3 used violet coloring to depict Laminin, it would be more consistent to change the blue coloring used to depict Laminin in panel G to the same violet coloring used for Laminin in the other panels of Fig. 3.
4. It would be beneficial to readers to briefly describe what cell type the transgenic reporters label when mentioned in the Results section to help readers unfamiliar with zebrafish.

Significance

This work is written well and provides new information on heart development in zebrafish, and provides additional information on the role of the epicardium in supporting the integrity of the ventricular wall during trabeculation.

Uma circunstância importante que diferencia a OSCIP da OS é que aquelas não foram idealizadas para substituir a Administração Pública, mediante “absorção” das atividades exercidas por órgãos ou entidades administrativas que o poder público pretendia extinguir.

• ADI 1923
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. AYRES BRITTO
• Redator(a) do acórdão: Min. LUIZ FUX
• Julgamento: 16/04/2015
• Publicação: 17/12/2015

Ementa AÇÃO DIRETA DE INCONSTITUCIONALIDADE. CONSTITUCIONAL. ADMINISTRATIVO. TERCEIRO SETOR. MARCO LEGAL DAS ORGANIZAÇÕES SOCIAIS. LEI Nº 9.637/98 E NOVA REDAÇÃO, CONFERIDA PELA LEI Nº 9.648/98, AO ART. 24, XXIV, DA LEI Nº 8.666/93. MOLDURA CONSTITUCIONAL DA INTERVENÇÃO DO ESTADO NO DOMÍNIO ECONÔMICO E SOCIAL. SERVIÇOS PÚBLICOS SOCIAIS. SAÚDE (ART. 199, CAPUT), EDUCAÇÃO (ART. 209, CAPUT), CULTURA (ART. 215), DESPORTO E LAZER (ART. 217), CIÊNCIA E TECNOLOGIA (ART. 218) E MEIO AMBIENTE (ART. 225). ATIVIDADES CUJA TITULARIDADE É COMPARTILHADA ENTRE O PODER PÚBLICO E A SOCIEDADE. DISCIPLINA DE INSTRUMENTO DE COLABORAÇÃO PÚBLICO-PRIVADA. INTERVENÇÃO INDIRETA. ATIVIDADE DE FOMENTO PÚBLICO. INEXISTÊNCIA DE RENÚNCIA AOS DEVERES ESTATAIS DE AGIR. MARGEM DE CONFORMAÇÃO CONSTITUCIONALMENTE ATRIBUÍDA AOS AGENTES POLÍTICOS DEMOCRATICAMENTE ELEITOS. PRINCÍPIOS DA CONSENSUALIDADE E DA PARTICIPAÇÃO. INEXISTÊNCIA DE VIOLAÇÃO AO ART. 175, CAPUT, DA CONSTITUIÇÃO. EXTINÇÃO PONTUAL DE ENTIDADES PÚBLICAS QUE APENAS CONCRETIZA O NOVO MODELO. INDIFERENÇA DO FATOR TEMPORAL. INEXISTÊNCIA DE VIOLAÇÃO AO DEVER CONSTITUCIONAL DE LICITAÇÃO (CF, ART. 37, XXI). PROCEDIMENTO DE QUALIFICAÇÃO QUE CONFIGURA HIPÓTESE DE CREDENCIAMENTO. COMPETÊNCIA DISCRICIONÁRIA QUE DEVE SER SUBMETIDA AOS PRINCÍPIOS CONSTITUCIONAIS DA PUBLICIDADE, MORALIDADE, EFICIÊNCIA E IMPESSOALIDADE, À LUZ DE CRITÉRIOS OBJETIVOS (CF, ART. 37, CAPUT). INEXISTÊNCIA DE PERMISSIVO À ARBITRARIEDADE. CONTRATO DE GESTÃO. NATUREZA DE CONVÊNIO. CELEBRAÇÃO NECESSARIAMENTE SUBMETIDA A PROCEDIMENTO OBJETIVO E IMPESSOAL. CONSTITUCIONALIDADE DA DISPENSA DE LICITAÇÃO INSTITUÍDA PELA NOVA REDAÇÃO DO ART. 24, XXIV, DA LEI DE LICITAÇÕES E PELO ART. 12, §3º, DA LEI Nº 9.637/98. FUNÇÃO REGULATÓRIA DA LICITAÇÃO. OBSERVÂNCIA DOS PRINCÍPIOS DA IMPESSOALIDADE, DA PUBLICIDADE, DA EFICIÊNCIA E DA MOTIVAÇÃO. IMPOSSIBILIDADE DE EXIGÊNCIA DE LICITAÇÃO PARA OS CONTRATOS CELEBRADOS PELAS ORGANIZAÇÕES SOCIAIS COM TERCEIROS. OBSERVÂNCIA DO NÚCLEO ESSENCIAL DOS PRINCÍPIOS DA ADMINISTRAÇÃO PÚBLICA (CF, ART. 37, CAPUT). REGULAMENTO PRÓPRIO PARA CONTRATAÇÕES. INEXISTÊNCIA DE DEVER DE REALIZAÇÃO DE CONCURSO PÚBLICO PARA CONTRATAÇÃO DE EMPREGADOS. INCIDÊNCIA DO PRINCÍPIO CONSTITUCIONAL DA IMPESSOALIDADE, ATRAVÉS DE PROCEDIMENTO OBJETIVO. AUSÊNCIA DE VIOLAÇÃO AOS DIREITOS CONSTITUCIONAIS DOS SERVIDORES PÚBLICOS CEDIDOS. PRESERVAÇÃO DO REGIME REMUNERATÓRIO DA ORIGEM. AUSÊNCIA DE SUBMISSÃO AO PRINCÍPIO DA LEGALIDADE PARA O PAGAMENTO DE VERBAS, POR ENTIDADE PRIVADA, A SERVIDORES. INTERPRETAÇÃO DOS ARTS. 37, X, E 169, §1º, DA CONSTITUIÇÃO. CONTROLES PELO TRIBUNAL DE CONTAS DA UNIÃO E PELO MINISTÉRIO PÚBLICO. PRESERVAÇÃO DO ÂMBITO CONSTITUCIONALMENTE DEFINIDO PARA O EXERCÍCIO DO CONTROLE EXTERNO (CF, ARTS. 70, 71, 74 E 127 E SEGUINTES). INTERFERÊNCIA ESTATAL EM ASSOCIAÇÕES E FUNDAÇÕES PRIVADAS (CF, ART. 5º, XVII E XVIII). CONDICIONAMENTO À ADESÃO VOLUNTÁRIA DA ENTIDADE PRIVADA. INEXISTÊNCIA DE OFENSA À CONSTITUIÇÃO. AÇÃO DIRETA JULGADA PARCIALMENTE PROCEDENTE PARA CONFERIR INTERPRETAÇÃO CONFORME AOS DIPLOMAS IMPUGNADOS.

1. A atuação da Corte Constitucional não pode traduzir forma de engessamento e de cristalização de um determinado modelo pré-concebido de Estado, impedindo que, nos limites constitucionalmente assegurados, as maiorias políticas prevalecentes no jogo democrático pluralista possam pôr em prática seus projetos de governo, moldando o perfil e o instrumental do poder público conforme a vontade coletiva.

2. Os setores de saúde (CF, art. 199, caput), educação (CF, art. 209, caput), cultura (CF, art. 215), desporto e lazer (CF, art. 217), ciência e tecnologia (CF, art. 218) e meio ambiente (CF, art. 225) configuram serviços públicos sociais, em relação aos quais a Constituição, ao mencionar que “são deveres do Estado e da Sociedade” e que são “livres à iniciativa privada”, permite a atuação, por direito próprio, dos particulares, sem que para tanto seja necessária a delegação pelo poder público, de forma que não incide, in casu, o art. 175, caput, da Constituição.

3. A atuação do poder público no domínio econômico e social pode ser viabilizada por intervenção direta ou indireta, disponibilizando utilidades materiais aos beneficiários, no primeiro caso, ou fazendo uso, no segundo caso, de seu instrumental jurídico para induzir que os particulares executem atividades de interesses públicos através da regulação, com coercitividade, ou através do fomento, pelo uso de incentivos e estímulos a comportamentos voluntários.

4. Em qualquer caso, o cumprimento efetivo dos deveres constitucionais de atuação estará, invariavelmente, submetido ao que a doutrina contemporânea denomina de controle da Administração Pública sob o ângulo do resultado (Diogo de Figueiredo Moreira Neto).

5. O marco legal das Organizações Sociais inclina-se para a atividade de fomento público no domínio dos serviços sociais, entendida tal atividade como a disciplina não coercitiva da conduta dos particulares, cujo desempenho em atividades de interesse público é estimulado por sanções premiais, em observância aos princípios da consensualidade e da participação na Administração Pública.

6. A finalidade de fomento, in casu, é posta em prática pela cessão de recursos, bens e pessoal da Administração Pública para as entidades privadas, após a celebração de contrato de gestão, o que viabilizará o direcionamento, pelo Poder Público, da atuação do particular em consonância com o interesse público, através da inserção de metas e de resultados a serem alcançados, sem que isso configure qualquer forma de renúncia aos deveres constitucionais de atuação.

7. Na essência, preside a execução deste programa de ação institucional a lógica que prevaleceu no jogo democrático, de que a atuação privada pode ser mais eficiente do que a pública em determinados domínios, dada a agilidade e a flexibilidade que marcam o regime de direito privado.

8. Os arts. 18 a 22 da Lei nº 9.637/98 apenas concentram a decisão política, que poderia ser validamente feita no futuro, de afastar a atuação de entidades públicas através da intervenção direta para privilegiar a escolha pela busca dos mesmos fins através da indução e do fomento de atores privados, razão pela qual a extinção das entidades mencionadas nos dispositivos não afronta a Constituição, dada a irrelevância do fator tempo na opção pelo modelo de fomento – se simultaneamente ou após a edição da Lei.

9. O procedimento de qualificação de entidades, na sistemática da Lei, consiste em etapa inicial e embrionária, pelo deferimento do título jurídico de “organização social”, para que Poder Público e particular colaborem na realização de um interesse comum, não se fazendo presente a contraposição de interesses, com feição comutativa e com intuito lucrativo, que consiste no núcleo conceitual da figura do contrato administrativo, o que torna inaplicável o dever constitucional de licitar (CF, art. 37, XXI).

10. A atribuição de título jurídico de legitimação da entidade através da qualificação configura hipótese de credenciamento, no qual não incide a licitação pela própria natureza jurídica do ato, que não é contrato, e pela inexistência de qualquer competição, já que todos os interessados podem alcançar o mesmo objetivo, de modo includente, e não excludente.

11. A previsão de competência discricionária no art. 2º, II, da Lei nº 9.637/98 no que pertine à qualificação tem de ser interpretada sob o influxo da principiologia constitucional, em especial dos princípios da impessoalidade, moralidade, publicidade e eficiência (CF, art. 37, caput). É de se ter por vedada, assim, qualquer forma de arbitrariedade, de modo que o indeferimento do requerimento de qualificação, além de pautado pela publicidade, transparência e motivação, deve observar critérios objetivos fixados em ato regulamentar expedido em obediência ao art. 20 da Lei nº 9.637/98, concretizando de forma homogênea as diretrizes contidas nos inc. I a III do dispositivo.

12. A figura do contrato de gestão configura hipótese de convênio, por consubstanciar a conjugação de esforços com plena harmonia entre as posições subjetivas, que buscam um negócio verdadeiramente associativo, e não comutativo, para o atingimento de um objetivo comum aos interessados: a realização de serviços de saúde, educação, cultura, desporto e lazer, meio ambiente e ciência e tecnologia, razão pela qual se encontram fora do âmbito de incidência do art. 37, XXI, da CF.

13. Diante, porém, de um cenário de escassez de bens, recursos e servidores públicos, no qual o contrato de gestão firmado com uma entidade privada termina por excluir, por consequência, a mesma pretensão veiculada pelos demais particulares em idêntica situação, todos almejando a posição subjetiva de parceiro privado, impõe-se que o Poder Público conduza a celebração do contrato de gestão por um procedimento público impessoal e pautado por critérios objetivos, por força da incidência direta dos princípios constitucionais da impessoalidade, da publicidade e da eficiência na Administração Pública (CF, art. 37, caput).

14. As dispensas de licitação instituídas no art. 24, XXIV, da Lei nº 8.666/93 e no art. 12, §3º, da Lei nº 9.637/98 têm a finalidade que a doutrina contemporânea denomina de função regulatória da licitação, através da qual a licitação passa a ser também vista como mecanismo de indução de determinadas práticas sociais benéficas, fomentando a atuação de organizações sociais que já ostentem, à época da contratação, o título de qualificação, e que por isso sejam reconhecidamente colaboradoras do Poder Público no desempenho dos deveres constitucionais no campo dos serviços sociais. O afastamento do certame licitatório não exime, porém, o administrador público da observância dos princípios constitucionais, de modo que a contratação direta deve observar critérios objetivos e impessoais, com publicidade de forma a permitir o acesso a todos os interessados.

15. As organizações sociais, por integrarem o Terceiro Setor, não fazem parte do conceito constitucional de Administração Pública, razão pela qual não se submetem, em suas contratações com terceiros, ao dever de licitar, o que consistiria em quebra da lógica de flexibilidade do setor privado, finalidade por detrás de todo o marco regulatório instituído pela Lei. Por receberem recursos públicos, bens públicos e servidores públicos, porém, seu regime jurídico tem de ser minimamente informado pela incidência do núcleo essencial dos princípios da Administração Pública (CF, art. 37, caput), dentre os quais se destaca o princípio da impessoalidade, de modo que suas contratações devem observar o disposto em regulamento próprio (Lei nº 9.637/98, art. 4º, VIII), fixando regras objetivas e impessoais para o dispêndio de recursos públicos.

16. Os empregados das Organizações Sociais não são servidores públicos, mas sim empregados privados, por isso que sua remuneração não deve ter base em lei (CF, art. 37, X), mas nos contratos de trabalho firmados consensualmente. Por identidade de razões, também não se aplica às Organizações Sociais a exigência de concurso público (CF, art. 37, II), mas a seleção de pessoal, da mesma forma como a contratação de obras e serviços, deve ser posta em prática através de um procedimento objetivo e impessoal.

17. Inexiste violação aos direitos dos servidores públicos cedidos às organizações sociais, na medida em que preservado o paradigma com o cargo de origem, sendo desnecessária a previsão em lei para que verbas de natureza privada sejam pagas pelas organizações sociais, sob pena de afronta à própria lógica de eficiência e de flexibilidade que inspiraram a criação do novo modelo.

18. O âmbito constitucionalmente definido para o controle a ser exercido pelo Tribunal de Contas da União (CF, arts. 70, 71 e 74) e pelo Ministério Público (CF, arts. 127 e seguintes) não é de qualquer forma restringido pelo art. 4º, caput, da Lei nº 9.637/98, porquanto dirigido à estruturação interna da organização social, e pelo art. 10 do mesmo diploma, na medida em que trata apenas do dever de representação dos responsáveis pela fiscalização, sem mitigar a atuação de ofício dos órgãos constitucionais.

19. A previsão de percentual de representantes do poder público no Conselho de Administração das organizações sociais não encerra violação ao art. 5º, XVII e XVIII, da Constituição Federal, uma vez que dependente, para concretizar-se, de adesão voluntária das entidades privadas às regras do marco legal do Terceiro Setor.

20. Ação direta de inconstitucionalidade cujo pedido é julgado parcialmente procedente, para conferir interpretação conforme à Constituição à Lei nº 9.637/98 e ao art. 24, XXIV, da Lei nº 8666/93, incluído pela Lei nº 9.648/98, para que:

21. (i) o procedimento de qualificação seja conduzido de forma pública, objetiva e impessoal, com observância dos princípios do caput do art. 37 da CF, e de acordo com parâmetros fixados em abstrato segundo o que prega o art. 20 da Lei nº 9.637/98;
22. (ii) a celebração do contrato de gestão seja conduzida de forma pública, objetiva e impessoal, com observância dos princípios do caput do art. 37 da CF;
23. (iii) as hipóteses de dispensa de licitação para contratações (Lei nº 8.666/93, art. 24, XXIV) e outorga de permissão de uso de bem público (Lei nº 9.637/98, art. 12, §3º) sejam conduzidas de forma pública, objetiva e impessoal, com observância dos princípios do caput do art. 37 da CF;
24. (iv) os contratos a serem celebrados pela Organização Social com terceiros, com recursos públicos, sejam conduzidos de forma pública, objetiva e impessoal, com observância dos princípios do caput do art. 37 da CF, e nos termos do regulamento próprio a ser editado por cada entidade;
25. (v) a seleção de pessoal pelas Organizações Sociais seja conduzida de forma pública, objetiva e impessoal, com observância dos princípios do caput do art. 37 da CF, e nos termos do regulamento próprio a ser editado por cada entidade; e
26. (vi) para afastar qualquer interpretação que restrinja o controle, pelo Ministério Público e pelo TCU, da aplicação de verbas públicas.

Carica le immagine della tua attività

Mandaci una descrizione completa dei servizi offerti utilizzando delle parole chiave che rappresentino la tua attività

missing the combining vowel for each word in this list. Need to be consistent with previous chapters and follow the description of the header.

arche/o (first, beginning)

cervic/o (cervix)

colp/o (vagina)

endometri/o (endometrium)

episi/o (vulva)

gyn/o (woman)

gynec/o (woman)

hymen/o (hymen)

hyster/o (uterus)

mamm/o (breast)

mast/o (breast)

men/o (menstruation)

metr/o (uterus)

metr/i (uterus)

oophor/o (ovary)

pelv/i (pelvis, pelvic bones, pelvic cavity)

perine/o (perineum)

salping/o (uterine tube, fallopian tube)

trachel/o (cervix)

vagin/o (vagina)

vulv/o (vulva)

missing the combining vowel for each word in this list. Need to be consistent with previous chapters and follow the header description

albumin/o (albumin)

azot/o (urea, nitrogen)

blast/o (developing cell, germ cell)

cyst/o (bladder, sac)

glomerul/o (glomerulus)

glyc/o (sugar)

glycos/o (sugar)

hydr/o (water)

lith/o (stone, calculus)

meat/o (meatus)

nephr/o (kidney)

noct/i (night)

olig/o (few, scanty)

py/o (pus)

pyel/o (renal pelvis)

ren/o (kidney)

ur/o (urine, urinary tract)

ureter/o (ureter)

urethr/o (urethra)

urin/o (urine, urinary tract)

vesic/o (bladder, sac)

errores intencionales o accindentales?

Q: HOW IS INFORMATION STRUCTURED AND PRESENTED? A: Valdez starts her article with an abstract giving a brief description of what she will be discussing in the rest of the article, in the abstract she highlights two figures that are referenced throughout the article. Since the topic has to do with race she gives the subject its own paragraph going into detail about how it emerged and why it is used in science. She writes a paragraph telling the origins of how the issue of improvising race came to her attention. - The subjects of her article are explained in detail before she goes into the main topic of her article. I surmise she did this so the reader will have a better understanding of her position on the topic. - The main topic of her article is improvising race and the issues this presents in clinical studies. So before she delves into the topic she gives prerequisite information.

Once she gives the necessary background information she goes into detail about the UK census and how it contributes to improvisation of race. - In other words after giving the necessary background information she goes into the main topic of her article.

In the conclusion she gives a summary of her findings

Q: CONVENTIONS, ANALYTICAL APPROACHES, AND DISCOURSE A: Valdez approaches this topic with the goal of solving a problem that is not apparent. She uses a scenario in which the issue occurs and highlights the fact that those involved in the study did not notice the issue until she pointed showing that there is a clear disconnect between the research subjects and the scientists who came up with the classification system they are being asked to use.

Q: HOW IS EVIDENCE IDENTIFIED AND KNOWLEDGE CREATED? A: The evidence to support Valdez's hypothesis came from the people involved in the clinical trials

Q: WHAT INFORMATION IS PRIORITIZED? A: Any information that is important to the main topic there is no "fluff" and the author never goes on a tangent that is not relevant to the topic.

Q: HOW IS EXPERTISE/VALIDITY ESTABLISHED? A: Valdez's credentials are mentioned in the notes section of the article.

Q: WHY DO YOU WISH TO EXPLORE THIS TOPIC FURTHER? A: This topic is something I have noticed myself especially when it comes to "mixed" people or people whose origins deviate from the norm.

Here Valdez defines the topic of her article: racial improvisation.

Valdez points out the arbitrary nature of ethnic classification systems.

Valdez is exploring how race can be hard to properly identify due to its arbitrary nature.

``` @Test void test() { PlacesListManagerInterface plm; PlacesListManagerInterface plmret; PlacesListInterface pli; ObjectRegistryInterface ori;

    try{
        System.out.println("Localizar ReplicaManager...");
        plm  = (PlacesListManagerInterface) Naming.lookup("rmi://localhost:2024/replicamanager");

        Place p1 = new Place("3510", "Viseu");
        System.out.println("Invocar addPlace() no ReplicaManager para 3510...");
        plm.addPlace(p1);

        Place p2 = new Place("1000", "Lisboa");
        System.out.println("Invocar addPlace() no ReplicaManager para 1000...");
        plm.addPlace(p2);

        Place p3 = new Place("4000", "Lisboa");
        System.out.println("Invocar addPlace() no ReplicaManager para 4000...");
        plm.addPlace(p3);

        System.out.println("Obter o endereço do ReplicaManager no Registry() para o ObjectID 1000 ...");
        ori = (ObjectRegistryInterface) Naming.lookup("rmi://localhost:2023/registry");
        String addrRM = ori.resolve("1000");

        System.out.println("Pedir ao ReplicaManager para devolver um PlaceManager para o ObjectID 1000 ...");
        plm = (PlacesListManagerInterface) Naming.lookup(addrRM);
        boolean pl1done = false;
        boolean pl2done = false;
        boolean pl3done = false;
        String plAddress = null;
        for(int i=0; i<10 ; i++) {
            plAddress = plm.getPlaceListAddress("1000");
            System.out.println("\tDevolveu " + plAddress);
            if(plAddress.equals("rmi://localhost:2025/placelist")) pl1done=true;
            else if(plAddress.equals("rmi://localhost:2026/placelist")) pl2done=true;
                 else if(plAddress.equals("rmi://localhost:2027/placelist")) pl3done=true;
        }

        if(!(pl1done==pl2done==pl3done==true)) fail("Não está a devolver um PlaceManager aleatório...");

        System.out.println("Invocar getPlace() no PlaceManager");
        pli  = (PlacesListInterface) Naming.lookup(plAddress);
        String locality = pli.getPlace("1000").getLocality();

        System.out.println("\tDevolveu " + locality);
        assertEquals("Lisboa", locality);


    } catch(Exception e) { fail("Erro\n" + e.getMessage()); }
}

```

This can be very dangerous if a kid is watching because then they can think the other way about Douglass and know that what they been taught was a lie so then they wont know what really is the truth.

Procedimento a ser seguido na modalidade de Leilão, havendo especificidades que afastam a aplicação do art. 17

• Informativo nº 792
• 24 de outubro de 2023.
• Processo: RMS 68.504-SC, Rel. Ministra Regina Helena Costa, Primeira Turma, por unanimidade, julgado em 10/10/2023, DJe 16/10/2023

Ramo do Direito DIREITO ADMINISTRATIVO

Paz, Justiça e Instituições EficazesTema <br /> Licitação na modalidade de leilão. Discricionariedade administrativa na forma de contratação de leiloeiro oficial pelo poder público. Art. 31, caput e § 1º da Lei n. 14.133/2021. Divulgação pública e permanente de edital de credenciamento em sítio eletrônico. Obrigação decorrente do art. 79, parágrafo único, I, da Lei n. 14.133/2021. Inaplicabilidade aos chamamentos públicos realizados sob a égide da Lei n. 8.666/1993.

DESTAQUE - A Administração Pública é obrigada a divulgar, permanentemente, edital de credenciamento em sítio eletrônico somente após a vigência da Nova Lei de Licitações e Contratações Administrativas.

INFORMAÇÕES DO INTEIRO TEOR - De acordo com o art. 31 da Lei n. 14.133/2021, os procedimentos licitatórios na modalidade leilão podem ser conduzidos por servidor público ou, alternativamente, ser cometidos a leiloeiro oficial, facultando-se à autoridade competente juízo discricionário entre o certame levado a efeito por agente integrante dos quadros da Administração ou por terceiro que atenda às prescrições do Decreto n. 21.981/1932, o qual regulamenta a profissão de leiloeiro.

• Outrossim, caso a escolha do responsável pela realização do leilão recaia sobre auxiliar do comércio, a norma contida no § 1º do art. 31 da Nova Lei de Licitações e Contratações Administrativas autoriza a seleção do profissional mediante pregão ou, ainda, por meio de credenciamento sem, no entanto, a fixação de critérios de precedência condicionada entre quaisquer dos instrumentos, razão pela qual inviável extrair de citada disposição normativa o dever legal de selecionar leiloeiros oficiais mediante divulgação de edital de chamamento público.

• Nesse contexto, embora o art. 79, parágrafo único, I, da Lei n. 14.133/2021 imponha a manutenção pública de edital de credenciamento em sítio eletrônico, de modo a permitir ao cadastramento permanente de novos interessados obstando, por conseguinte, a fixação prévia de balizas temporais limitando o acesso de novos postulantes, especificamente quanto à contratação de leiloeiros oficiais, tal normatividade somente incide quando presente prova cabal da opção administrativa por essa modalidade de seleção pública na vigência da Nova Lei de Licitações e Contratações Administrativas, porquanto ausente igual obrigação nas disposições constantes da Lei n. 8.666/1993.

flashcard 33 - hemo/ptysis should be: hem/o/ptysis

flashcard 37 set 1 - hypopnea - has the correct phonetic pronunciation but the audio is incorrect. We must have missed this one in edition 1.

FC 54 - set 1 - nasopharyngeal - my reference only has RIN in capital letters; make je small letters for nasopharyngeal

(nā-zō-fa-RIN-jē-ăl)

In valores assoluto o in un anno?

Indeed

Hmm, it seems that the proposed algorithm may terminate prior to reaching n, having found the latest dependency.

Additionally, algorithm can be restructured to complete in one pass, as "go through the log until you have found all latest deps". Then algorithm will have time complexity up to O(n). If I'm not mistaken.

As an alternative, perhaps keeping indexes per each variable may provide interesting tradeoff of time for space. UPD: this technique is described in 3.3.1.

Embora o art. trate de exceção à indelegabilidade da competência tributária, deve ser entendido esta norma como previsão de delegação da capacidade tributária ativa. Isto é, enquanto a competência tributária diz respeito à criação de tributo, a capacidade tributária quer dizer sobre a arrecadação e fiscalização em relação aos tributos já instituídos.

Exemplo disso é a contribuição referente aos Conselhos Profissionais do art. 149 da Constituição Federal. Embora seja competência privativa da União, são os conselhos profissionais quem arrecadam e fiscalizam o pagamento do tributo.

Outro exemplo é o ITR. Embora seja a União quem institua e discipline a tributação do território rural, pode ser delegado ao município a competência para fiscalizar e arrecadar.

Ou seja, a criação de tributo, a competência tributária, é indelegável. Mas não é indelegável a capacidade tributária.

Tema Repetitivo 193

• Os Estados da Federação são partes legítimas para figurar no pólo passivo das ações propostas por servidores públicos estaduais, que visam o reconhecimento do direito à isenção ou à repetição do indébito relativo ao imposto de renda retido na fonte. -PROCESSO CIVIL E TRIBUTÁRIO. RECURSO ESPECIAL REPRESENTATIVO DE CONTROVÉRSIA. ART. 543-C, DO CPC. RESTITUIÇÃO. IMPOSTO DE RENDA RETIDO NA FONTE. LEGITIMIDADE PASSIVA DO ESTADO DA FEDERAÇÃO. REPARTIÇÃO DA RECEITA TRIBUTÁRIA.
• Os Estados da Federação são partes legítimas para figurar no pólo passivo das ações propostas por servidores públicos estaduais, que visam o reconhecimento do direito à isenção ou à repetição do indébito relativo ao imposto de renda retido na fonte. Precedentes.
• "O imposto de renda devido pelos servidores públicos da Administração direta e indireta, bem como de todos os pagamentos feitos pelos Estados e pelo Distrito Federal, retidos na fonte, irão para os cofres da unidade arrecadadora, e não para os cofres da União, já que, por determinação constitucional "pertencem aos Estados e ao Distrito Federal."

SÚMULA 447/STJ

• Os Estados e o Distrito Federal são partes legítimas na ação de restituição de imposto de renda retido na fonte proposta por seus servidores.

REsp 947206 / RJ

Tema Repetitivo 229 - A ação de repetição de indébito (...) visa à restituição de crédito tributário pago indevidamente ou a maior, por isso que o termo a quo é a data da extinção do crédito tributário, momento em que exsurge o direito de ação contra a Fazenda Pública, sendo certo que, por tratar-se de tributo sujeito ao lançamento de ofício, o prazo prescricional é quinquenal, nos termos do art. 168, I, do CTN

PROCESSO CIVIL E TRIBUTÁRIO. RECURSO ESPECIAL REPRESENTATIVO DE CONTROVÉRSIA. ART. 543-C, DO CPC. IPTU, TCLLP E TIP. INCONSTITUCIONALIDADE DA COBRANÇA DO IPTU PROGRESSIVO, DA TCLLP E DA TIP. AÇÃO ANULATÓRIA DE LANÇAMENTO FISCAL. CUMULADA COM REPETIÇÃO DE INDÉBITO. PRESCRIÇÃO. TERMO A QUO. ILEGITIMIDADE DO NOVO ADQUIRENTE QUE NÃO SUPORTOU O ÔNUS FINANCEIRO. VIOLAÇÃO AO ARTIGO 535 DO CPC. INOCORRÊNCIA. REDUÇÃO DOS HONORÁRIOS ADVOCATÍCIOS. SÚMULA 07 DO STJ. 1. O prazo prescricional adotado em sede de ação declaratória de nulidade de lançamentos tributários é qüinqüenal, nos moldes do art. 1º do Decreto 20.910/32. (Precedentes: AgRg no REsp 814.220/RJ, Rel. Ministra ELIANA CALMON, SEGUNDA TURMA, julgado em 19/11/2009, DJe 02/12/2009; AgRg nos EDcl no REsp 975.651/RJ, Rel. Ministro MAURO CAMPBELL MARQUES, SEGUNDA TURMA, julgado em 28/04/2009, DJe 15/05/2009; REsp 925.677/RJ, Rel. Ministro LUIZ FUX, PRIMEIRA TURMA, julgado em 21/08/2008, DJe 22/09/2008; AgRg no Ag 711.383/RJ, Rel. Min. DENISE ARRUDA, DJ 24.04.2006; REsp 755.882/RJ, Rel. Ministro FRANCISCO FALCÃO, DJ 18.12.2006)

1. Isto porque o escopo da demanda é a anulação total ou parcial de um crédito tributário constituído pela autoridade fiscal, mediante lançamento de ofício, em que o direito de ação contra a Fazenda Pública decorre da notificação desse lançamento.
2. A ação de repetição de indébito, ao revés, visa à restituição de crédito tributário pago indevidamente ou a maior, por isso que o termo a quo é a data da extinção do crédito tributário, momento em que exsurge o direito de ação contra a Fazenda Pública, sendo certo que, por tratar-se de tributo sujeito ao lançamento de ofício, o prazo prescricional é quinquenal, nos termos do art. 168, I, do CTN. (Precedentes: REsp 1086382/RS, Rel. Ministro LUIZ FUX, PRIMEIRA SEÇÃO, julgado em 14/04/2010, DJe 26/04/2010; AgRg nos EDcl no REsp 990.098/SP, Rel. Ministro BENEDITO GONÇALVES, PRIMEIRA TURMA, julgado em 09/02/2010, DJe 18/02/2010; AgRg no REsp 759.776/RJ, Rel. Ministro HERMAN BENJAMIN, SEGUNDA TURMA, julgado em 17/03/2009, DJe 20/04/2009; AgRg no REsp 1072339/SP, Rel. Ministro CASTRO MEIRA, SEGUNDA TURMA, julgado em 03/02/2009, DJe 17/02/2009)
3. In casu, os ora Recorridos ajuizaram ação anulatória dos lançamentos fiscais que constituíram créditos tributários relativos ao IPTU, TCLLP e TIP, cumuladamente com ação de repetição de indébito relativo aos mesmos tributos, referente aos exercícios de 1995 a 1999, sendo certo que o pedido principal é a restituição dos valores pagos indevidamente, razão pela qual resta afastada a regra do Decreto 20.910/32. É que a demanda foi ajuizada em 31/05/2000, objetivando a repetição do indébito referente ao IPTU, TCLLP, TIP e TCLD, dos exercícios de 1995 a 1999, ressoando inequívoca a inocorrência da prescrição quanto aos pagamentos efetuados posteriormente a 31/05/1995, consoante decidido na sentença e confirmado no acórdão recorrido.
4. O direito à repetição de indébito de IPTU cabe ao sujeito passivo que efetuou o pagamento indevido, ex vi do artigo 165, do Codex Tributário. "Ocorrendo transferência de titularidade do imóvel, não se transfere tacitamente ao novo proprietário o crédito referente ao pagamento indevido. Sistema que veda o locupletamento daquele que, mesmo tendo efetivado o recolhimento do tributo, não arcou com o seu ônus financeiro (CTN, art. 166). Com mais razão, vedada é a repetição em favor do novo proprietário que não pagou o tributo e nem suportou, direta ou indiretamente, o ônus financeiro correspondente." (REsp 593356/RJ, Relator p/ acórdão Ministro Teori Albino Zavascki, publicado no DJ de 12.09.2005).
5. O artigo 123, do CTN, prescreve que, "salvo disposições de lei em contrário, as convenções particulares, relativas à responsabilidade pelo pagamento de tributos, não podem ser opostas à Fazenda Pública, para modificar a definição legal do sujeito passivo das obrigações tributárias correspondentes".
6. Outrossim, na seção atinente ao pagamento indevido, o Código Tributário sobreleva o princípio de que, em se tratando de restituição de tributos, é de ser observado sobre quem recaiu o ônus financeiro, no afã de se evitar enriquecimento ilícito, salvo na hipótese em que existente autorização expressa do contribuinte que efetivou o recolhimento indevido, o que abrange a figura da cessão de crédito convencionada. (EREsp 708237/RJ, Rel. Ministro LUIZ FUX, PRIMEIRA SEÇÃO, DJ 27/08/2007). (Outros precedentes: REsp 892.997/RJ, Rel. Ministra ELIANA CALMON, SEGUNDA TURMA, julgado em 18/09/2008, DJe 21/10/2008; AgRg nos EREsp 778.162/SP, Rel. Ministro LUIZ FUX, PRIMEIRA SEÇÃO, julgado em 25/06/2008, DJe 01/09/2008; EREsp 761.525/RJ, Rel. Ministro HUMBERTO MARTINS, PRIMEIRA SEÇÃO, julgado em 26/03/2008, DJe 07/04/2008; AgRg no REsp 965.316/RJ, Rel. Ministro FRANCISCO FALCÃO, PRIMEIRA TURMA, julgado em 11/09/2007, DJ 11/10/2007)
7. In casu, as instâncias ordinárias decidiram pela legitimidade de todos os adquirentes para a ação de repetição de indébito relativo a créditos tributários anteriores à data da aquisição do imóvel, utilizando-se, contudo, de fundamentação inconclusiva quanto à existência ou não de autorização do alienante do imóvel, que efetivamente suportou o ônus do tributo.
8. A exegese da cláusula da escritura que transfere diretamente a ação ao novel adquirente deve ser empreendida no sentido de que esse direito é ação sobre o imóvel, referindo-se à transmissão da posse e da propriedade, como v.g., se o alienante tivesse ação possessória em curso ou a promover, não se aplicando aos tributos cuja transferência do jus actionis deve ser específica, o que não ocorreu in casu em relação a um dos autores.
9. O reexame dos critérios fáticos, sopesados de forma eqüitativa e levados em consideração para fixar os honorários advocatícios, nos termos das disposições dos parágrafos 3º e 4º do artigo 20, do CPC, em princípio, é inviável em sede de recurso especial, nos termos da jurisprudência dominante desta Corte. Isto porque a discussão acerca do quantum da verba honorária encontra-se no contexto fático-probatório dos autos, o que obsta o revolvimento do valor arbitrado nas instâncias ordinárias por este Superior Tribunal de Justiça. (Precedentes: AgRg no Ag 1107720/SP, Rel. Ministro HERMAN BENJAMIN, SEGUNDA TURMA, julgado em 16/03/2010, DJe 26/03/2010; AgRg no REsp 1144624/RR, Rel. Ministro HUMBERTO MARTINS, SEGUNDA TURMA, julgado em 15/12/2009, DJe 02/02/2010; REsp 638.974/SC, Rel. Ministro CARLOS FERNANDO MATHIAS (JUIZ CONVOCADO DO TRF 1ª REGIÃO), SEGUNDA TURMA, julgado em 25.03.2008, DJ 15.04.2008; AgRg no REsp 941.933/SP, Rel. Ministro JORGE MUSSI, QUINTA TURMA, julgado em 28.02.2008, DJ 31.03.2008; REsp 690.564/BA, Rel. Ministro JOÃO OTÁVIO DE NORONHA, SEGUNDA TURMA, julgado em 15.02.2007, DJ 30.05.2007).
10. O art. 535 do CPC resta incólume se o Tribunal de origem, embora sucintamente, pronuncia-se de forma clara e suficiente sobre a questão posta nos autos. Ademais, o magistrado não está obrigado a rebater, um a um, os argumentos trazidos pela parte, desde que os fundamentos utilizados tenham sido suficientes para embasar a decisão.
11. Recurso especial parcialmente provido, para reconhecer a ilegitimidade ativa ad causam da autora Ruth Raposo Pereira. Acórdão submetido ao regime do art. 543-C do CPC e da Resolução STJ 08/2008. Embargos de declaração dos recorridos prejudicados. (REsp n. 947.206/RJ, relator Ministro Luiz Fux, Primeira Seção, julgado em 13/10/2010, DJe de 26/10/2010.)

Art. 155. A concessão da moratória em caráter individual não gera direito adquirido e será revogado de ofício, sempre que se apure que o beneficiado não satisfazia ou deixou de satisfazer as condições ou não cumprira ou deixou de cumprir os requisitos para a concessão do favor, cobrando-se o crédito acrescido de juros de mora:

• I - com imposição da penalidade cabível, nos casos de dolo ou simulação do beneficiado, ou de terceiro em benefício daquele;
• II - sem imposição de penalidade, nos demais casos.
• Parágrafo único: No caso do inciso I deste artigo, o tempo decorrido entre a concessão da moratória e sua revogação não se computa para efeito da prescrição do direito à cobrança do crédito; no caso do inciso II deste artigo, a revogação só pode ocorrer antes de prescrito o referido direito.

PROCESSO CIVIL E TRIBUTÁRIO. AGRAVO INTERNO NO RECURSO ESPECIAL. ENUNCIADO ADMINISTRATIVO N. 3/STJ. ICMS. DECADÊNCIA DA OBRIGAÇÃO TRIBUTÁRIA. CREDITAMENTO INDEVIDO. DOLO. TERMO INICIAL. ARTIGO 173, I, DO CTN. PRECEDENTES. AGRAVO INTERNO PROVIDO. 1. Deveras, a obrigação tributária não declarada pelo contribuinte no tempo e modo determinados pela legislação de regência está sujeita ao procedimento de constituição do crédito pelo fisco, por meio do lançamento substitutivo, o qual deve se dar no prazo decadencial previsto no art. 173, I, do CTN, quando não houver pagamento antecipado, ou no art. 150, § 4º, do CTN, quando ocorrer o recolhimento de boa-fé, ainda que em valor menor do que aquele que a Administração entende devido, pois, nesse caso, a atividade exercida pelo contribuinte, de apurar, pagar e informar o crédito tributário, está sujeita à verificação pelo ente público, sem a qual ela é tacitamente homologada. A propósito, essa orientação também tem aplicação quando o pagamento parcial do tributo decorre de creditamento tido pelo fisco como indevido, hipótese dos autos. 2. Com efeito, no presente caso, conforme as provas contidas às fls. 649 (e-STJ), se depreendeu que os créditos apurados pelo contribuinte na composição do saldo devedor de ICMS, não eram passíveis de inclusão na escrituração contábil para a apuração do imposto devido, pois eram frutos de valores acostados em ação judicial ajuizada pelo contribuinte e outrora julgada improcedente. Portanto, era de conhecimento do contribuinte, a impossibilidade de se esciturar valores creditórios inexistentes, ao se apurar o saldo de ICMS no creditamento fiscal. 3. No presente caso, a Corte estadual compreendeu que o creditamento indevido não foi realizado de boa-fé, reconhecendo que a situação dos autos se enquadra na hipótese de exceção à aplicação do art. 150, § 4º, do CTN, relacionada com a existência de fraude, dolo ou simulação por parte do contribuinte, de forma que o prazo decadencial para lançamento do crédito segue a regra do art. 173, I, do CTN. Precedentes. 4. Agravo Interno provido. (AgInt no REsp n. 1.866.709/SP, relator Ministro Mauro Campbell Marques, Segunda Turma, julgado em 30/8/2021, DJe de 3/9/2021.)

TRIBUTÁRIO. ICMS. CREDITAMENTO INDEVIDO. LANÇAMENTO. DECADÊNCIA. CONTAGEM. TERMO INICIAL. PAGAMENTO PARCIAL DE BOA-FÉ: FATO GERADOR. MÁ-FÉ. RECONHECIMENTO. AUSÊNCIA. 1. A decadência para a realização de lançamento de ICMS pago a menor, inclusive quando fundado em creditamento indevido, deve ser contada de acordo com a regra contida no art. 150, § 4º, do CTN, exceto nos casos de dolo ou má-fé, em que deverá ser observado o disposto no art. 173, I, do CTN. 2. Hipótese em que, diversamente do assentado pelo ente público agravante, as instâncias ordinárias não afirmaram que a contribuinte agiu de má-fé ao realizar o creditamento glosado, mas, apenas, que não logrou comprovar a realização da operação estampada na nota fiscal posteriormente declarada inidônea. 3. A comprovação da operação comercial é condição para o adquirente de boa-fé proceder ao creditamento do ICMS destacado na correspondente nota fiscal posteriormente declarada inidônea (Súmula 509 do STJ). 4. A falta da referida comprovação não é suficiente para a caracterização de má-fé na conduta do contribuinte, cujo reconhecimento pressupõe juízo de valor fundado em prova específica, sendo inadmissível a presunção desse elemento subjetivo. 5. Agravo interno desprovido. (AgInt no REsp n. 1.978.830/SP, relator Ministro Gurgel de Faria, Primeira Turma, julgado em 25/4/2022, DJe de 27/4/2022.)

La ejecución de este código genera como resultado:

Como nos damos cuenta, hay algunos caractéres no imprimibles, debido a que falta instalar las tipografías en el sistema operativo, que las hacen visibles en resultado.

Que un caracter no sea imprimible dentro del Glamorous Toolkit, no quiere decir que no se pueda ver cuando se exporta a otros entornos, por ejemplo a la web.

Reviewer #1 (Public Review):

The authors deploy a combination of their own previously developed computational methods and databases (SIGNOR and CellNOptR) to model the FLT3 signaling landscape in AML and identify synergistic drug combinations that may overcome the resistance AML cells harboring ITD mutations in the TKI domain of FLT3 to FLT3 inhibitors. I did not closely evaluate the details of these computational models since they are outside of my area of expertise and have been previously published. The manuscript has significant issues with data interpretation and clarity, as detailed below, which, in my view, call into question the main conclusions of the paper.

The authors train the model by including perturbation data where TKI-resistant and TKI-sensitive cells are treated with various inhibitors and the activity (i.e. phosphorylation levels) of the key downstream nodes are evaluated. Specifically, in the Results section (p. 6) they state "TKIs sensitive and resistant cells were subjected to 16 experimental conditions, including TNFa and IGF1 stimulation, the presence or absence of the FLT3 inhibitor, midostaurin, and in combination with six small-molecule inhibitors targeting crucial kinases in our PKN (p38, JNK, PI3K, mTOR, MEK1/2 and GSK3)". I would appreciate more details on which specific inhibitors and concentrations were used for this experiment. More importantly, I was very puzzled by the fact that this training dataset appears to contain, among other conditions, the combination of midostaurin with JNK inhibition, i.e. the very combination of drugs that the authors later present as being predicted by their model to have a synergistic effect. Unless my interpretation of this is incorrect, it appears to be a "self-fulfilling prophecy", i.e. an inappropriate use of the same data in training and verification/test datasets.

My most significant criticism is that the proof-of-principle experiment evaluating the combination effects of midostaurin and SP600125 in FLT3-ITD-TKD cell line model does not appear to show any synergism, in my view. The authors' interpretation of the data is that the addition of SP600125 to midostaurin rescues midostaurin resistance and results in increased apoptosis and decreased viability of the midostaurin-resistant cells. Indeed, they write on p.9: "Strikingly, the combined treatment of JNK inhibitor (SP600125) and midostaurin (PKC412) significantly increased the percentage of FLT3ITD-TKD cells in apoptosis (Fig. 4D). Consistently, in these experimental conditions, we observed a significant reduction of proliferating FLT3ITD- TKD cells versus cells treated with midostaurin alone (Fig. 4E)." However, looking at Figs 4D and 4E, it appears that the effects of the midostaurin/SP600125 combination are virtually identical to SP600125 alone, and midostaurin provides no additional benefit. No p-values are provided to compare midostaurin+SP600125 to SP600125 alone but there seems to be no appreciable difference between the two by eye. In addition, the evaluation of synergism (versus additive effects) requires the use of specialized mathematical models (see for example Duarte and Vale, 2022). That said, I do not appreciate even an additive effect of midostaurin combined with SP600125 in the data presented.

In my view, there are significant issues with clarity and detail throughout the manuscript. For example, additional details and improved clarity are needed, in my view, with respect to the design and readouts of the signaling perturbation experiments (Methods, p. 15 and Fig 2B legend). For example, the Fig 2B legend states: "Schematic representation of the experimental design: FLT3 ITD-JMD and FLT3 ITD-JMD cells were cultured in starvation medium (w/o FBS) overnight and treated with selected kinase inhibitors for 90 minutes and IGF1 and TNFa for 10 minutes. Control cells are starved and treated with PKC412 for 90 minutes, while "untreated" cells are treated with IGF1 100ng/ml and TNFa 10ng/ml with PKC412 for 90 minutes.", which does not make sense to me. The "untreated" cells appear to be treated with more agents than the control cells. The logic behind cytokine stimulation is not adequately explained and it is not entirely clear to me whether the cytokines were used alone or in combination. Fig 2B is quite confusing overall, and it is not clear to me what the horizontal axis (i.e. columns of "experimental conditions", as opposed to "treatments") represents. The Method section states "Key cell signaling players were analyzed through the X-Map Luminex technology: we measured the analytes included in the MILLIPLEX assays" but the identities of the evaluated proteins are not given in the Methods. At the same time, the Results section states "TKIs sensitive and resistant cells were subjected to 16 experimental conditions" but these conditions do not appear to be listed (except in Supplementary data; and Fig 2B lists 9 conditions, not 16). In my subjective view, the manuscript would benefit from a clearer explanation and depiction of the experimental details and inhibitors used in the main text of the paper, as opposed to various Supplemental files/figures. The lack of clarity on what exactly were the experimental conditions makes the interpretation of Fig 2 very challenging. In the same vein, in the PCA analysis (Fig 2C) there seems to be no reference to the cytokine stimulation status while the authors claim that PC2 stratifies cells according to IGF1 vs TNFalpha. There are numerous other examples of incomplete or confusing legends and descriptions which, in my view, need to be addressed to make the paper more accessible.

I am not sure that I see significant value in the patient-specific logic models because they are not supported by empirical evidence. Treating primary cells from AML patients with relevant drug combinations would be a feasible and convincing way to validate the computational models and evaluate their potential benefit in the clinical setting.

eso eso eso eso eso

son puntos de vista opuestos

muy similar a lo que hemos leido ya

las diferencias socioeconómicas me interesan muchísimo... ¿la gente de clase alta también prefiere otras variedades a la variedad chilena? unas veces escuché a mi mamá chilena usando la theta, pero no sé si fue por su herencia español o por su deseo de aparecer como si fuera de clase alta-alta

SI PODEMOS

Trabalho intermitente. Lei nº 13.467/2017. Transcendência jurídica reconhecida. Adoção apenas em situações específicas. Interpretação que contrasta com a literalidade dos arts. 443, § 2º, e 452-A da CLT.

“(...) II) RECURSO DE REVISTA – RITO SUMARÍSSIMO – TRABALHO INTERMITENTE – MATÉRIA NOVA – TRANSCENDÊNCIA JURÍDICA – VIOLAÇÃO DO ART. 5º, II, DA CF – DESRESPEITO PATENTE À LEI 13.467/17, QUE INTRODUZIU OS ARTS. 443, § 3º, E 452- A NA CLT. 1. Constitui matéria nova no âmbito deste Tribunal, a ensejar o conhecimento de recurso de revista com base em sua transcendência jurídica (CLT, art. 896-A, § 1º, IV), aquela concernente ao regramento do trabalho intermitente, introduzido em nosso ordenamento jurídico pela Lei 13.467/17. 2. Discutida a matéria em recurso oriundo de processo submetido ao rito sumaríssimo, apenas por violação direta de dispositivo constitucional se pode conhecer do apelo, nos termos do § 9º do art. 896 da CLT. 3. É pacifica a jurisprudência do TST no sentido de que, excepcionalmente, pode-se conhecer de recurso de revista em rito sumaríssimo por violação ao princípio da legalidade insculpido no art. 5º, II, da CF, como forma de controle jurisdicional das decisões dos TRTs que deixarem flagrantemente de aplicar dispositivo legal que rege a matéria em debate (Precedentes de todas as Turmas, em variadas questões). 4. In casu, o 3º Regional reformou a sentença, que havia julgado improcedente a reclamatória, por entender que o trabalho intermitente "deve ser feito somente em caráter excepcional, ante a precarização dos direitos do trabalhador, e para atender demanda intermitente em pequenas empresas" e que "não é cabível ainda a utilização de contrato intermitente para atender posto de trabalho efetivo dentro da empresa". 5. Pelo prisma da doutrina pátria, excessos exegéticos assomam tanto nas fileiras dos que pretendem restringir o âmbito de aplicação da nova modalidade contratual, como nas dos que defendem sua generalização e maior flexibilidade, indo mais além do que a própria lei prevê. 6. Numa hermenêutica estrita, levando em conta a literalidade dos arts. 443, § 3º, e 452-A da CLT, que introduziram a normatização do trabalho intermitente no Brasil, tem-se como "intermitente o contrato de trabalho no qual a prestação de serviços, com subordinação, não é contínua, ocorrendo com alternância de períodos de prestação de serviços e de inatividade, determinados em horas, dias ou meses, independentemente do tipo de atividade do empregado e do empregador, exceto para os aeronautas, regidos por legislação própria" (§ 3º). Ou seja, não se limita a determinadas atividades ou empresas, nem a casos excepcionais. Ademais, fala-se em valor horário do salário mínimo ou daquele pago a empregados contratados sob modalidade distinta de contratação (CLT, art. 452-A). 7. Contrastando a decisão regional com os comandos legais supracitados, não poderia ser mais patente o desrespeito ao princípio da legalidade. O 3º Regional, refratário, como se percebe, à reforma trabalhista, cria mais parâmetros e limitações do que aqueles impostos pelo legislador ao trabalho intermitente, malferindo o princípio da legalidade, erigido pelo art. 5º, II, da CF como baluarte da segurança jurídica. 8. Ora, a introdução de regramento para o trabalho intermitente em nosso ordenamento jurídico deveu-se à necessidade de se conferir direitos básicos a uma infinidade de trabalhadores que se encontravam na informalidade (quase 50% da força de trabalho do país), vivendo de "bicos", sem carteira assinada e sem garantia de direitos trabalhistas fundamentais. Trata-se de uma das novas modalidades contratuais existentes no mundo, flexibilizando a forma de contratação e remuneração, de modo a combater o desemprego. Não gera precarização, mas segurança jurídica a trabalhadores e empregadores, com regras claras, que estimulam a criação de novos postos de trabalho. 9. Nesses termos, é de se acolher o apelo patronal, para restabelecer a sentença de improcedência da reclamatória trabalhista. Recurso de revista conhecido e provido.”

(TST-RR-10454-06.2018.5.03.0097, 4ª Turma, rel. Min. Ives Gandra da Silva Martins Filho, julgado em 7.8.2019) Informativo TST - nº 201

• ADI 1721
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CARLOS BRITTO
• Julgamento: 11/10/2006
• Publicação: 29/06/2007

Ementa EMENTA: AÇÃO DIRETA DE INCONSTITUCIONALIDADE. ARTIGO 3º DA MEDIDA PROVISÓRIA Nº 1.596-14/97, CONVERTIDA NA LEI Nº 9.528/97, QUE ADICIONOU AO ARTIGO 453 DA CONSOLIDAÇÃO DAS LEIS DO TRABALHO UM SEGUNDO PARÁGRAFO PARA EXTINGUIR O VÍNCULO EMPREGATÍCIO QUANDO DA CONCESSÃO DA APOSENTADORIA ESPONTÂNEA. PROCEDÊNCIA DA AÇÃO. 1. A conversão da medida provisória em lei prejudica o debate jurisdicional acerca da "relevância e urgência" dessa espécie de ato normativo. 2. Os valores sociais do trabalho constituem: a) fundamento da República Federativa do Brasil (inciso IV do artigo 1º da CF); b) alicerce da Ordem Econômica, que tem por finalidade assegurar a todos existência digna, conforme os ditames da justiça social, e, por um dos seus princípios, a busca do pleno emprego (artigo 170, caput e inciso VIII); c) base de toda a Ordem Social (artigo 193). Esse arcabouço principiológico, densificado em regras como a do inciso I do artigo 7º da Magna Carta e as do artigo 10 do ADCT/88, desvela um mandamento constitucional que perpassa toda relação de emprego, no sentido de sua desejada continuidade. 3. A Constituição Federal versa a aposentadoria como um benefício que se dá mediante o exercício regular de um direito. E o certo é que o regular exercício de um direito não é de colocar o seu titular numa situação jurídico-passiva de efeitos ainda mais drásticos do que aqueles que resultariam do cometimento de uma falta grave (sabido que, nesse caso, a ruptura do vínculo empregatício não opera automaticamente). 4. O direito à aposentadoria previdenciária, uma vez objetivamente constituído, se dá no âmago de uma relação jurídica entre o segurado do Sistema Geral de Previdência e o Instituto Nacional de Seguro Social. Às expensas, portanto, de um sistema atuarial-financeiro que é gerido por esse Instituto mesmo, e não às custas desse ou daquele empregador. 5. O Ordenamento Constitucional não autoriza o legislador ordinário a criar modalidade de rompimento automático do vínculo de emprego, em desfavor do trabalhador, na situação em que este apenas exercita o seu direito de aposentadoria espontânea, sem cometer deslize algum. 6. A mera concessão da aposentadoria voluntária ao trabalhador não tem por efeito extinguir, instantânea e automaticamente, o seu vínculo de emprego. 7. Inconstitucionalidade do § 2º do artigo 453 da Consolidação das Leis do Trabalho, introduzido pela Lei nº 9.528/97.

Observar Lei nº 605/49.

• Sum-146/TST. TRABALHO EM DOMINGOS E FERIADOS, NÃO COM-PENSADO

• O trabalho prestado em domingos e feriados, não compensado, deve ser pago em dobro, sem prejuízo da remuneração relativa ao repouso semanal.

• Precedentes:

• ERR 198.573/95.7

• [...] Não vislumbro qualquer vulneração ao art. 9º da Lei 605/49. Isto porque a melhor interpretação do referido dispositivo legal é exatamente no sentido de que deve ser pago em dobro a remuneração do trabalho realizado em dia de feriado.
• Ademais, a mens legis, é no sentido de que o empregado descanse pelo menos 1 (um) dia em cada semana.
• Assim, não se concebe que fosse estabelecer a lei o pagamento do trabalho em dia que deveria ser destinado ao repouso, da mesma maneira que o trabalho realizado em dias normais.

• [...] Em outras palavras, quando o empregador exige trabalho do empregado em dia de feriado, deverá remunerar de forma dobrada o trabalho prestado neste dia, sem prejuízo do pagamento do repouso semanal remunerado.

• RR 1482-81/79

• [...] Domina no TST jurisprudência consoante a decisão recorrida. É ilegal absorção das 11 horas de repouso entre jornadas pelas vinte e quatro horas de descanso semanal remunerado.

SUM-110 JORNADA DE TRABALHO. INTERVALO No regime de revezamento, as horas trabalhadas em seguida ao repouso semanal de 24 horas, com prejuízo do intervalo mínimo de 11 horas consecutivas para descanso entre jornadas, devem ser remuneradas como extraordinárias, inclusive com o respectivo adicional.

Recurso de Revista Repetitivo nº 9

Questão Submetida a Julgamento - A majoração do valor do repouso semanal remunerado, decorrente da integração das horas extras habituais, deve repercutir no cálculo das demais parcelas salariais?

Tese Firmada - A majoração do valor do repouso semanal remunerado, decorrente da integração das horas extras habituais, deve repercutir no cálculo das férias, da gratificação natalina, do aviso prévio e do FGTS, sem que se configure a ocorrência de bis in idem.

Situação do Tema: TRANSITADO JULGADO

O pagamento do salário é, em regra, mensal. No entanto, tal com os vendedores, a remuneração tida por comissão poderá ser ajustada para prazo não superior ao pagamento trimestral, conforme previsão da Lei 3.207/57

-> https://www.planalto.gov.br/ccivil_03/leis/l3207.htm

Art. 473 - O empregado poderá deixar de comparecer ao serviço sem prejuízo do salário: - I - até 2 (dois) dias consecutivos, em caso de falecimento do cônjuge, ascendente, descendente, irmão ou pessoa que, declarada em sua carteira de trabalho e previdência social, viva sob sua dependência econômica; - II - até 3 (três) dias consecutivos, em virtude de casamento; <br /> - III - por 5 (cinco) dias consecutivos, em caso de nascimento de filho, de adoção ou de guarda compartilhada; - IV - por um dia, em cada 12 (doze) meses de trabalho, em caso de doação voluntária de sangue devidamente comprovada;<br /> - V - até 2 (dois) dias consecutivos ou não, para o fim de se alistar eleitor, nos têrmos da lei respectiva; - VI - no período de tempo em que tiver de cumprir as exigências do Serviço Militar referidas na letra "c" do art. 65 da Lei nº 4.375, de 17 de agosto de 1964 (Lei do Serviço Militar). - VII - nos dias em que estiver comprovadamente realizando provas de exame vestibular para ingresso em estabelecimento de ensino superior - VIII - pelo tempo que se fizer necessário, quando tiver que comparecer a juízo - IX - pelo tempo que se fizer necessário, quando, na qualidade de representante de entidade sindical, estiver participando de reunião oficial de organismo internacional do qual o Brasil seja membro. <br /> - X - pelo tempo necessário para acompanhar sua esposa ou companheira em até 6 (seis) consultas médicas, ou em exames complementares, durante o período de gravidez; - XI - por 1 (um) dia por ano para acompanhar filho de até 6 (seis) anos em consulta médica. <br /> - XII - até 3 (três) dias, em cada 12 (doze) meses de trabalho, em caso de realização de exames preventivos de câncer devidamente comprovada. <br /> - Parágrafo único. O prazo a que se refere o inciso III do caput deste artigo será contado a partir da data de nascimento do filho.

¿son los mensajes binarios una forma de identificar los datos procesados? o solo los datos?

Los arreglos dinámicos y estáticos son importantes porque inspeccionan los movimientos o los arreglos realizados

las cadenas son un tipo de caracteres donde se puede denominar una palabra, una letra o algo que sea importante para los documentos o 'anotaciones', por ejemplo.

The fact that worms/arachnids are a key element of this reading is really fascinating—and it sort of seems to come out of nowhere. The spider, just as in these lines, is mentioned in the Webster reading, of course:

O men, / That lie upon your death-beds, and are haunted / With howling wives! ne'er trust them; they 'll re-marry / Ere the worm pierce your winding-sheet, ere the spider / Make a thin curtain for your epitaphs.

The "worm" is mentioned by Webster, too, though it is not brought up in Eliot's poem. However, it does share a common trait with a spider—silk: just as a worm can produce silk (i.e. the silkworm), so does a spider ("spider's silk.") Silk as a material may be too specific for what Eliot is referencing, but nonetheless, it can form the material for the "draperies" and the "seals" mentioned in these lines.

It's clear to see the depiction of contrast between each creature. On the one hand, the worm doesn't produce the silk for the draperies, the seals, the winding-sheets, or the curtains—but breaks them. In this way, the creatures may be shorthands for humans that deal with a body after death. A winding-sheet is a "a cloth in which a body is wrapped for burial" (Wikipedia). According to Webster, the worm quickly pierces this cloth—in a way, quickly breaking the period of rest for the dead—just as, per Eliot, the "lean solicitor" breaks the "seals." The worm is akin to the solicitor—that who manages the will/other documents of the dead.

On the other hand, the spider is "beneficent"—it is good, because it doesn't break but "drapes" and "makes a thin curtain" for the dead. One can look at what a drape is, exactly, in its purpose: drapes are often referenced to curtains, which are almost always translucent or opaque so as to block out sunlight. In like fashion, the "drapes" of "memories"—or the "thin curtains" over "epitaphs"—seek to block out the memories of someone who is dead; or blocking out the fact that they even lived a life (which is recorded by an epitaph). On the other hand, the spider can represent the most vile and evil of acts. As Isabel Su points out in a historical annotation, "male spiders basically trap/stalk sexually immature females to ensure that they are the first ones to mate with them, and then female spiders eat their partners after copulation." The theme of sexual violence is very present.

In addition, there's an interesting play between life and death here. The death, and the draping of memories, seems to erase the record that life was present at all. Instead, the solicitor—the worm—brings the dead "back to life" by invoking the memory that they even existed in the first place. Even more interesting is what this has to do with datta—with giving. Is the solicitor truly the giver of life even though all it gives are the "memories"? In some ways, this would be supported by the Bradley text: if life is merely an illusion, the only way for our souls to communicate would be if we were to believe, or remember, that they were present in the first place. The only way for one's life to truly have existed would be if other people saw it as such—if they were remembered.

Author Response

Reviewer #1 (Public Review):

I believe it is important for the authors to clarify how the time frames to test for group differences of ERP components were defined. Were the components defined based on a grand average across lesions and controls or based or on the maximum range for both groups? As the paper is written currently this is unclear to me. It is also unclear why the group comparisons between controls and lateral PFC group were based only on the control group. To ensure no inadvertent biases towards the larger control group were introduced and ensure the studies findings were reliable, it would be appreciated if the authors could clarify this.

We thank the reviewer for the helpful comment. We recognize the need for a clearer definition of time frames for testing group differences in the ERP components and apologize for any ambiguity in the previous version of the manuscript.

Regarding the time frames to test for group differences of ERP components for the OFC and control groups, they were determined based on the combined maximum range for both groups. The time range for each group and each ERP component was derived from the statistical analysis of the condition contrasts run for each group. For instance, for the Local Deviance MMN, the condition contrast (i.e., Control condition versus Local Deviance condition) for the CTR group revealed a MMN component from 67 to128 ms, while the same condition contrast for the OFC group revealed a MMN from 73 to131 ms. The time frame used for the group comparison on the MMN time window was 50 to 150 ms to capture component activity for both groups. In the same way, for the Local Deviance P3a, the condition contrast (i.e., Control condition versus Local Deviance condition) for the CTR group revealed a P3a component ranging from 141 to 313 ms, while the same condition contrast for the OFC group revealed a P3a from 145 to 344 ms. The time frame used for the group comparison on the P3a time window encompassed 140 to 350 ms to capture component activity for both groups.

In the “Results” section of the main manuscript, together with the results from the cluster-based permutation independent samples t-tests, we provide the time frames in which the latter were computed for each ERP component. These segments have been highlighted with yellow in the revised manuscript. Moreover, in the section “Materials and methods - Statistical analysis of event-related potentials” of the main manuscript [page 37, paragraph 2], we provide a revised description of how the time frames for group differences of ERPs were defined. The revised description states: “In a second step, to check for differences in the ERPs between the two main study groups, we ran the same cluster-based permutation approach contrasting each of the four conditions of interest between the two groups using independent samples t-tests. The cluster-based permutation independent samples t-tests were computed in the latency range of each component, which was determined based on the maximum range for both groups combined. The latency range for each group and component was based on the time frames derived from the statistical analysis of task condition contrasts.”

Regarding the comparisons between the lateral PFC and control groups, they were not based solely on the control group condition contrast. This was miswritten. The approach to define time frames to test for ERP differences between the CTR and the lateral PFC group was the same as the one used to test differences between CTR and OFC groups. We apologize for any confusion this may have caused. We have revised the erroneous statements in the Supplementary File 1 [highlighted text, page 9-10].

An additional potential weakness of the paper, and one that if addressed would increase our confidence that neural differences arise because of the specific lesion effect, is the lack of evidence that the lesion and control groups do not differ on measures that could inadvertently bias the neural data. For example, while the groups did not differ on demographics and a range of broad cognitive functions, were there any differences between the number or distribution of bad/noisy channels in each subject between the two groups? Were there differences in the number of blinks/saccades or distribution of blinks or saccades across the conditions in each subject across the two groups.

We thank the reviewer for this suggestion. We have completed a number of measurements and tests to ensure that the OFC lesion group and the control group did not differ on measures that could affect the neural data. First, we computed the number of bad/noisy channels for each subject and group, and found that the two groups did not differ significantly. Second, we computed the number of trials remaining after removing the noisy segments across conditions for each subject and group, and found no significant differences between the groups. Third, the number of blinks/saccades across conditions for each subject and group showed no significant group differences. Altogether, the results indicate that the neural differences observed in our study arose because of the specific lesion effect.

These additional EEG measures and the statistical test results are included in the Supplementary File 1 [page 15-16] and Supplementary File 1g. We have also added text in the section “Materials and methods - EEG acquisition and pre-processing” of the main manuscript [page 35, paragraph 3], which states: “To ensure the validity of the neural data analysis, potential sources of bias were assessed between the healthy control participants and the OFC lesion patients. Specifically, no significant differences were observed between the two groups in terms of the number of noisy channels, the number of noisy trials, or the number of blinks across the task blocks and the experimental conditions.”

On a similar note, while I appreciate this is a well established task could the authors clarify whether task difficulty is balanced across the different conditions? The authors appear to have used the counting task to ensure equal attention is paid across conditions although presumably the blocks differ in the number of deviant tones and therefore in the task difficulty. Typically, tasks to maintain attention are orthogonal to the main task and equally challenging across the different blocks. Is there a way to reassure readers that this has not affected the neural results?

Thank you for pointing this out. Indeed, the experimental blocks differ in the number of deviant tones and therefore in the task difficulty. Thus, it is a very good suggestion to look for behavioral performance differences across the different blocks. In the present set of analyses, two block types were used: Regular (xX) and Irregular (xY). In regular blocks, where the repeated sequence is xxxxx, participants were required to count the rare/uncommon sequences, i.e., xxxxy and xxxxo. In irregular blocks, where the repeated sequence is xxxxy, participants were required to count the rare/uncommon sequences, i.e., xxxxx and xxxxo. We have now updated the behavioral analysis. First, by excluding the omission block’s counting performance, and second, by calculating the counting performance separately for the two blocks. The new behavioral analysis revealed that participants from both groups performed better in the irregular block compared to the regular block. However, there was no statistically significant difference between the counting performances of the two groups.

The new results are reported on page 5 of the main manuscript, section “Results - Behavioral performance”, paragraph 1: “Participants from both groups performed the task properly with an average error rate of 9.54% (SD 8.97) for the healthy control participants (CTR) and 10.55% (SD 6.18) for the OFC lesion patients (OFC). There was no statistically significant difference between the counting performance of the two groups [F(24) = 0.11, P = 0.75]. Participants from both groups performed better in the irregular block (CTR: 8.39 ± 8.24%; OFC: 7.50 ± 7.34%) compared to the regular block (CTR: 10.69 ± 11.36%; OFC: 13.60 ± 10.97%) [F(24) = 3.55, P = 0.07]. There was no block X group interaction effect [F(24) = 0.73, P = 0.40].”

As with many patient lesion studies, while the comparison directly against the healthy age matched controls is critical it would have strengthened the authors claims if they could show differences between the brain damaged control group. Given the previous literature that also links lateral PFC with prediction error detection, I understand that this region is potentially not the clearest brain damaged control group and therefore another lesion group might have strengthened claims of specificity. Furthermore, the authors do not offer an explanation for why no differences between lateral PFC and control groups were found when others have previously reported them. Identifying those differences would strengthen our understanding of the involvement of different structures in this task/function.

We thank the reviewer for raising this crucial issue. We recognize the importance of addressing the lack of neurophysiological differences between the lateral PFC lesion group and the control group. First, it is important to clarify that the lateral PFC lesion control group was initially included not as a control for specific lateral PFC lesions but rather a broader control group to account for potentially general effects of frontal brain damage. However, considering that previous studies have implicated specific areas of the lateral PFC (e.g., inferior frontal gyrus; IFG) in predictive processing, we also think that a more thorough justification of these null findings is needed.

Intracranial EEG studies examining local and global level prediction error detection pointed to the role of inferior frontal gyrus (IFG) as a frontal source supporting top-down predictions in MMN generation (Dürschmid et al., 2016; Nourski et al., 2018; Phillips et al., 2016; Rosburg et al., 2005). However, other intracranial studies reported unclear (Bekinschtein et al., 2009) or weak (Dürschmid et al., 2016) frontal MMN effects. El Karoui et al. (2015) observed late ERP responses in the lateral PFC related to global deviants but no MMN to local deviants, and it was not clear where in the PFC these responses occurred, not showing responses in the IFG. Additionally, studies employing dynamic causal modeling of MMN consistently modeled frontal sources in the IFG region (Garrido et al., 2008; Garrido et al., 2009; Phillips et al., 2015). A review by Deouell (2007) highlighted the potential contributions of both IFG and middle frontal gyrus to MMN generation, suggesting that the specific source might vary depending on characteristics of the deviant stimuli, such as pitch or duration.

In Alho et al. (1994) lesion study, diminished MMN to local-level deviants was found after lesion to the lateral PFC, with the lesion cohort exhibiting a hemisphere ratio of 7/3 for left and right hemispheres, respectively, which is different from our cohort's ratio of 4/6. Furthermore, all individuals in that study had infarcts in the middle cerebral artery, resulting in a more uniform lesion location compared to our cohort. Notably, the lesions observed in our lateral PFC group appeared to be situated in more superior brain regions and towards the MFG compared to the predominantly reported involvement of the IFG in previous studies. Another factor that might contribute to the lack of significant effects is the heterogeneity of the lesions in our lateral PFC group (see Supplementary Figures 2, 3 and 4). Especially for the left hemisphere cohort, the individual lesions did not share a consistent anatomical location. The right hemisphere cohort had a greater lesion overlap, but overall, the lesions were not centered in the IFG area with highest overlap being in the MFG area. This distinction in lesion location might contribute to the absence of effects observed in our study.

Regarding the global effect, often reflected in the P300 component, it appears that the neural sources responsible for processing global deviance exhibit a more distributed pattern. This means that the brain regions involved in detecting and processing global deviations may not be as localized or concentrated as those implicated in local deviance processing. Given that the neural mechanisms underlying global deviance detection and processing are likely to involve a wider network of brain regions, they may be less susceptible to disruptions caused by focal lesions in the lateral PFC.

In response to your comment, we have expanded the “Discussion” to address this point by adding a new section titled “Lack of findings in the lateral PFC lesion group” [page 21]. In this section, we first present some of the findings implicating specific areas of the lateral PFC in the generation of MMN and in predictive processing, and then offer an account of the potential reasons behind the lack of neurophysiological differences between the lateral PFC and control groups.

Finally, while the authors have already cited widely across multiple fields, again speaking to the likely large impact the study will make, there does appear to be an unexplored conceptual link between the conclusions here that the OFC supports "the formation of predictions that define the current task by using context and temporal structure to allow old rules to be disregarded so that new ones can be rapidly acquired" and that lesions of the lateral portions of the OFC disrupt the assignment of credit or value to a stimuli that occurred temporally close to the outcome (Walton et al 2010, Noonan et al 2010, PNAS, Rudebeck et al 2017 Neuron, Noonan et al 2017, JON, Wittmann et al 2023 PlosB, note the wider imaging literature in line with this work Jocham et al 2014 Neuron and Wang et al bioRxiv). Without the OFC monkeys and humans appear to rely on an alternative, global learning mechanism that spreads the reinforcing properties of the outcome to stimuli that occurred further back in time. Could the authors speculate on how these two strains of evidence might converge? For example, does the OFC only assign credit in the event of a prediction error or does one mechanism subsume another?

We thank the reviewer for this comment regarding the unexplored conceptual link between our study’s conclusion, which suggests that the OFC facilitates the detection of prediction errors, and the findings of other research that delves into the OFC’s role in assignment of credit to stimuli. We find this comment very interesting and appreciate the opportunity to speculate on the potential functional convergence of these two processes within the OFC.

The OFC is a critical neural hub implicated in learning, decision-making, and adaptive behavior. The detection of prediction errors and the assignment of credit to stimuli are mechanisms linked with the OFC, which play an important role in all these functions (Noonan et al., 2012; Schultz & Dickinson, 2000; Sul et al., 2010; Tobler et al., 2006; Walton et al., 2010; Walton et al., 2011). Prediction errors involve recognizing discrepancies between expected and actual outcomes, which engages the OFC in rapidly updating stimulus valuations to align with newfound information (Holroyd & Coles, 2002; Kakade & Dayan, 2002). Signaling of errors provides a powerful mechanism whereby OFC facilitates adaptive learning and enables the brain to adjust its expectations based on novel experiences (Schultz, 2015; Seymour et al., 2004). Credit assignment, on the other hand, refers to properly identifying the causes of prediction errors. Without proper credit assignment, one might have intact error signaling mechanisms, but lose the ability to learn appropriately. This is especially true when multiple possible antecedents may be related to the error or when past choices have been unpredictable. In such situations, it is important to assign credit to the most recent choice and not get distracted by previous alternatives (Stalnaker et al., 2015).

These mechanisms within the OFC appear interrelated yet distinct. While prediction errors could trigger credit assignment, the OFC's ability to continually assess stimuli's values extends beyond instances of prediction errors. The OFC is involved in continuously evaluating and updating the values of stimuli based on ongoing experiences (Padoa-Schioppa & Assad, 2006; Tremblay & Schultz, 1999). This process enables the brain to learn from both unexpected outcomes and regular, predictable interactions with the environment. In situations where outcomes are not solely determined by prediction errors, the assignment of credit remains important. Complex decision-making involves considering a variety of factors beyond just prediction errors, such as contextual information and long-term consequences. Clarifying the convergence of these mechanisms within the OFC holds profound implications for understanding the intricacies of learning dynamics and the orchestration of adaptive responses to the environment.

While we recognize the value of this discussion, we believe it extends beyond the primary focus of our study. Consequently, we have made the decision not to incorporate it into the current manuscript.

One remaining weakness, which plagues all patient studies, is that of anatomical specificity. The authors have analysed what is, for the field, a large group of patients, and while the lesions appear to be relatively focused on the OFC the individuals vary in the degree to which different subregions within the OFC are damaged. This is increasingly important as evidence over the last 10 years has identified functional roles of these specific structures (Rushworth et al 2011, Neuron, Rudebeck et al 2017 Neuron). It would be important to ultimately know whether the detection of prediction errors was specific to a particular OFC subregion, a general mechanism across this area of cortex, or whether different subregions were more involved during different contexts or types of stimuli/contexts/tasks etc. Some comments on this would be appreciated.

The reviewer raised an important point here. It would have been interesting to explore this aspect. However, one challenge with focal lesion studies is to establish large patient cohorts. The group size of our study, which is relatively large compared to other studies of focal PFC lesions, does not allow us to perform any exploratory lesion-symptom mapping analyses. A larger patient sample will provide a stronger basis for drawing conclusions about the critical role of a particular OFC subregion to the detection of prediction errors and allow statistical approaches to lesion subclassification and brain-behavior analysis (e.g., voxel-based lesion-symptom mapping (Bates et al., 2003; Lorca-Puls et al., 2018)).

Considering the average percentage of damaged tissue in our study, the medial part of OFC or Brodmann area 11 is affected more by the lesion (approx. 33%), followed by the anterior-most region of the prefrontal cortex or Brodmann area 10 (approx. 25%), and the lateral portions of the OFC or Brodmann area 47 (approx. 12%). From our analysis, it is difficult to conclude whether the detection of prediction errors in our study was specific to a certain OFC area, or whether different subregions were involved more than others during different types of stimuli/contexts processing.

To provide a more balanced interpretation of our findings, we incorporated a section in the “Discussion”, titled “Limitations and future directions” [page 24-25], which delves into the limitations of our study and lesion studies generally with respect to anatomical specificity and the challenge to establish large patient cohorts.

Reviewer #2 (Public Review):

The current version of the manuscript is overall very long and verbose, for example, the introduction is 5 pages long and includes up to 102 references. In my view this is way too much. I suppose authors wish to be very detailed, but somehow they get an opposite effect, the main message of the introduction and aims get diluted.

We thank the reviewer for the feedback on our manuscript's length and content. This prompted us to carefully reconsider the balance between providing necessary context and ensuring the clarity of our main message. Our intention was to establish a strong foundation for our research by presenting relevant literature and setting the stage for our aims. In our revised manuscript, we have condensed the Introduction while retaining the key elements necessary to understand the context and motivations behind our research. Specifically, the current version of the “Introduction” is three pages long and includes 83 references.

I wonder if the presentation rate used, SOA; 150 is too fast and the stimuli too short 50 ms. Please prove a rationale for this.

We appreciate the reviewer's thoughtful consideration of the stimulus duration and presentation rate (SOA) used in our study. We understand the importance of providing a rationale for our choices to ensure the validity of our experimental design. The decision to use a SOA of 150 ms and stimuli of 50 ms duration was grounded in established practices and relevant literature in the field. Similar presentation rates and stimulus durations were employed in previous studies using similar auditory oddball paradigms, investigating rapid cognitive processes in combination with event-related potentials (ERPs). For instance, Bekinschtein et al. (2009) first introduced the task by using a SOA of 150 ms and stimulus duration of 50 ms, demonstrating that this combination is sensitive to detecting auditory deviations and eliciting early and late ERP components. Additionally, Wacongne et al. (2011), Chennu et al. (2013), Uhrig et al. (2014), and El Karoui et al. (2015) employed similar task designs with the same SOA and stimulus duration in combination with scalp EEG, fMRI and intracranial recordings, further supporting the validity of this approach. Other studies, employing the same paradigm, such as Chao et al. (2018) and Doricchi et al. (2021), used a SOA of 200 ms but kept the same stimulus duration of 50 ms.

One of the conditions is 'omissions', but results are not reported, so either authors do not mention this at all, or they report these data, which would be probably interesting.

We thank the reviewer for the nice reminder. The “omissions” condition is indeed an integral part of our study, and we acknowledge its potential significance. However, we have decided to publish the detailed analysis of the 'omissions' condition in a separate paper, because we think that such analysis and discussion would make the current paper quite dense and complicated. We apologize for any confusion that might arise from the absence of the 'omissions' results in this manuscript. On page 33 of the main manuscript, we state the reason for not including the “omissions” condition in the current analysis: “In the present set of analyses, the Omission blocks were not further examined, because such analysis and discussion would make the current paper overly dense and complicated.”

The Discussion is very long and in some aspect even too speculative. For example, in the conclusions authors claim that the OFC contributes to a top-down predictive process that modulates the deviance detection system in the primary auditory cortices and may be involved in connecting PEs at lower hierarchical areas with predictions at higher areas. I am not sure the current data support this. This would-be probably more appropriate if they could compare results from OFC and AC etc. so it is a more dynamic study.

We thank the reviewer for this observation. We have made revisions to shorten and refine the discussion, with a primary focus on presenting and interpreting the key results in a more concise and straightforward manner (See tracked changes in the revised manuscript).

However, the overall length of the Discussion has not been reduced significantly because we have introduced two additional sections within the Discussion (i.e., “Lack of findings in the lateral PFC lesion group” and “Limitations and future directions”) in response to reviewers’ request to address the lack of finding in the lateral PFC lesion group and certain limitations associated with the employed lesion method.

We also agree that the claim mentioned by the reviewer is overly too speculative and therefore revised the sentence as follows [page 38, “Conclusion”]: “We suggest that the OFC likely contributes to a top-down predictive process that modulates the deviance detection system in lower sensory areas.”

At the beginning of Discussion, the authors mention that overall, these findings provide novel information about the role of the OFC in detecting violation of auditory prediction at two levels of stimuli abstraction/time scale. I think this needs to be detailed more specifically rather than mention they provide novel results.

We understand the importance of providing readers with precise descriptions about the novelty of our study. Therefore, we have revised the statement to provide more detailed information about the novel contributions offered by our study. The revised text states as follows [“Discussion”, page 18,]: “These findings indicate that the OFC is causally involved in the detection of local and local + global auditory PEs, thus providing a novel perspective on the role of OFC in predictive processing.”

I am not sure I like to have a section as a general discussion within the discussion itself, probably this heading should be reformatted to be more specific to what is discussed.

As suggested by the reviewer, we reformatted the heading to “OFC and hierarchical predictive processing” [page 22-24] to better capture the essence of the content covered in this section of the “Discussion”. Here, we discuss the functional relevance of our EEG findings under the umbrella of the predictive coding framework and the potential role of OFC in predictive processes (See tracked changes in the revised manuscript).

Reviewer #3 (Public Review):

The central claim of the study is that hierarchical predictive processing is altered in OFC patients. However, OFC patients were able to identify global deviants as well as controls. Thus, hierarchical predictive processing itself seems to be unaltered, even though its neural correlates were different. This begs the question of what exactly the functional meaning of the EEG findings is. From the evidence presented this is difficult to determine for three reasons (See comments below).

We thank the reviewer for the detailed observations and valuable comments. The reviewer points out that hierarchical predictive processing is unaltered even though the neural correlates were altered, because OFC patients were able to identify global deviants as accurately as control participants. We respectfully disagree with the reviewer’s claim for two reasons: 1) The primary purpose of the behavioral data in this study was not to measure the participants’ deviant detection performance, but to confirm that they were paying attention to the global rule of each block. However, we agree that an effect of lesion on behavioral performance would strengthen the claim of altered high-level predictive processing. Your point highlights the importance of looking more carefully at our behavioral results. In a follow up study, which we are currently running, we explore the behavioral nuances of our task by measuring reaction times of correct deviant detections. 2) Earlier lesion studies reported typical performance on simple oddball tasks for patients with focal frontal lesions that did not significantly differ from control participants. However, despite normal task execution and neuropsychological profiles, patients with LPFC and OFC lesions present distinct neurophysiological evidence of alterations in novelty processing (Knight, 1984, 1997; Knight & Scabini, 1998; Løvstad et al., 2012; Yamaguchi & Knight, 1991).

Regarding the central claim of our study being that hierarchical predictive processing is altered in OFC patients, we have tried not to make strong claims about our results showing altered hierarchical predictive processing. For example, the conclusion of the abstract states: “the altered magnitudes and time courses of MMN/P3a responses after lesions to the OFC indicate that the neural correlates of detection of auditory regularity violation is impacted at two hierarchical levels of stimuli abstraction.” Thus, we do not claim that detection of regularity violation is directly impaired (e.g., OFC patients were able to identify global deviants as well as healthy controls) but that the neural correlates of deviants’ detection are altered, and therefore impaired.

Finally, we have gone through all the comments/reasons, which the reviewer believes are difficult to determine the functional meaning of our EEG findings, and addressed them one by one (see comments below). We hope that the revised manuscript has been improved accordingly and provides a more critical view on the extent to which the findings support hierarchical predictive coding.

It is possible that the shifts in scalp potentials are due to volume conduction differences linked to post-lesion changes in neural tissue and anatomy rather than differences in information processing per se.

We appreciate your comment regarding the potential influence of volume conduction differences on the observed shifts in scalp potentials in our study. We acknowledge that there are special challenges in interpreting ERP findings in brain lesion populations (Kutas et al., 2012; Rugg, 1995). To reliably interpret changes in the ERPs in lesion patients as reflecting impairments in certain cognitive processes, it is necessary to identify factors that might possibly affect the results and to apply the appropriate control measures. As noted by the reviewer, structural pathology, and the replacement of neural tissue by cerebrospinal fluid following tumor resection, likely causes inhomogeneities in the volume conduction of electrical activity and resulting changes in current flow patterns. Moreover, post-craniotomy skull defects can cause local inhomogeneities in the resistive properties of the skull (Løvstad & Cawley, 2011; Rugg, 1995). Both types of biophysical changes might alter the amplitude levels and/or topography (by altering the configuration of the generators) of surface-recorded ERPs (e.g., Swick (2005)). Consequently, caution is warranted when comparing the ERPs and their scalp distributions of intact and brain-lesioned groups. It is difficult to directly quantify the consequences of brain lesions on tissue conductivity. To conclude that ERP differences between patients and controls reflect functional abnormalities in particular cognitive processes, and not primarily nonspecific effects of structural brain damage, it is helpful to demonstrate that they are specific to certain ERP components/stages of information processing and task conditions. Changes confined to one or a subset of ERP components, that additionally may not manifest across all task conditions, can give some indication concerning the specificity of ERP changes (Kutas et al., 2012; Swaab, 1998). In our study, group differences pertaining to ERP amplitudes were limited to specific task conditions and not across all data. This condition-dependent pattern suggests that the observed shifts are related to the specific cognitive processes engaged during those task conditions rather than being a global artifact of volume conduction. If volume conduction was the main driver, we would expect these group differences to be more uniformly present across task conditions. Another piece of evidence against volume conduction effects is the scalp potentials’ latency differences between the two groups observed for the Local + Global deviance detection. Group differences in the latencies of ERPs, such as the MMN and P3a, cannot be attributed to volume conduction alone (Hämäläinen et al., 1993). These differences in the timing of neural responses strongly indicate genuine variations in cognitive processing.

To provide a more balanced interpretation of our findings, we have incorporated a section in the “Discussion” that delves into the limitations of our study and lesion studies generally with respect to volume conduction and amplitude changes, titled “Limitations and future directions” [page 24-25].

It is unclear from the analyses whether the P3a amplitude differences are true amplitude differences or a byproduct of latency differences. The reason is that the statistical method used (cluster based permutations) might yield significant effects when the latency of a component is shifted, even if peak amplitudes are the same. Complementary analyses on mean or peak amplitudes could resolve this issue.

We thank the reviewer for raising an important concern about the use of cluster-based permutation tests and their potential to yield significant effects when the latency of a component is shifted. We acknowledge this concern and recognize the need for complementary analyses to address this issue. To provide a clearer understanding of the nature of the observed ERP amplitude differences, we conducted complementary analyses on mean amplitudes of the MMN and P3a components on the midline sensors for the conditions where significant group differences were observed. For the MMN component elicited by the Local Deviance, we found group amplitude differences on the electrodes AFz (p = 0.021), Fz (p = 0.008), CPz (p = 0.015), and Pz (p < 0.001). Surprisingly, we also found amplitude differences for the P3a component elicited by the Local Deviance on the electrodes AFz (p < 0.001), Fz (p < 0.001), FCz (p < 0.001), and Cz (p = 0.002) that were not observed previously with the cluster-based permutation analysis. For the MMN component elicited by the Local+Global Deviance, our analysis showed group amplitude differences on the electrodes AFz (p = 0.007), FCz (p = 0.051), Cz (p = 0.004), CPz (p = 0.002), and Pz (p < 0.001). However, as the reviewer rightly pointed out, the group differences for the P3a elicited by the Local + Global Deviance seem to be a byproduct of latency differences, as we did not find amplitude differences on any of the midline electrodes. Overall, this complementary analysis shows that the OFC patients had an attenuated MMN/P3a to local level prediction violation, and an attenuated and delayed MMN followed by a delayed P3a to the combined local and global level prediction violation. The new analysis is added in the Supplementary File 1 [page 5-7] and Supplementary File 1c and 1d.

The MMN, P3a and P3b components are difficult to map to the hierarchical PC theory. Traditionally, the MMN is ascribed to lower level processing while P3a and P3b are ascribed to higher level processing. However, the picture is more complicated. For example, the current results show that the MMN is enhanced in local + global surprise while the P3a is elicited by local surprise. Furthermore, the P3a is classically interpreted as reflecting attention reorientation and the P3b as reflecting the conscious detection of task-relevant targets. How attention and conscious awareness fit in hierarchical PC is not entirely clear.

Indeed, the relationships between MMN, P3a and P3b components and the predictive coding (PC) framework can be intricate. However, numerous studies employed the PC theory to interpret these common electrophysiological signatures as prediction error (PE) signals (Garrido et al., 2007, 2009; Lieder et al., 2013) and dissociations between these ERPs supported that there are successive levels of predictive processing (Chennu et al., 2013; El Karoui et al., 2015; Wacongne et al., 2011).

In terms of hierarchical PC (Friston, 2005), the temporally constrained MMN has been traditionally linked with first-level predictive processing, known as the local effect of short-term stimulus deviance. PE signals at this level feed forward to a temporally extended, attention-dependent system that extracts longer-term patterns. PE signals at the higher level are usually indexed by the P300, identified as the global effect of longer-term stimulus deviance. The P300 reflects a more attention-driven process, emerging in response to novel or low-probability “target” stimuli that violate broader contextual expectations (Polich, 2007), such as those that form over multiple trials. Because the MMN, P3a and P3b also appear to exhibit varying degrees of sensitivity to preconscious and conscious perceptual predictions (Sculthorpe et al., 2009), they could serve as measures for examining the concept of a predictive neural hierarchy.

Indeed, the MMN has been viewed as sensitive to local violation and essentially blind to higher-order regularities. However, this is a simplified view. For example, Wacongne et al. (2011) showed that violating a low-level perceptual expectation triggers the MMN, violating contextual expectations triggers the higher-level P3, and when both expectations are simultaneously violated, a larger response is evoked compared to either one alone. These findings, which are consistent with the results of our study, show that the local and global effects are not fully independent but interact in an early time window, indexed by enhanced and temporally extended MMN responses. They provide support not just for a hierarchical model, but for a predictive rather than a feedforward one. Moreover, the MMN has been found to be relatively insensitive to attention, because it is elicited in situations in which the subjects’ attention is directed away from the stimuli and there are no task demands (Chennu et al., 2013). Given that early MMN is a pre-attentive automatic ERP component (Näätänen et al., 2001; Pegado et al., 2010; Tiitinen et al., 1994), and given that it has been observed in comatose and vegetative state patients (Bekinschtein et al., 2009; Fischer et al., 2004; Naccache et al., 2004), the finding that even early MMN is impaired in OFC patients indicate that patients may suffer from a deficit in sensory predictive processing that is independent of attention and conscious awareness.

The picture is more complicated when it comes to the predictive roles of P3a and P3b components. Following the MMN, a positive polarity P300 complex, sensitive to the detection of unpredicted auditory events, has been reported (Chennu et al., 2013; Doricchi et al., 2021; Kompus et al., 2020; Liaukovich et al., 2022). However, the two types of P300 (P3a and P3b) have not been clearly fitted into the hierarchical PC theory. The P3a is considered to be part of the brain's mechanism for detecting PEs (Wessel et al., 2012; Wessel et al., 2014) and may indicate that the brain is reallocating attentional resources to process and learn from these unexpected events. The P3a is typically interpreted as reflecting an involuntary attentional reorienting process (Escera & Corral, 2007; Ungan et al., 2019), which may relate to the operations of the ventral attention network (Corbetta et al., 2008; Corbetta & Shulman, 2002; Nieuwenhuis et al., 2005). Predictive coding emphasizes the role of contextual information in generating predictions with P3a being influenced by the context in which an unexpected event occurs (Schomaker et al., 2014). In the hierarchy of predictive processing, the P3a may reflect PEs at different hierarchical levels, depending on the complexity of the prediction and the degree to which it deviates from the sensory input. On the other hand, the P3b is linked to higher-level cognitive processes that involve updating long-term predictions based on incoming sensory information. It is highly dependent on attention, conscious awareness and active engagement with the task (Bekinschtein et al., 2009; Del Cul et al., 2007; Sergent et al., 2005; Strauss et al., 2015). It is thought to play a role in integrating the unexpected sensory input into the current context, potentially leading to updates of predictions in working memory (Chao et al., 1995; Donchin & Coles, 1988; Polich, 2007).

Hierarchical PC theory is continually evolving, and the relationship between these ERP components and attention or conscious awareness remains an active area of research. We acknowledge the need for further investigation to better understand how attention and conscious awareness fit within this framework. In light of your comment, we provide a more comprehensive discussion about the functional meaning of the EEG findings in our “Discussion - OFC and hierarchical predictive processing” [page 22-24].

The fact that lateral PFC patients show unaltered neural responses contradicts prominent views from PC identifying this region as a generator of the MMN and a source of predictions sent to temporal auditory areas.

We appreciate the reviewer's comment and want to acknowledge that another reviewer raised this concern previously. We have provided a detailed response to this issue in our previous response (see Response to Reviewer #1 Comment 4). We have expanded the “Discussion” to address this point by adding a new section titled “Lack of findings in the lateral PFC lesion group” [page 21]. In this section, we first present some of the findings implicating specific areas of the lateral PFC in the generation of MMN and in predictive processing, and then offer an account of the potential reasons behind the lack of neurophysiological differences between the lateral PFC and control groups.

For these reasons, a more critical view on the extent to which the findings support hierarchical predictive coding is needed.

By responding to the reviewer’s previous comments (i.e., the reasons why the reviewer thinks it is difficult to determine the functional meaning of the EEG findings), we believe that we have offered a more critical view on this matter.

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Tema Repetitivo nº 1.013

PREVIDENCIÁRIO. RECURSO ESPECIAL REPRESENTATIVO DA CONTROVÉRSIA. ARTS. 1.036 E SEGUINTES DO CPC/2015. TEMA REPETITIVO 1.013/STJ. BENEFÍCIO POR INCAPACIDADE. APOSENTADORIA POR INVALIDEZ E AUXÍLIO-DOENÇA. DEMORA NA IMPLEMENTAÇÃO DO BENEFÍCIO. EXERCÍCIO DE ATIVIDADE REMUNERADA PELO SEGURADO. NECESSIDADE DE SUBSISTÊNCIA DO SEGURADO. FUNÇÃO SUBSTITUTIVA DA RENDA NÃO CONSUBSTANCIADA. POSSIBILIDADE DE RECEBIMENTO CONJUNTO DA RENDA DO TRABALHO E DAS PARCELAS RETROATIVAS DO BENEFÍCIO ATÉ A EFETIVA IMPLANTAÇÃO. TESE REPETITIVA FIXADA. IDENTIFICAÇÃO E DELIMITAÇÃO DA CONTROVÉRSIA

1. O tema repetitivo ora controvertido consiste em definir a "possibilidade de recebimento de benefício, por incapacidade, do Regime Geral de Previdência Social, de caráter substitutivo da renda (auxílio-doença ou aposentadoria por invalidez), concedido judicialmente em período de abrangência concomitante àquele em que o segurado estava trabalhando e aguardava o deferimento do benefício. "

2. Os fatos constatados no presente Recurso Especial consistem cronologicamente em: a) o segurado teve indeferido benefício por incapacidade (auxílio-doença ou aposentadoria por invalidez) na via administrativa; b) para prover seu sustento, trabalhou após o indeferimento e entrou com ação judicial para a concessão de benefício por incapacidade; c) a ação foi julgada procedente para conceder o benefício desde o requerimento administrativo, o que acabou por abranger o período de tempo em que o segurado trabalhou; e d) o debate, travado ainda na fase ordinária, consiste no entendimento do INSS de que o benefício por incapacidade concedido judicialmente não pode ser pago no período em que o segurado estava trabalhando, ante seu caráter substitutivo da renda e à luz dos arts. 42, 46 e 59 da Lei 8.213/1991.

3. A presente controvérsia e, consequentemente, a tese repetitiva que for fixada não abrangem as seguintes hipóteses:

4. 3.1. O segurado está recebendo regularmente benefício por incapacidade e passa a exercer atividade remunerada incompatível com sua incapacidade, em que não há o caráter da necessidade de sobrevivência como elemento que justifique a cumulação, e a função substitutiva da renda do segurado é implementada de forma eficaz. Outro aspecto que pode ser analisado sob perspectiva diferente é o relativo à boa-fé do segurado. Há jurisprudência das duas Turmas da Primeira Seção que analisa essa hipótese, tendo prevalecido a compreensão de que há incompatibilidade no recebimento conjunto das verbas. A exemplo: AgInt no REsp 1.597.369/SC, Rel. Ministra Regina Helena Costa, Primeira Turma, DJe 13.4.2018; REsp 1.454.163/RJ, Rel. Ministro Mauro Campbell Marques, Segunda Turma, DJe 18.12.2015; e REsp 1.554.318/SP, Rel. Ministro Herman Benjamin, Segunda Turma, DJe 2.9.2016.
5. 3.2. O INSS alega o fato impeditivo do direito (o exercício de trabalho pelo segurado) somente na fase de cumprimento da sentença, pois há elementos de natureza processual prejudiciais à presente tese a serem considerados, notadamente a aplicabilidade da tese repetitiva fixada no REsp 1.253.513/AL (Rel. Ministro Castro Meira, Primeira Seção, DJe de 20.8.2012). RESOLUÇÃO DA TESE CONTROVERTIDA
6. Alguns benefícios previdenciários possuem a função substitutiva da renda auferida pelo segurado em decorrência do seu trabalho, como mencionado nos arts. 2º, VI, e 33 da Lei 8.213/1991. Em algumas hipóteses, a substitutividade é abrandada, como no caso de ser possível a volta ao trabalho após a aposentadoria por tempo de contribuição (art. 18, § 2º, da Lei 8.213/1991). Em outras, a substitutividade resulta na incompatibilidade entre as duas situações (benefício e atividade remunerada), como ocorre com os benefícios auxílio-doença por incapacidade e aposentadoria por invalidez.
7. Desses casos de auxílio-doença e aposentadoria por invalidez, é pressuposto que a incapacidade total para o trabalho seja temporária ou definitiva, respectivamente.
8. Como consequência, o Regime Geral de Previdência Social arca com os citados benefícios por incapacidade para consubstanciar a função substitutiva da renda, de forma que o segurado que não pode trabalhar proveja seu sustento.
9. A cobertura previdenciária, suportada pelo regime contributivo solidário, é o provimento do sustento do segurado enquanto estiver incapaz para o trabalho.
10. É decorrência lógica da natureza dos benefícios por incapacidade, substitutivos da renda, que a volta ao trabalho seja, em regra, causa automática de cessação desses benefícios, como se infere do requisito da incapacidade total previsto nos arts. 42 e 59 da Lei 8.213/1991, com ressalva ao auxílio-doença.
11. No caso de aposentadoria por invalidez, o art. 42 da Lei de Benefícios da Previdência Social (LBPS) estabelece como requisito a incapacidade "para o exercício de atividade que lhe garanta a subsistência", e, assim, a volta a qualquer atividade resulta no automático cancelamento do benefício (art. 46).
12. Já o auxílio-doença estabelece como requisito (art. 59) que o segurado esteja "incapacitado para o seu trabalho ou para a sua atividade habitual". Desse modo, a função substitutiva do auxílio-doença é restrita às duas hipóteses, fora das quais o segurado poderá trabalhar em atividade não limitada por sua incapacidade.
13. Alinhada a essa compreensão, já implícita desde a redação original da Lei 8.213/1991, a Lei 13.135/2015 incluiu os §§ 6º e 7º no art. 60 daquela, com a seguinte redação (grifos acrescentados): "§ 6º O segurado que durante o gozo do auxílio-doença vier a exercer atividade que lhe garanta subsistência poderá ter o benefício cancelado a partir do retorno à atividade. § 7º Na hipótese do § 6º, caso o segurado, durante o gozo do auxílio-doença, venha a exercer atividade diversa daquela que gerou o benefício, deverá ser verificada a incapacidade para cada uma das atividades exercidas."
14. Apresentado esse panorama legal sobre o tema, importa estabelecer o ponto diferencial entre a hipótese fática dos autos e aquela tratada na lei: aqui o segurado requereu o benefício, que lhe foi indeferido, e acabou trabalhando enquanto não obteve seu direito na via judicial; já a lei trata da situação em que o benefício é concedido, e o segurado volta a trabalhar.
15. A presente controvérsia cuida de caso, portanto, em que falhou a função substitutiva da renda, base da cobertura previdenciária dos benefícios auxílio-doença e aposentadoria por invalidez.

16. O provimento do sustento do segurado não se materializou, no exato momento da incapacidade, por falha administrativa do INSS, que indeferiu incorretamente o benefício, sendo inexigível do segurado que aguarde a efetivação da tutela jurisdicional sem que busque, pelo trabalho, o suprimento da sua subsistência.

17. Por culpa do INSS, resultado do equivocado indeferimento do benefício, o segurado teve de trabalhar, incapacitado, para o provimento de suas necessidades básicas, o que doutrinária e jurisprudencialmente convencionou-se chamar de sobre-esforço. Assim, a remuneração por esse trabalho tem resultado inafastável da justa contraprestação pecuniária.

18. Na hipótese, o princípio da vedação do enriquecimento sem causa atua contra a autarquia previdenciária, pois, por culpa sua - indeferimento equivocado do benefício por incapacidade -, o segurado foi privado da efetivação da função substitutiva da renda laboral, objeto da cobertura previdenciária, inerente aos mencionados benefícios.

19. Como tempero do elemento volitivo do segurado, constata-se objetivamente que, ao trabalhar enquanto espera a concessão de benefício por incapacidade, está ele atuando de boa-fé, cláusula geral hodiernamente fortalecida na regência das relações de direito.

20. Assim, enquanto a função substitutiva da renda do trabalho não for materializada pelo efetivo pagamento do auxílio-doença ou da aposentadoria por invalidez, é legítimo que o segurado exerça atividade remunerada para sua subsistência, independentemente do exame da compatibilidade dessa atividade com a incapacidade laboral.

21. No mesmo sentido do entendimento aqui defendido: AgInt no AREsp 1.415.347/SP, Rel. Ministro Francisco Falcão, Segunda Turma, Je de 28.10.2019; REsp 1.745.633/PR, Rel. Ministro Francisco Falcão, Segunda Turma, DJe de 18.3.2019; AgInt no REsp 1.669.033/SP, Rel. Ministro Napoleão Nunes Maia Filho, Primeira Turma, DJe de 30.8.2018; REsp 1.573.146/SP, Rel. Ministro Napoleão Nunes Maia Filho, Primeira Turma, DJe de 13.11.2017; AgInt no AgInt no AREsp 1.170.040/SP, Rel. Ministro Sérgio Kukina, Primeira Turma, DJe de 10.10.2018; AgInt no REsp 1.620.697/SP, Rel. Ministro Benedito Gonçalves, Primeira Turma, DJe de 2.8.2018; AgInt no AREsp 1.393.909/SP, Rel. Ministro Gurgel de Faria, Primeira Turma, DJe de 6.6.2019; e REsp 1.724.369/SP, Rel. Ministro Herman Benjamin, Segunda Turma, DJe de 25.5.2018. FIXAÇÃO DA TESE REPETITIVA

22. O Tema Repetitivo 1.013/STJ é assim resolvido: "No período entre o indeferimento administrativo e a efetiva implantação de auxílio-doença ou de aposentadoria por invalidez, mediante decisão judicial, o segurado do RPGS tem direito ao recebimento conjunto das rendas do trabalho exercido, ainda que incompatível com sua incapacidade laboral, e do respectivo benefício previdenciário pago retroativamente." RESOLUÇÃO DO CASO CONCRETO

23. Ao Recurso Especial deve-se negar provimento, pois o Tribunal de origem julgou o presente caso no mesmo sentido do entendimento aqui proposto (fl. 142-143/e-STJ): "A permanência do segurado no exercício das atividades laborativas decorre da necessidade de prover sua subsistência enquanto a administração ou o Judiciário não reconheça sua incapacidade, não obstando a concessão do benefício vindicado durante a incapacidade."
24. Consubstanciado o que previsto no Enunciado Administrativo 7/STJ, o recorrente é condenado ao pagamento de honorários advocatícios de 10% (dez por cento) sobre o valor total da verba sucumbencial fixada nas instâncias ordinárias, com base no § 11 do art. 85 do CPC/2015. CONCLUSÃO 23. Recurso Especial não provido, sob o rito dos arts. 1.036 e seguintes do CPC/2015. (REsp n. 1.786.590/SP, relator Ministro Herman Benjamin, Primeira Seção, julgado em 24/6/2020, DJe de 1/7/2020.)

This is something I would like o improve on from last project

Manganês é um elemento químico de símbolo Mn e número atômico 25. É um metal de transição encontrado na tabela periódica. O manganês é conhecido por sua alta resistência à corrosão e sua capacidade de formar ligas com outros metais. É amplamente utilizado na indústria, especialmente na produção de aço, onde é adicionado para melhorar a resistência e a dureza. Também é utilizado em baterias recarregáveis, na produção de fertilizantes e na fabricação de pigmentos e corantes.

Nióbio é um elemento químico que existe na natureza. É uma substância que pode ser usada em várias coisas diferentes. Pense em nióbio como uma ferramenta muito útil. Quando usado, ele pode tornar outras coisas melhores. Por exemplo, o nióbio pode ser adicionado a um tipo de metal chamado aço. Isso torna o aço mais forte, resistente à corrosão e capaz de suportar condições difíceis. O nióbio também é usado em outras coisas, como em baterias especiais e em equipamentos que precisam suportar altas temperaturas. Em resumo, o nióbio é um elemento que nos ajuda a melhorar materiais e torná-los mais úteis em várias situações.

Reminiscent of Plato's republic.

similar to scarcity book?

Author Response:

We are sorry that both eLife and the Reviewers feel that our submitted studies are currently insufficient to support our hypothesis that loss of H2-O function affects thymic Treg selection. As this is the first study directly evaluating loss of H2-O in the thymus we do not feel that we overstated our finding as suggested by Reviewer 1. We hope that a revised version of the manuscript can satisfy the reviewers’ criticisms.

-Reviewer 1 is asking us to address the presumed discrepancies between our previous work (Welsh et al 2020, https://doi.org/10.1371/journal.pbio.3000590) and data from Lee et al. 2021 (https://doi.org/10.4049/jimmunol.2100650) in this current manuscript, which does not report on the development of EAE in DO-KO and DO-WT mice. All experiments here are on naïve mice. As such, we wish to justify our lack of discussion of Lee et al (2021) findings.

Lee et al (2021) reported the effects of DO on both EAE and SLE development, they used mainly H2-Oβ KO mice. As we have never used these CRISPR generated mice, we cannot have a direct in-house comparison. However, we did note that reported disease curve for female H2-Oβ KO mice had a similar trend indicating increased EAE disease development, similar to what we have reported back in our 2020 paper (Welsh et al PLoS Biology). In the single experiment that utilized H2-Oβ KO mice for EAE development, Lee et al found a different disease trend than ours. However, Lee et al (2021)’s tested only 4-5 mice per group in the single experiment and their measurement of the disease development solely relied on visual assessment of the limbs and tail functionality. Our study verified EAE disease development by multiple approached including analyses of MOG-specific tetramer staining of the CNS CD4 lymphocyte infiltrate, and in vivo NIRF whole-body imaging on diseased DO-WT and DO-KO mice using an antibody probe specific to MBP. We had repeated our experiments on the disease development greater than 15 times using 5-8 mice per group. Below is an excerpt from our Results Section of Welsh et al PLoS Biology, clearly explaining how many experiments were performed and the number of mice per group per experiment:

“From these studies, we found that DO-KO mice had an accelerated onset of disease compared to DO-WT mice (Fig 7A). Disease symptoms (Score 1) appeared around Day 8–10 and quickly progressed to advanced disease (Score 3–4) by Day 14–16 in DO-KO. In contrast, DO-WT mice started showing symptoms around Day 12 and progressed to advanced disease scores by Day 20. Total cell infiltration into the CNS tissue was slightly higher in DO-KO mice, but no change in total brain weight was observed (S5 Fig). To further correlate the state of disease with CD4 infiltration, we performed in vivo NIRF whole-body imaging on diseased DO-WT and DO-KO mice using an antibody (Ab) probe specific to myelin basic protein (MBP). The Ab reacts with MBP only when the myelinated glia cells are damaged during disease development [56]. Thus, by detecting demyelination, whole-body imaging allowed us to fully visualize the co-localization of CD4 T cells at the sites of demyelination occurring in diseased mice. Interestingly, when mice of various disease scores were imaged, we found increased co-localization of infiltrating CD4 T cells with anti-MBP staining in DO-KO mice, but not in DO-WT mice (Fig 7B). These data not only confirmed the flow cytometric findings that diseased DO-KO mice have a greater influx of lymphocytes into their CNS tissue (S5 Fig), it also verified the massive demyelination that occurs during the disease”

And again in the Legend to Figure 7;

“Representative curves showing the time course of disease development in DO-KO (red) and DO-WT mice (white). N = 5 mice per group, representative of >15 repeat experiments. Score system: 0 = no symptoms, 1 = limp tail, 2 = limp tail + partial hind limb paralysis, 3 = limp tail + total hind limb paralysis, 4 = limp tail + total hind limb paralysis + partial forelimb paralysis. Data represented as mean ± SEM.”

Despite clarity of the description of our experiments, Lee et al have publicly slandered us and grossly misrepresented our work by stating the following:

“A recent study (11-Welsh et al) found that B6.Oa−/− mice were more susceptible to EAE than control B6J animals. However, that conclusion was based on a single experiment, in which control B6J mice developed very mild EAE disease with an average score of 1, which is far lower than the disease scores published by other groups (30–32) and also observed in our study. Thus, in this inducible model of autoimmunity, H2-O deficiency does not contribute to either disease development or severity.”

-Another important variable between our studies and Lee et al (Lee et al 2021) was the use of a commercially available disease induction kit versus our immunization solutions that followed the established protocols by Nancy Ruddle et al (J Exp Med. 1997 Oct 20; 186(8): 1233–1240. doi: 10.1084/jem.186.8.1233). Notoriously, EAE disease development could vary widely based upon the quantities and purity of, a) MOG peptide, b) amount of tuberculosis antigen in the CFA, c) quantity of pertussis toxin and injection strategies, as well as many other uncontrollable factors. While a comparison these two results are irrelevant to our current study, we will be more than happy to compare our results from the previously published work with Lee et al. in the discussion.

-We want to emphasize that we did follow Hogquists et al’s gating strategy for detecting auditing vs deleted thymocytes by subdividing total thymocytes into “Non-signaled” (TCR-β-, CD5-/inter) and “Signaled” (TCR-β+ CD5+/hi) populations before further gating on only medulla localized CD4 T cells. The “CCR7+ CD4+” label in Figure 1 was meant to orient the reader without overwhelming the figure with numerous flow plots. To address this concern, we will be including (1) updated Supplemental figures showing the complete gating strategy, (2) updated figure legends and text to emphasize the fact that auditing/deletion gating came from CD4 T cells which passed positive selection (i.e. TCR-β+ CD5+/hi), and (3) including representative flow plots for all Figure 1 panels to the revise manuscript.

-Also, regarding “discrepancies between our data and Liljedahl et al 1998”;

H2-O KO mice used by Liljedahl et al were on a 129/Ola genomic background. The H2-O KO mice used for both of our papers have been completely backcrossed to C57BL/6J. Clearly, non-MHC genes contribute to the impacts of MHC proteins, yet how the 129/Ola genomic background could affect the H2-O genes remains to be discovered. And (B), no data was shown supporting their published statement below:

“The proportions of B cells as well as of CD4+ and CD8+ T cells in the lymph node, spleen, and thymus were similar in H2-Oa–deficient and wild-type mice (data not shown)”. (Liljedahl et al 1998).

Reviewer 2:

scRNA-Seq analysis was performed by the Computational Biology Computing Core at Johns Hopkins School of Medicine. We missed including this acknowledgement as our core facility does not request authorship or acknowledgements. The sentence has been edited for the correct terminology.

-About truncated bar graph, in the entire paper we have only two bar graphs, neither of which is truncated. So, we are puzzled by the reviewer’s comment as to what figure he/she is referring to. -We would like to remind the Reviewer 2 that since DO works together with DM and functions differently on peptide of different sequences, the reported data on cumulative effects of DO in vivo have notoriously been rather minor. Especially, since our current study focuses on the naïve mice, major changes were not expected.

-Regarding leaving out gating strategies, we missed out on providing the gating strategies for all the figure in the original version. However, full FACS gating strategies have now been provided in the new supplemental figures and representative FACS plots have been added to ALL main figures.

Reviewer #2 (Public Review):

Summary:

The manuscript's main claim is that the absence of H2-O, a component of the MHC II presentation pathway, promotes regulatory T cell development and function.

Unfortunately, the submitted material is not sufficient for proper evaluation of the manuscript, both in terms of the significance of the findings and the strength of the supporting evidence.

Major issues include:

- the scRNAseq (shown in Fig. 5) is too rudimentary to allow any conclusion. Statements in the text (eg "Principle Component Analysis (PCA) of the normalized scRNA-seq data identified 11 distinct CD4 T cell clusters", line 166) suggest that additional expertise should be leveraged for these analyses.

- Most flow cytometry data (Figs. 1 and 2) shows marginal (at best) differences on y-axis truncated bar graphs, with no original data plot, gating strategies, etc., severely challenging conclusions drawn from this data.

eLife assessment

This paper seeks to understand how the presentation of peptides by medullary thymic epithelial cells may be regulated by the MHCII peptide loading modulator, H2-O, and how this may affect the selection of regulatory T (Treg) cells. Further work is needed to ensure that the findings are robust: currently the analysis of data is inadequate and inconsistencies in the reported findings are not placed in context with results from other groups. The current version does not provide sufficient support for the claims regarding the effects on Treg cell selection.

Reviewer #1 (Public Review):

The non-classical MHCII-like protein H2-M is essential for the loading of peptides on MHCII. The discovery that DM was partnered with a second MHCII-like protein, H2-O, which squelched or modified its activity was confounding. It was immediately speculated that H2-O was likely diminished self-peptide presentation. This led to the hypothesis that H2-O was involved in preventing unwanted CD4 T cell activation, thereby making autoimmunity less likely. 25 years of analysis of H2-O deficient mice have, indeed, shown that the self-peptide repertoire in the absence of H2-O is modestly altered. Demonstrating that autoimmunity results from this altered peptide repertoire has been decidedly less convincing. Old mice are reported to have increased serum anti-nuclear antibody titers, but mice prone to type 1 diabetes (T1D) and systemic lupus erythematosus (SLE) were not impacted by the loss of H2-O (Lee et al, 2021). Induction of the multiple sclerosis-like disease, EAE, in mice, was also shown to not be impacted by Lee et al 2021, although in a previous paper (Welsh et al 2020), the authors of this current manuscript suggest otherwise. Unfortunately, these discrepancies are not acknowledged by the authors, and the papers are, for the most part, not referenced.

In addition to antigen-presenting cells, H2-O is also found in MHCII-expressing medullary epithelial cells, suggesting it might play a role in T-cell selection. Direct data to support this idea, however, has, at most, shown a minimal impact. In this manuscript, the authors follow up on their previous paper (Welsh et al, 2020) to further evaluate changes to T cell development. The conclusions are that H2-O impacts Treg development and changes the frequency and homeostasis of CD4 T cells. Although these would be interesting results, the data analysis is flawed, the presentation is incomplete, and the conclusions are exaggerated.

T-cell development analysis shown in Figs. 1 and 2 use the discovery from the Hogquist lab (Breed et all 2019) that thymocytes destined for clonal deletion can be differentiated from those still "auditioning" for selection by FACS for expression of cleaved caspase 3. Detection relies on complex FACS analysis that requires the exclusion of multiple populations, followed by accurate gating on CD5+TCRb+ cells (see Hogquist Fig. 1A). The authors apparently neglected to use the essential gating steps, but rather only used CD4 and CCR7 expression (Fig. 1A). This deviation from the Hogquist approach makes interpretation of Figs 1 and 2 meaningless. Even if this is an oversight in the description of the experiments, key conclusions are drawn from minimal changes to CD69 expression. CD69 is expressed as a continuum in the thymus (a "shoulder") making gating somewhat subjective and prone to variation from experiment to experiment. At the minimum, FACS data should be shown to indicate how these changes were measured, plus variations from mouse to mouse should be plotted, with statistics. FACS data needs to be shown to define how the complex semi-mature, M1, and M2 populations were defined (see Hogquist Fig. 2) from which key conclusions are drawn.

To make the data more robust, 1) cell numbers must be included for all experiments;

2) rather than normalizing results to "the average H2-O WT levels", the actual data should be included;

3) figures should be more completely labeled/described;

4) FACS gating strategies should be clearly laid out (again, see Hogquist for examples). Furthermore, efforts must be made to explain why results are so different from analyses of H2-O deficient mice that have been published by many other groups. For example, the reported "dramatic increase in the proportion of CD3+CD4+ T cells" is not consistent with previous reports starting with Lars Karlsson's initial report (Liljedahl et al 1998). Extensive spontaneous activation of CD4 T cells has also not been reported in other papers that have studied these mice. Again, the paper is not placed in the context of the long, very thorough analysis of both the H2-O deficient mice and the study of H2-O/DO and H2-M/DM in general.

T O AUDIENCES . The writers were addressing particular audiences in the first and early second centuries.

Características claves del periodismo de investigacion

Yep

Would add after "this is most effective when it is a regular habit. Try to return to your notes before more than a week has gone by and quiz yourself or add some more questions to follow up" (this is terrible, but you see..)

我如何搭建和使用 Surge Snell 协议

This is a clever way to differentiate between cytokinetic bridges and the TNT-like connections, but I don't know that it fully rules out that the TNT-like connections could be related to division. Is there a way to block division and see if you still get TNT-like connections?

https://scicrunch.org/resolver/RRID:SCR_018568

https://twitter.com/scite

Descripción

Plataforma que por medio de inteligencia artificial permite analizar las citas de un artículo académico con base en la sección del artículo en la que se hizo, la intención clasificada en cuatro categorías: se apoya, se menciona, se contrasta o no sin clasificación.

Las estadísticas no mienten, si bien las mujeres abarcan un buen porcentaje de migrantes en nuestro país ¿porque no visibilizarlas? a demás muchas de estas mujeres no viajan solas, traen con sigo a sus hijos o hijas y verdaderamente están en un completo estado de vulnerabilidad.

A pesar de que el mundo político moderno valora las encuestas o el mero concepto de "los datos", no siempre se realizan adecuadamente. Las noticias están repletas de estudios y estadísticas para moldear las opiniones públicas, pero con la falta de ciertas preguntas clave se puede contar una historia completamente distinta a la realidad.

Al realizar encuestas, todo comunica y al no incluir ciertos aspectos como el género, se deja fuera mucha información que es de suma importancia para la investigación o estudio de un tema.

I wonder if there was ever a question of reliability and truth from people talking on the radio

DOI: 10.1186/s12967-023-04542-4

Resource: (Abcam Cat# ab52903, RRID:AB_882596)

Curator: @scibot

SciCrunch record: RRID:AB_882596

What is this?

I learned that science and cooking is always connected. Even if we don't think about it in every day life like when water evaporates or freezes it is chemistry. But what I found most interesting that I learned is how water immobilization works, or to put it more simply the science behind Jell-O. When you add gelatin to water it traps the water molecules in place which creates the sort of liquid and solid hybrid we find with Jell-O.

porque existem dois como o ácido hydroxybenzpoic

O concurso de preferência entre os entes federados na cobrança judicial dos créditos tributários e não tributários, previsto no parágrafo único do art. 187 da Lei 5.172/1966 (Código Tributário Nacional) e no parágrafo único do art. 29 da Lei 6.830/1980 (Lei de Execuções Fiscais), não foi recepcionado pela Constituição Federal de 1988 (CF/1988). (ADPF 357/DF, relatora Min. Cármen Lúcia, julgamento em 24.6.2021)

Súmula CARF nº 1

• Importa renúncia às instâncias administrativas a propositura pelo sujeito passivo de ação judicial por qualquer modalidade processual, antes ou depois do lançamento de ofício, com o mesmo objeto do processo administrativo, sendo cabível apenas a apreciação, pelo órgão de julgamento administrativo, de matéria distinta da constante do processo judicial.

• RE 233582
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. MARCO AURÉLIO
• Redator(a) do acórdão: Min. JOAQUIM BARBOSA
• Julgamento: 16/08/2007
• Publicação: 16/05/2008

Ementa EMENTA: CONSTITUCIONAL. PROCESSUAL TRIBUTÁRIO. RECURSO ADMINISTRATIVO DESTINADO À DISCUSSÃO DA VALIDADE DE DÍVIDA ATIVA DA FAZENDA PÚBLICA. PREJUDICIALIDADE EM RAZÃO DO AJUIZAMENTO DE AÇÃO QUE TAMBÉM TENHA POR OBJETIVO DISCUTIR A VALIDADE DO MESMO CRÉDITO. ART. 38, PAR. ÚN., DA LEI 6.830/1980. - O direito constitucional de petição e o princípio da legalidade não implicam a necessidade de esgotamento da via administrativa para discussão judicial da validade de crédito inscrito em Dívida Ativa da Fazenda Pública. É constitucional o art. 38, par. ún., da Lei 6.830/1980 (Lei da Execução Fiscal - LEF), que dispõe que "a propositura, pelo contribuinte, da ação prevista neste artigo [ações destinadas à discussão judicial da validade de crédito inscrito em dívida ativa] importa em renúncia ao poder de recorrer na esfera administrativa e desistência do recurso acaso interposto". Recurso extraordinário conhecido, mas ao qual se nega provimento.

Author Response

Reviewer #1 (Public Review):

Summary:

Rai1 encodes the transcription factor retinoic acid-induced 1 (RAI1), which regulates expression of factors involved in neuronal development and synaptic transmission. Rai1 haploinsufficiency leads to the monogenic disorder Smith-Magenis syndrome (SMS), which is associated with excessive feeding, obesity and intellectual disability. Consistent with findings in human subjects, Rai1+/- mice and mice with conditional deletion of Rai1 in Sim+ neurons, which are abundant in the paraventricular nucleus (PVN), exhibit hyperphagia, obesity and increased adiposity. Furthermore, RAI1-deficient mice exhibit reduced expression of brain-derived neurotrophic factor (BDNF), a satiety factor essential for the central control of energy balance. Notably, overexpression of BDNF in PVN of RAI1-deficient mice mitigated their obesity, implicating this neurotrophin in the metabolic dysfunction these animals exhibit. In this follow up study, Javed et al. interrogated the necessity of RAI1 in BDNF+ neurons promoting metabolic health.

Consistent with previous reports, the authors observed reduced BDNF expression in the hypothalamus of Rai1+/- mice. Moreover, proteomics analysis indicated impairment in neurotrophin signaling in the mutants. Selective deletion of Rai1 in BDNF+ neurons in the brain during development resulted in increased body weight, fat mass and reduced locomotor activity and energy expenditure without changes in food intake. There was also a robust effect on glycemic control, with mutants exhibiting glucose intolerance. Selective depletion of RAI1 in BDNF+ neurons in PVN in adult mice also resulted in increased body weight, reduced locomotor activity, and glucose intolerance without affecting food intake. Blunting RAI1 activity also leads to increases and decreases in the inhibitory tone and intrinsic excitability, respectively, of BDNF+ neurons in the PVN.

Strengths:

Overall, the experiments are well designed and multidisciplinary approaches are employed to demonstrate that RAI1 deficits in BDNF+ neurons diminish hypothalamic BDNF signaling and produce metabolic dysfunction. The most significant advance relative to previous reports is the finding from electrophysiological studies showing that blunting RAI1 activity leads to increases and decreases the inhibitory tone and intrinsic excitability, respectively, of BDNF+ neurons in the PVN. Furthermore, that intact RAI1 function is required in BDNF+ neurons for the regulation of glucose homeostasis.

Weaknesses:

Some of the data need to be reconciled with previous findings by others. For example, the authors report that more than 50% of BDNF+ neurons in PVN also express pTrkB whereas about 20% of pTrkB+ cells contain BDNF, raising the possibility that autocrine mechanisms might be at play. This is in conflict with a previous study by An et al, (2015) showing that these cell populations are largely non-overlapping in the PVN.

We fully agree with this assessment. Given the difficulty of using immunostaining to characterize the expression of membrane proteins in vivo, and the specificity of the pTrkB antibody in different tissues remains unknown, it is difficult to interpret the signals we observed. We have excluded the data because the histological analysis of p-TRKB and BDNF autocrine/paracrine signalling is not a focus of the present study. Future studies using a more advanced genetic method (i.e., Ntrk2CreER/+; Ai9 mouse line as used by An et al., 2015) is more suitable and should be used in the future to investigate the function of Rai1 in the TRKB+ neurons.

Another issue that deserves more in-depth discussion is that diminished BDNF function appears to play a minor part driving deficits in energy balance regulation. Accordingly, both global central depletion of Rai1 in BDNF+ neurons during development and deletion of Rai1 in BDNF+ neurons in the adult PVN elicited modest effects on body weight (less than 18% increase) and did not affect food intake. This contrasts with mice with selective Bdnf deletion in the adult PVN, which are hyperphagic and dramatically obese (90% heavier than controls). Therefore, the results suggest that deficits in RAI1 in PVN or the whole brain only moderately affect BDNF actions influencing energy homeostasis and that other signaling cascades and neuronal populations play a more prominent role driving the phenotypes observed in Rai1+/- mice, which are hyperphagic and 95% heavier than controls. The results from the proteomic analysis of hypothalamic tissue of Rai1 mutant mice and controls could be useful in generating alternative hypotheses. Depleting RAI1 in BDNF+ neurons had a robust effect compromising glycemic control. However, as the approach does not necessarily impact BDNF exclusively, there should be a larger discussion of alternative mechanisms.

We thank the reviewer for these insightful comments. We want to highlight that global deletion of Rai1 from BDNF neurons did induce food intake increase in male mice (Fig 2figure supplement 4K). We have incorporated the following paragraphs into the discussion section.

Lines 364-384: “Notably, mice lacking one copy of Rai1 in the BDNF-producing cells do not exhibit obesity, whereas SMS patients and SMS mice show pronounced obesity (Burns et al., 2010; Huang et al., 2016; Smith et al., 2005). This indicates that although reduced Bdnf expression and BDNF-producing neurons contribute to regulating body weight, additional molecular changes and other hypothalamic populations also play important roles in regulating body weight homeostasis in SMS. Our RPPA data suggest that mTOR signalling is also misregulated in addition to the reduced activation of the neurotrophin downstream cascades. Hypothalamic mTORC1 is crucial to regulate glucose release from the liver, peripheral lipid metabolism, and insulin sensitivity (Burke et al., 2017; Caron et al., 2016; Smith et al., 2015), while mTORC2 regulates glucose tolerance and fat mass (Kocalis et al., 2014). How the impaired mTOR signalling contributes to energy homeostasis defects in SMS and the therapeutic potential of targeting this pathway to treat SMS-related obesity remains unclear and warrants future investigation.

What additional Rai1-dependent hypothalamic cell types residing in brain regions other than PVH regulate obesity in SMS? Other important cell types such as TRKB neurons within the PVH (An et al., 2020) and several RAI1-expressing hypothalamic nuclei including the arcuate nucleus, ventromedial nucleus of the hypothalamus (VMH), and lateral hypothalamus all play important roles in regulating energy homeostasis. POMC- and AGRP-expressing neurons within the arcuate nucleus are known to regulate food intake and glucose and insulin homeostasis (Quarta et al., 2021; Vohra et al., 2022). Therefore, Rai1 function in these neurons could contribute to obesity in SMS, a topic that awaits future investigation.”

Reviewer #2 (Public Review):

Understanding disease conditions often yields valuable insights into the physiological regulation of biological functions, as well as potential therapeutic approaches. In previous investigations, the author's research group identified abnormal expression of brain-derived neurotrophic factor (BDNF) in the hypothalamus of a mouse model exhibiting Smith-Magenis syndrome (SMS), which is caused by heterozygous mutations of the Rai1 gene. Human SMS is associated with distinct facial characteristics, sleep disturbances, behavioral issues, and intellectual disabilities, often accompanied by obesity. Conditional knockout (cKO) of the Bdnf gene from the paraventricular hypothalamus (PVH) in mice led to hyperphagic obesity, while overexpression of the Bdnf gene in the PVH of Rai1 heterozygous mice restored the SMS-like obese phenotype. Based on these preceding findings, the authors of the present study discovered that homozygous Rai1 cKO restricted to Bdnf-expressing cells, or Rai1 gene knockdown solely in Bdnf-positive neurons in the PVH, induced obesity along with intricate alterations in adipose tissue composition, energy expenditure, locomotion, feeding patterns, and glucose tolerance, some of which varied between sexes. Additionally, the authors demonstrated that a brain-penetrating drug capable of activating the TrkB pathway, a downstream signaling pathway of BDNF, partially alleviated the SMS-like obesity phenotype in female mice with Rai1 heterozygous mutations. Although the specific (neural) cell type responsible for this TrkB signaling remains an open question, the present study unequivocally highlights the importance of Rai1 gene function in PVH Bdnf neurons for the obesity phenotype, providing valuable insights into potential therapeutic strategies for managing obesity associated with SMS.

In the proteomic analysis (Fig. 1), the authors elucidated that multiple phospho-protein signaling pathways, including Akt and mTOR pathways, exhibited significant attenuation in the SMS model mice. Of significance, the manifestation of haploinsufficiency of the Rai1 gene exclusively within the BDNF+ cells demonstrated negligible impact on body weight (Fig. 2supple 3D), despite observing a reduction in BDNF levels in the heterozygous Rai1 mutant (Fig. 1A). Conversely, the homozygous Rai1 cKO in the BDNF+ cells prominently displayed an obesity phenotype, suggesting substantial dissimilarities in the gene expression profiles between Rai1 heterozygous and homozygous conditions within the BDNF+ cell population. It would be advantageous to precisely identify the responsible differentially expressed genes, possibly including Bdnf itself, in the homozygous cKO model. The observed reduction in the excitability of PVH BDNF+ cells (Fig. 3) is presumably attributed to aberrant gene expression other than Bdnf itself, which may serve as a prospective target for gene expression analysis. Notably, the Rai1 homozygous cKO mice in BDNF+ cells exhibited some sexual dimorphisms in feeding and energy expenditures, as evidenced by Fig. 2 and related figures. Exploring the potential relevance of these sexual differences to human SMS cases and investigating the underlying cellular/molecular mechanisms in the future would provide valuable insights.

Although the CRISPR-mediated knockdown of the Rai1 gene (Fig. 4) appears to be highly effective, given the broad transduction of AAV serotype 9, it may be helpful to exclude the possibility of other brain regions adjacent to the PVH, such as the DMH or VMH, being affected by this viral procedure. If the PVH-specificity is established, the majority of Rai1 cKO effects in Bdnf+ cells are primarily attributed to PVH-Bdnf+ cells based on the similarity of phenotypes observed. With regards to the apparent rescue of the body weight phenotype in Rai1 heterozygous mutants using a selective TrkB activator, the specific biological processes, and neurons responsible for this effect remain unclear to this reviewer. Elucidating these aspects would be significant when considering potential applications to human SMS cases.

We appreciate the reviewer's insightful comments. We agree that the logical next step would be to identify the profile of the differentially expressed genes in our homozygous conditional knockout model. We have included the following paragraphs in the discussion.

Lines 364-384: “Notably, mice lacking one copy of Rai1 in the BDNF-producing cells do not exhibit obesity, whereas SMS patients and SMS mice show pronounced obesity (Burns et al., 2010; Huang et al., 2016; Smith et al., 2005). This indicates that although reduced Bdnf expression and BDNF-producing neurons contribute to regulating body weight, additional molecular changes and other hypothalamic populations also play important roles in regulating body weight homeostasis in SMS. Our RPPA data suggest that mTOR signalling is also misregulated in addition to the reduced activation of the neurotrophin downstream cascades. Hypothalamic mTORC1 is crucial to regulate glucose release from the liver, peripheral lipid metabolism, and insulin sensitivity (Burke et al., 2017; Caron et al., 2016; Smith et al., 2015), while mTORC2 regulates glucose tolerance and fat mass (Kocalis et al., 2014). How the impaired mTOR signalling contributes to energy homeostasis defects in SMS and the therapeutic potential of targeting this pathway to treat SMS-related obesity remains unclear and warrants future investigation.

What additional Rai1-dependent non-PVH hypothalamic cell types regulate obesity in SMS? Other important cell types such as TRKB neurons within the PVH (An et al., 2020) and several RAI1expressing hypothalamic nuclei including the arcuate nucleus, ventromedial nucleus of the hypothalamus (VMH), and lateral hypothalamus all play important roles in regulating energy homeostasis. POMC- and AGRP-expressing neurons within the arcuate nucleus are known to regulate food intake and glucose and insulin homeostasis (Quarta et al., 2021; Vohra et al., 2022). Therefore, Rai1 function in these neurons could contribute to obesity in SMS, a topic that awaits future investigation.”

Lines 409-418: “It is plausible that RAI1 regulates the expression of genes encoding inward rectifier K+ channels, which regulate neuronal activity and potentially energy homeostasis. For instance, KIR6 (a family of ATP-sensitive potassium channels, KATP) is widely expressed in the hypothalamus. Deleting the hypothalamic KIR6.2 subunit impairs KATP channel function and glucose tolerance (Miki et al., 2001; Parton et al., 2007). Moreover, reduced expression of hypothalamic GIRK4 (encoding an inwardly rectifying potassium channel) causes obesity (Perry et al., 2008). GABAergic neurotransmission from arcuate AGRP-expressing neurons to the PVH neurons is important to increase appetite by favouring hyperphagia (Atasoy et al., 2012). Disrupting the composition of these ion channels could contribute to reduced PVHBDNF neuronal firing, which awaits further investigations.”

Moreover, to facilitate the future exploration of the potential relevance of sexual differences to human SMS cases, we have incorporated the following explanation in the discussion section.

Lines 419-426: “Female mice with a conditional knockout of Rai1 from BDNF-producing neurons do not display a noteworthy difference in food intake. Conversely, their male counterparts exhibit a significant increase in food intake. Although SMS individuals of both genders tend to overeat, male patients who are obese show significantly higher food consumption than their female counterparts (Gandhi et al., 2022). This observation raises the possibility that Rai1 regulates eating behaviours through multiple cell types in the hypothalamus and that a male-specific involvement of BDNF-producing neurons in regulating food intake, potentially provides a neurobiological basis for the observed pattern in SMS patients (Gandhi et al., 2022).”

To exclude the possibility of other brain regions adjacent to the PVH (such as VMH and arcuate nucleus) being affected by our AAV-CRISPR-mediated Rai1 knockout, we have analyzed other hypothalamic regions including VMH and arcuate nucleus from the same slides used to confirm PVH viral expression and we confirmed that the AAV was not expressed in these regions. We have incorporated a representative image (Figure 4 suppl 1F) depicting limiting AAV expression in these nuclei.

Regarding LM22A-4: It is possible that LM22A-4 functions directly through binding to TRKB or indirectly engages TRKB downstream molecules through activating other receptors such as GPCR. LM22A-4 appears to engage neurotrophin downstream PI3KAKT pathway, which was identified by our RPPA analysis to be downregulated in the hypothalamus of Rai1-deficient mice. Reduced AKT activity is associated with insulin resistance and obesity in mice. Restoration of functional activity of AKT by LM22A-4 could be the primary mode of action for this drug in the brain. However, since we observed that this drug only partially rescued the body weight defect, future research exploring more potent TrkB agonists or utilizing a combination therapy that targets both the neurotrophin and mTOR pathways might yield improved responses to the pharmacological interventions. We have included the following paragraph in the discussion:

Lines 451-461: “ We recognize that while several in vivo studies have demonstrated the potential of LM22A-4 in targeting neurotrophin downstream signalling (Kron et al., 2014; Li et al., 2017), an in vitro analysis failed to demonstrate the ability of LM22A-4 to activate TrkB directly (Boltaev et al., 2017). Therefore, the precise mechanism by which LM22A-4 enhances AKT cascades in the mammalian brain remains unclear and awaits further investigations. In the hypothalamus of SMS mice, LM22A-4 could indirectly engage neurotrophin downstream PI3KAKT pathway through the G protein-coupled receptor-dependent transactivation of the TRKB receptor (Domeniconi & Chao, 2010) or other unknown mechanisms. Moreover, while LM22A4 may have potential side effects, we found that wild-type mice treated with LM22A-4 did not show a further decrease in body weight, suggesting limited side effects regarding body weight regulation.”

Overall, the present study represents a valuable addition to the authors' series of high-quality molecular genetic investigations into the in vivo functions of the Rai1 gene. This reviewer particularly commends their diligent efforts to enhance our comprehension of SMS and contribute to the future development of more effective therapies for this syndrome.

We thank the reviewer for finding our study valuable in advancing the understanding of RAI1 function.

Reviewer #3 (Public Review):

Summary:

Smith-Magenis syndrome (SMS) is associated with obesity and is caused by deletion or mutations in one copy of the Rai1 gene which encodes a transcriptional regulator. Previous studies have shown that Bdnf gene expression is reduced in the hypothalamus of Rai1 heterozygous mice. This manuscript by Javed et al. further links SMS-associated obesity with reduced Bdnf gene expression in the PVH.

Strengths:

The authors show that deletion of the Rai1 gene in all BDNF-expressing cells or just in the PVH BDNF neurons postnatally caused obesity. Interestingly, mutant mice displayed sexual dimorphism in the cause for the obesity phenotype. Overall, the data are well presented and convincing except the data from LM22A-4.

Weaknesses:

1) The most serious concern is about data from LM22A-4 administration experiments (Figure 5 and associated supplemental figures). A rigorous study has demonstrated that LM22A-4 does not activate TrkB (Boltaev et al., Science Signaling, 2017), which is consistent with unpublished results from many labs in the neurotrophin field. It is tricky to interpret body weight data from pharmacological studies because compounds always have some side effects, which can reduce body weight non-specifically.

We thank this reviewer for their valuable comments. Indeed, the precise mechanism by which LM22A-4 exerts its effect is not entirely clear and there has been mixed evidence regarding its identity as a TRKB agonist in vitro. We have refrained from stating LM22A-4 as a partial agonist of TRKB, and instead have focused on highlighting the potential of this drug in activating neurotrophin downstream signalling through increasing AKT phosphorylation in vivo. We have modified the title to remove TRKB, and the following changes have been made in the discussion:

Lines 451-461: “ We recognize that while several in vivo studies have demonstrated the potential of LM22A-4 in targeting neurotrophin downstream signalling (Kron et al., 2014; Li et al., 2017), an in vitro analysis failed to demonstrate the ability of LM22A-4 to activate TrkB directly (Boltaev et al., 2017). Therefore, the precise mechanism by which LM22A-4 enhances AKT cascades in the mammalian brain remains unclear and awaits further investigations. In the hypothalamus of SMS mice, LM22A-4 could indirectly engage neurotrophin downstream PI3KAKT pathway through the G protein-coupled receptor-dependent transactivation of the TRKB receptor (Domeniconi & Chao, 2010) or other unknown mechanisms. Moreover, while LM22A4 may have potential side effects, we found that wild-type mice treated with LM22A-4 did not show a further decrease in body weight, suggesting limited side effects regarding body weight regulation.”

2) The resolution of all figures are poor, and thus I could not judge the quality of the micrographs.

We have updated with higher resolution images.

3) Citation of the literature is not precise. The study by An et al. (2015) shows that deletion of the Bdnf gene in the PVH leads to obesity due to increased food intake and reduced energy expenditure (not just hyperphagic obesity; Line 72). Furthermore, the study by Unger et al. (2017) carried out Bdnf deletion in the VMH and DMH using AAV-Cre and did not discuss SF1 neurons at all (Line 354). The two studies by Yang et al. (Mol Endocrinol, 2016) and Kamitakahara et al. (Mol Metab, 2015) did use SF1-Cre to delete the Bdnf gene and did not observe any obesity phenotype.

We thank the reviewer for bringing this to our attention. We have revised the text to ensure accurate representation of the cited publications. The following changes have been made: Lines 348-350: “ Although BDNF is required in the VMH and DMH to regulate body weight (Unger et al., 2007), embryonic deletion of Bdnf from the SF1-lineage populations including the VMH did not result in obesity (Kamitakahara et al., 2016; Yang et al., 2016).”

4) Animal number is not described in many figure legends.

We thank the reviewer for pointing it out. We have revised the manuscript to incorporate the missing animal numbers.

Reviewer #1 (Recommendations For The Authors):

Additional points:

1) The data provided indicating increased inhibitory tone onto BDNF neurons in PVN of Rai1 mutant mice are not convincing that inhibitory drive is significantly affected.

We have modified the sentences as follows, we have also deleted these conclusions from the abstract and discussion:

Lines 215-220: “We observed a slight rightward shift of the probability of miniature inhibitory postsynaptic current (mIPSC) frequency in cKO PVHBDNF neurons, although the average frequency (Fig 3K) was not significantly different between groups. The probability of mIPSC amplitude also showed a right shift without a significant change (Fig 3L, Figure 3—figure supplement 1D). However, we observes a significant increased area under the curve (Fig 3M).”

2) Fig. 3C - Was outlier analysis performed for these data? One of the data points for the control group looks like an outlier that might be skewing the data.

We performed an outlier analysis and found that indeed one data point was an outlier, after removing this data point, the data remained statistically significant (*p<0.05) and the new manuscript has been updated.

Reviewer #2 (Recommendations For The Authors):

1) The manuscript would benefit from improved usage and precise descriptions of statistics. The authors often provided only general statements such as "one or two-way ANOVA" without specifying the exact statistical tests used. It is important to differentiate between one-way and two-way ANOVA, particularly when using the latter, by clearly indicating the within-group effects and interaction effects. The representation of p-values associated with ANOVA using asterisks requires clarification, specifying which statistics indicate ANOVA results and which ones correspond to post hoc analysis. It is advisable to assess the normality of the distribution before employing t-tests or consider non-parametric comparisons such as Wilcoxon's rank sum test if normality assumptions are not met. Additionally, it is essential to specify whether the tests are one-sided or two-sided and whether they are paired or unpaired. In some figure panels, such as Fig. 2H and K, the statistical tests used were not indicated at all.

We have clarified the exact statistical tests in the figure legend for each figure.

2) Rearranging the figures to facilitate a direct comparison of the sexual phenotypes (Fig. 2 and Fig. 2-supple 4) within the same figures would greatly improve reader comprehension.

We have decided to keep the figure arrangement because of the focus on female mice in the main figures.

3) To improve the comprehension of the figures and text, the following points should be addressed:

• Fig. 1D: The definition of the expression level in the color code is not clear.

Explanation for the color code has been added in the method section.<br /> Lines 652-656: “The vertical axis of the dendrogram represents the dissimilarity (measured as distance) between protein expressions, and the horizontal axis represents the individual test samples. The colour code (ranging from red to yellow to green) specifies the expression levels of different proteins, where red indicates nifies low expression, yellow indicates intermediate expression, and green indicates high expression.”

• Fig. 1F: One parenthesis is missing from the figure label.

Fixed

• Fig. 2C: It is unclear why there are so many dots for just n = 3 animals. It would be better to specify the conditions or use "animals" as a unit of measurement.

The dots represent percentage cells quantified per sliced from 3 animals. It has been clarified in the figures.

• Fig. 2F: There seems to be an unnecessary label "I" in the middle of the panel.

Fixed

• It is not completely clear if the data in Fig. 2E-L were all obtained at 26 weeks of age.

To clarify, following line has been added to the method section:

Lines 517-518: “After the 25th week, mice were subjected to body composition analysis.”

• In Fig. 2-Supple 1, the legend should read "G-J." Additionally, please provide a definition for the arrowheads.

Line 1086: “yellow arrowheads indicate Ai9 marked BDNF cells co-expressing endogenous BDNF.”

• It is not completely clear if the data in Fig. 3 were all obtained from female mice.

It is explained in the legend of Fig 3.

• The description of the number of animals seems to be missing in Fig. 4

The description for the number of animals has been added in the figure legend. Line 1004: “(Ctrl group: n=5, Exp group: n =5)”

• On line 280-281, "Fig 4A." should be corrected to "Fig. 5A."

Corrected.

• In Fig. 5C-E, it is uncertain if multiple pairwise comparisons for three groups are statistically appropriate. At the very least, multiple comparisons should be corrected.

We performed two-way ANOVA where mean body weight of age-matched groups were compared with each other (i.e. between control saline-injected and SMS saline-injected, SMS saline-injected and LM22A-4 -saline injected, and Control saline-injected and SMS LM22A-4 injected). We used Šidák’s multiple comparisons test, where statistical significance was indicated with p<0.05, p < 0.01, p<0.001, **p < 0.0001. We have clarified this in the figure 5 legends.

• The unit of measurement should be standardized across figures, if possible, to facilitate better side-by-side comparisons. For example, most bodyweight figures use "g" (grams), but "mg" (milligrams) is used in Fig. 5.

All measurements are corrected to be consistent (in grams).

• It is unclear if nM (not mM) of glucose was actually measured in the glucose tolerance test (Fig. 2L and Fig. 4L).

Fixed.

Reviewer #3 (Recommendations For The Authors):

1) The authors can remove the LM22A-4 data without much detrimental effects on the conclusion of the manuscript. Otherwise, the authors have to demonstrate that LM22A-4 activates TrkB, does not have any toxicity, and does not cause aversion.

We thank this reviewer the valuable comments and we acknowledge the valid concern. Indeed, the precise mechanism by which LM22A-4 exert its effects is not clear and there has been mixed opinions regarding its function as TRKB agonist in in-vitro assays. To clarify, we have refrained from stating LM22A-4 as a partial agonist of TRKB, and instead have focused on highlighting the potential of this drug in activating neurotrophin downstream signalling through increased AKT phosphorylation, in-vivo.

We have also modified the title of our article to exclude the word “TRKB Signalling”. The new title is as follows:

“Smith-Magenis syndrome protein RAI1 regulates body weight homeostasis through hypothalamic BDNF-producing neurons and neurotrophin downstream signalling”

2) Line 50: "40% > 95th percentile weight, 40% > 85th percentile weight" should be "40% > 95th percentile weight, 80% > 85th percentile weight".

Corrected.

3) Abbreviations for brain-derived neurotrophic factor: Bdnf for gene and BDNF for protein.

Abbreviations have been corrected throughout the manuscript.

4) Need to specify the animal age when viruses were injected into the PVH to inactivate the Bdnf gene.

Line 235: Virus was injected at 3 weeks of age. It has been specified in the main text.

5) Line 832: "3 technical triplicates" can be simplified as "3 technical repeats" because 3 and triplicates are redundant.

Corrected.

6) Figure 2B: The "O" in cKO is misplaced.

Fixed.

7) Figure 3: The black legends in E and F should include Ctrl.

Fixed in the Figure 3.

Author Response

The data we produce are not criticized as such and thus, do not require revision; the criticisms concern our interpretation of them. General themes of the reviews are that i) genetic signatures do not matter for defining neuronal types (here sympathetic versus parasympathetic); ii) that a cholinergic postganglionic autonomic neuron must be parasympathetic; and iii) that some physiology of the pelvic region would deserve the label “parasympathetic”. We answered the latter argument in (Espinosa-Medina et al., 2018) to which we refer the interested reader; and we fully disagree with the first two. Of note, part of the last sentence of the eLife assessment is misleading and does not reflect the referees’ comments. Our paper analyses genetic differences between the cranial and sacral outflow and uses them to argue that they cannot be both parasympathetic. The eLife assessment acknowledges the “genetic differences” but concludes that, somehow, they don’t detract from a common parasympathetic identity. We take issue with this paradox, of course, but it is coherent with the referee’s comments. On the other hand, the eLife assessment alone pushes the paradox one step further by stating that “functional differences” between the cranial and sacral outflows can’t either prevent them from being both parasympathetic. We would also object to this, but the only “functional differences” used by the referees to dismiss our diagnostic of a sympathetic-like character (rather than parasympathetic) for the sacral outflow are between noradrenergic and cholinergic, and between sympathetic and parasympathetic (and we also disagree with those, see above, and below) —not between cranial and sacral.

We will thus use the opportunity offered by eLife to keep the paper as it is (with a few minor stylistic changes). We respond below to the referees’ detailed remarks and hope that the publication, as per eLife new model, of the paper, the referees’ comments and our response will help move the field forward.

Public review by Referee #1

“Consistently, the P3 cluster of neurons is located close to sympathetic neuron clusters on the map, echoing the conventional understanding that the pelvic ganglia are mixed, containing both sympathetic and parasympathetic neurons”.

The greater closeness of P3 than of P1/2/4 to the sympathetic cluster can be used to judge P1/2/4 less sympathetic than P3 (and more… something else), but not more parasympathetic. There is no echo of the “conventional understanding” here.

“A closer look at the expression showed that some genes are expressed at higher levels in sympathetic neurons and in P2 cluster neurons ” [We assume that the referee means “in sympathetic neurons and in P3 cluster neurons”] but much weaker in P1, P2, and P4 neurons such as Islet1 and GATA2, and the opposite is true for SST. Another set of genes is expressed weakly across clusters, like HoxC6, HoxD4, GM30648, SHISA9, and TBX20.

These statements are inaccurate; On the one hand, the classification is not based on impression by visual inspection of the heatmap, but by calculations, using thresholds. Admittedly, the thresholds have an arbitrary aspect, but the referee can verify (by eye inspection of heatmap) that genes which we calculate as being at “higher levels in sympathetic neurons and in P3 cluster neurons, but much weaker in P1, P2, and P4 neurons” or vice versa, i.e. noradrenergic or cholinergic neurons (genes from groups V and VI, respectively), have a much bigger difference than those cited by the referee, indeed are quasi-absent from the weaker clusters or ganglia. In addition, even by subjective eye inspection:

Islet is equally expressed in P4 and sympathetics.

SST is equally expressed in P1 and sympathetics.

Tbx20 is equally expressed in P2 and sympathetics.

HoxC6, HoxD4, GM30648, SHISA9 are equally expressed in all clusters and all sympathetic ganglia.

“Since the pelvic ganglia are in a caudal body part, it is not surprising to have genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa (to have genes expressed in sphenopalatine ganglia, but not in pelvic ganglia), according to well recognized rostro-caudal body patterning, such as nested expression of hox genes.”

We do not simply show “genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa”, i.e. a genetic distance between pelvic and sphenopalatine, but many genes expressed in all pelvic cells and sympathetic ones, i.e. a genetic proximity between pelvic and sympathetic. This situation can be deemed “unsurprising”, but it can only be used to question the parasympathetic nature of pelvic cells (as we do), or considered irrelevant (as the referee does, because genes would not define cell types, see our response to an equivalent stance by Referee#2). Concerning Hox genes, we do take them into account, and speculate in the discussion that their nested expression is key to the structure of the autonomic nervous system, including its division into sympathetic and parasympathetic outflows.

It is much simpler and easier to divide the autonomic nervous system into sympathetic neurons that release noradrenaline versus parasympathetic neurons that release acetylcholine, and these two systems often act in antagonistic manners, though in some cases, these two systems can work synergistically. It also does not matter whether or not pelvic cholinergic neurons could receive inputs from thoracic-lumbar preganglionic neurons (PGNs), not just sacral PGNs; such occurrence only represents a minor revision of the anatomy. In fact, it makes much more sense to call those cholinergic neurons located in the sympathetic chain ganglia parasympathetic.

This “minor revision of the anatomy” would make spinal preganglionic neurons which are universally considered sympathetic (in the thoraco-lumbar chord), synapse onto large numbers of parasympathetic neurons (in the paravertebral chains for sweat glands and periosteum, and in the pelvic ganglion), robbing these terms of any meaning.

Thus, from the functionality point of view, it is not justified to claim that "pelvic organs receive no parasympathetic innervation".

There never was any general or rigorous functional definition of the sympathetic and parasympathetic nervous systems — it is striking, almost ironic, that Langley, creator of the term parasympathetic and the ultimate physiologist, provides an exclusively anatomic definition in his Autonomic Nervous System, Part I. Hence, our definition cannot clash with any “functionality point of view”. In fact, as we briefly say in the discussion and explore in (Espinosa-Medina et al., 2018), it is the “sacral parasympathetic” paradigm which is unjustified from a functionality point of view, for implying a functional antagonism across the lumbo-sacral gap, which has been disproven repeatedly. It remains to be determined which neurons are antagonistic to which on the blood vessels of the external genitals; antagonism within one division of the autonomic nervous system would not be without precedent (e.g. there exist both vasoconstrictor and vasodilator sympathetic neurons, and both, inhibitor and activator enteric motoneurons). The way to this question is finally open to research, and as referee#2 says “it is early days”.

Public review by Referee #2

This work further documents differences between the cranial and sacral parasympathetic outflows that have been known since the time of Langley - 100 years ago.

We assume that the referee means that it is the “cranial and sacral parasympathetic outflows” which “have been known since the time of Langley”, not their differences (that we would “further document”): the differences were explicitly negated by Langley. As a matter of fact, the sacral and cranial outflows were first likened to each other by Gaskell, 140 years ago (Gaskell, 1886). This anatomic parallel (which is deeply flawed (Espinosa-Medina et al., 2018)) was inherited wholesale by Langley, who added one physiological argument (Langley and Anderson, 1895) (which has been contested many times (Espinosa-Medina et al., 2018) and references within).

In addition, the sphenopalatine and other cranial ganglia develop from placodes and the neural crest, while sympathetic and sacral ganglia develop from the neural crest alone.

Contrary to what the referee says, the sphenopalatine has no placodal contribution. There is no placodal contribution to any autonomic ganglion, sympathetic or parasympathetic (except an isolated claim concerning the ciliary ganglion (Lee et al., 2003)). All autonomic ganglia derive from the neural crest as determined a long time ago in chicken. For the sphenopalatine in mouse, see our own work (Espinosa-Medina et al., 2014).

One feature that seems to set the pelvic ganglion apart is […] the convergence of preganglionic sympathetic and parasympathetic synapses on individual ganglion cells (Figure 3). This unusual organization has been reported before using microelectrode recordings (see Crowcroft and Szurszewski, J Physiol (1971) and Janig and McLachlan, Physiol Rev (1987)). Anatomical evidence of convergence in the pelvic ganglion has been reported by Keast, Neuroscience (1995).

Contrary to what the referee says, we do not provide in Figure 3 any evidence for anatomic convergence, i.e. for individual pelvic ganglion cells receiving dual lumbar and sacral inputs. We simply show that cholinergic neurons figure prominently among targets of the lumbar pathway. This said, the convergence of both pathways on the same pelvic neurons, described in the references cited by the referee, is another major problem in the theory of the “sacral parasympathetic” (as we discussed previously (Espinosa-Medina et al., 2018)).

It should also be noted that the anatomy of the pelvic ganglion in male rodents is unique. Unlike other species where the ganglion forms a distributed plexus of mini-ganglia, in male rodents the ganglion coalesces into one structure that is easier to find and study. Interestingly the image in Figure 3A appears to show a clustering of Chat-positive and Th-positive neurons. Does this result from the developmental fusion of mini ganglia having distinct sympathetic and parasympathetic origins?

The clustering of Chat-positive and Th-positive cells could arise from a number of developmental mechanisms, that we have no idea of at the moment. This has no bearing on sympathetic and parasympathetic.

In addition, Brunet et al dismiss the cholinergic and noradrenergic phenotypes as a basis for defining parasympathetic and parasympathetic neurons. However, see the bottom of Figure S4 and further counterarguments in Horn (Clin Auton Res (2018)).

The bottom of Figure S4 simply indicates which cells are cholinergic and adrenergic. We have already expounded many times that noradrenergic and cholinergic do not coincide with sympathetic and parasympathetic. Henry Dale (Nobel Prize 1936) demonstrated this. Langley himself devoted several pages of his final treatise to this exception to his “Theory on the relation of drugs to nerve system” (Langley, 1921) (p43) (which was actually a bigger problem for him than it is for us, for reason which are too long to recount here; it is as if the theoretical difficulties experienced by Langley had been internalized to this day in the form of a dismissal of the cholinergic sympathetic neurons as a slightly scandalous but altogether forgettable oddity). (Horn, 2018), reviews the evidence that the thoracic cholinergic sympathetic phenotype is brought about by a secondary switch upon interaction with the target and argues that this would be a fundamental difference with the sacral “parasympathetic”. But in fact the secondary switch is preceded by co-expression of ChAT and VAChT with Th in most sympathetic neurons (reviewed in (Ernsberger and Rohrer, 2018)); and we have no idea of the dynamic in the pelvic ganglion. It may also be mentioned in this context that target-dependent specification of neuronal identity has also been demonstrated of other types of sympathetic neurons ((Furlan et al., 2016)

What then about neuropeptides, whose expression pattern is incompatible with the revised nomenclature proposed by Brunet et al.?

There was never any neuropeptide-inspired criterion for a nomenclature of the autonomic nervous system.

Figure 1B indicates that VIP is expressed by sacral and cranial ganglion cells, but not thoracolumbar ganglion cells.

Contrary to what the referee says, there are VIP-positive cells in our sympathetic data set and even strongly positive ones, except they are scattered and few (red bars on the UMAP). They correspond to cholinergic sympathetics, likely sudomotor, which are known to contain VIP (e.g.(Anderson et al., 2006)(Stanke et al., 2006)). In other words, VIP is probably part of what we call the cholinergic synexpression group (but was not placed in it by our calculations, probably because of a low expression level even in sympathetic noradrenergic cells).

The authors do not mention neuropeptide Y (NPY). The immunocytochemistry literature indicates that NPY is expressed by a large subpopulation of sympathetic neurons but never by sacral or cranial parasympathetic neurons.

Contrary to what the referee says, Keast (Keast, 1995) finds 3.7% of pelvic neurons double stained for NPY and VIP in male rats, and says (Keast, 2006) that in females “co-expression of NPY and VIP is common” ( thus in cholinergic neurons that the referee calls “parasympathetic”). Single cell transcriptomics is probably more sensitive than immunochemistry, and in our dichotomized data set (table S1), NPY is expressed in all pelvic clusters and all sympathetic ganglia. In other words, it is one more argument for their kinship. It does not appear in the heatmap because it ranks below the 100 top genes.

References

Anderson, C. R., Bergner, A. and Murphy, S. M. (2006). How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion? Neuroscience 140, 567–576.

Ernsberger, U. and Rohrer, H. (2018). Sympathetic tales: subdivisons of the autonomic nervous system and the impact of developmental studies. Neural Dev 13, 20.

Espinosa-Medina, I., Outin, E., Picard, C. A., Chettouh, Z., Dymecki, S., Consalez, G. G., Coppola, E. and Brunet, J. F. (2014). Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors. Science 345, 87–90.

Espinosa-Medina, I., Saha, O., Boismoreau, F. and Brunet, J.-F. (2018). The “sacral parasympathetic”: ontogeny and anatomy of a myth. Clin Auton Res 28, 13–21.

Furlan, A., La Manno, G., Lübke, M., Häring, M., Abdo, H., Hochgerner, H., Kupari, J., Usoskin, D., Airaksinen, M. S., Oliver, G., et al. (2016). Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control. 19, 1331–1340.

Gaskell, W. H. (1886). On the Structure, Distribution and Function of the Nerves which innervate the Visceral and Vascular Systems. J Physiol 7, 1-80.9.

Horn, J. P. (2018). The sacral autonomic outflow is parasympathetic: Langley got it right. Clin Auton Res 28, 181–185.

Jänig, W. (2006). The Integrative Action of the Autonomic Nervous System: Neurobiology of Homeostasis. Cambridge: Cambridge University Press.

Keast, J. R. (1995). Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion. Neuroscience 66, 655–662.

Keast, J. R. (2006). Plasticity of pelvic autonomic ganglia and urogenital innervation. International Review of Cytology - a Survey of Cell Biology, Vol 248 248, 141-+.

Langley, J. N. (1921). In The autonomic nervous system (Pt. I)., p. Cambridge: Heffer & Sons ltd.

Langley, J. N. and Anderson, H. K. (1895). The Innervation of the Pelvic and adjoining Viscera: Part II. The Bladder. Part III. The External Generative Organs. Part IV. The Internal Generative Organs. Part V. Position of the Nerve Cells on the Course of the Efferent Nerve Fibres. J Physiol 19, 71–139.

Lee, V. M., Sechrist, J. W., Luetolf, S. and Bronner-Fraser, M. (2003). Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve. Developmental biology 263, 176–190.

Stanke, M., Duong, C. V., Pape, M., Geissen, M., Burbach, G., Deller, T., Gascan, H., Parlato, R., Schütz, G. and Rohrer, H. (2006). Target-dependent specification of the neurotransmitter phenotype:cholinergic differentiation of sympathetic neurons is mediated in vivo by gp130 signaling. Development 133, 141–150.

Zeisel, A., Hochgerner, H., Lönnerberg, P., Johnsson, A., Memic, F., van der Zwan, J., Häring, M., Braun, E., Borm, L. E., La Manno, G., et al. (2018). Molecular Architecture of the Mouse Nervous System. Cell 174, 999-1014.e22.

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This useful manuscript challenges the utility of current paradigms for estimating brain-age with magnetic resonance imaging measures, but presents inadequate evidence to support the suggestion that an alternative approach focused on predicting cognition is more useful. The paper would benefit from a clearer explication of the methods and a more critical evaluation of the conceptual basis of the different models. This work will be of interest to researchers working on brain-age and related models.

Response: Thank you so much for providing high-quality reviews on our manuscript. We revised the manuscript to address all of the reviewers’ comments and provided full responses to each of the comments below.

Briefly, regarding clearer explanations of the methods, we added additional analyses (e.g., commonality analyses on ridge regression and on multiple regressions with a quadratic term for chronological age) to address some of the concerns and additional details in text and figures to ensure that the reader can fully understand our methodological procedures. Regarding the critical evaluation of the conceptual basis of the different models, we added discussions to help with interpretations and the scope of the generalisability of our findings. For instance, as opposed to treating Brain Cognition and Brain Age as separate biomarkers and comparing them in the ability to explain fluid cognition, we now treated the capability of Brain Cognition in capturing fluid cognition as the upper limit of Brain Age’s capability in capturing fluid cognition. In other words, we now examined the extent to which Brain Age missed the variation in the brain MRI that could explain fluid cognition (for this particular issue, please see our response to Reviewer 3 Public Review #4).

Reviewer 1:

This is a reasonably good paper and the use of a commonality analysis is a nice contribution to understanding variance partitioning across different covariates. I have some comments that I believe the authors ought to address which mostly relate to clarity and interpretation.

Reviewer 1 Public Review #1:

First, from a conceptual point of view, the authors focus exclusively on cognition as a downstream outcome. I would suggest the authors nuance their discussion to provide broader considerations of the utility of their method and on the limits of interpretation of brain-age models more generally. Further, I think that since brain-age models by construction confound relevant biological variation with the accuracy of the regression models used to estimate them, there may be limits to the interpretation of (e.g.) the brain-age gap is as a dimensionless biomarker. This has also been discussed elsewhere (see e.g. https://academic.oup.com/brain/article/143/7/2312/5863667). I would suggest that the authors consider and comment on these issues.

Response: Thank you Reviewer 1 for pointing out these important issues. We addressed them in our response to Reviewer 1 Recommendations For The Authors #1 (see below).

Reviewer 1 Public Review #2

Second, from a methods perspective, there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand how the stacked regression models were constructed. Stacked models can be prone to overfitting when combined with cross-validation. This is because the predictions from the first-level models (i.e. the features that are provided to the second level 'stacked' models) contain information about the training set and the test set. If cross-validation is not done very carefully (e.g. using multiple hold-out sets), information leakage can easily occur at the second level. Unfortunately, there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand what was actually done. Please provide more information to enable the reader to better understand the stacked regression models. If the authors are not using an approach that fully preserves training and test separability, they need to do so.

Response: Thank you Reviewer 1. We addressed this issue in our response to Reviewer 1 Recommendations For The Authors #2 (see below). Briefly, we now made it clearer that training models for both non-stacked and stacked models did not involve the test set, ensuring that there was no data leakage between training and test sets.

Reviewer 1 Public Review #3

Please also provide an indication of the different regression strengths that were estimated across the different models and cross-validation splits. Also, how stable were the weights across splits?

Response: Thank you Reviewer 1. We addressed this issue in our response to Reviewer 1 Recommendations For The Authors #3 (see below).

Reviewer 1 Public Review #4:

Please provide more details about the task designs, MRI processing procedures that were employed on this sample in addition to the regression methods, and bias-correction methods used. For example, there are several different parameterisations of the elastic net, please provide equations to describe the method used here so that readers can easily determine how the regularisation parameters should be interpreted.

Response: Thank you Reviewer 1. We addressed this issue in our response to Reviewer 1 Recommendations For The Authors #5-#6. Briefly, we followed your advice and add all of the suggested details.

Reviewer 2 (Public Review):

Reviewer 2 Public Review Overall:

In this study, the authors aimed to evaluate the contribution of brain-age indices in capturing variance in cognitive decline and proposed an alternative index, brain-cognition, for consideration. The study employs suitable data and methods, albeit with some limitations, to address the research questions. A more detailed discussion of methodological limitations in relation to the study's aims is required. For instance, the current commonality analysis may not sufficiently address potential multicollinearity issues, which could confound the findings. Importantly, given that the study did not provide external validation for the indices, it is unclear how well the models would perform and generalize to other samples. This is particularly relevant to their novel index, brain-cognition, given that brain-age has been validated extensively elsewhere. In addition, the paper's rationale for using elastic net, which references previous fMRI studies, seemed somewhat unclear. The discussion could be more nuanced and certain conclusions appear speculative.

Response Thank you for your encouragement. We have now added discussion of methodological limitations (see below). Regarding potential multicollinearity issues, we addressed this comment using Ridge regressions (see our response to Reviewer 2 Recommendations For The Authors #2). Regarding external validation, we now added discussions about how consistency between our results and several recent studies that investigated similar issues with Brain Age in different populations (see Reviewer 2 Recommendations For The Authors #1). Regarding Brain Cognition, we also added previous studies showing similarly high prediction for cognition functioning (Dubois et al., 2018; Pat, Wang, Anney, et al., 2022; Rasero et al., 2021; Sripada et al., 2020; Tetereva et al., 2022; for review, see Vieira et al., 2022). We added a discussion about Elastic Net (see Reviewer 1 Recommendations For The Authors #6)

Discussion

“There are several potential limitations of this study. First, we conducted an investigation relying only on one dataset, the Human Connectome Project in Aging (HCP-A) (Bookheimer et al., 2019). While HCP-A used state-of-the-art MRI methodologies, covered a wide age range from 36 to 100 years old and used several task-fMRI from different tasks that are harder to find in other bigger databases (e.g., UK Biobank from Sudlow et al., 2015), several characteristics of HCP-A might limit the generalisability of our findings. For instance, the tasks used in task-based fMRI in HCP-A are not used widely in clinical settings (Horien et al., 2020). This might make it challenging to translate the approaches used here. Similarly, HCP-A also excluded participants with neurological conditions, possibly making their participants not representative of the general population. Next, while HCP-A’s sample size is not small (n=725 and 504 people, before and after exclusion, respectively), other datasets provide a much larger sample size (Horien et al., 2020). Similarly, HCP-A does not include younger populations. But as mentioned above, a study with a larger sample in older adults (Cole, 2020) and studies in younger populations (8-22 years old) (Butler et al., 2021; Jirsaraie, Kaufmann, et al., 2023) also found small effects of the adjusted Brain Age Gap in explaining cognitive functioning. And the disagreement between the predictive performance of age-prediction models and the utility of Brain Age found here is largely in line with the findings across different phenotypes seen in a recent systematic review (Jirsaraie, Gorelik, et al., 2023).”

Reviewer 2 Public Review #1:

The authors aimed to evaluate how brain-age and brain-cognition indices capture cognitive decline (as mentioned in their title) but did not employ longitudinal data, essential for calculating 'decline'. As a result, 'cognition-fluid' should not be used interchangeably with 'cognitive decline,' which is inappropriate in this context.

Response Thank you for raising this issue. We now no longer used the word ‘cognitive decline’.

Reviewer 2 Public Review #2:

In their first aim, the authors compared the contributions of brain-age and chronological age in explaining variance in cognition-fluid. Results revealed much smaller effect sizes for brain-age indices compared to the large effects for chronological age. While this comparison is noteworthy, it highlights a well-known fact: chronological age is a strong predictor of disease and mortality. Has the brain-age literature systematically overlooked this effect? If so, please provide relevant examples. They conclude that due to the smaller effect size, brain-age may lack clinical significance, for instance, in associations with neurodegenerative disorders. However, caution is required when speculating on what brain-age may fail to predict in the absence of direct empirical testing. This conclusion also overlooks extant brain-age literature: although effect sizes vary across psychiatric and neurological disorders, brain-age has demonstrated significant effects beyond those driven by chronological age, supporting its utility.

Response For aim 1, we focused our claims on cognitive functioning and not on any clinical significance for neurodegenerative disorders. We now made it clearer that the small effects of the Corrected Brain Age Gap in explaining fluid cognition of aging individuals found here are consistent with a study with a larger sample in older adults (Cole, 2020) and studies in younger populations (8-22 years old) (Butler et al., 2021; Jirsaraie, Kaufmann, et al., 2023).

We believe this issue of the utility of brain age on cognitive functioning vs neurological/psychological disorders requires another consideration, namely the discrepancy in the training and test samples typically used for studies focusing on neurological/psychological disorders. We made this point in the discussion now (see below).

Discussion

“There is a notable difference between studies investigating the utility of Brain Age in explaining cognitive functioning, including ours and others (e.g., Butler et al., 2021; Cole, 2020, 2020; Jirsaraie, Kaufmann, et al., 2023) and those explaining neurological/psychological disorders (e.g., Bashyam et al., 2020; Rokicki et al., 2021). That is, those Brain Age studies focusing on neurological/psychological disorders often build age-prediction models from MRI data of largely healthy participants (e.g., controls in a case-control design or large samples in a population-based design), apply the built age-prediction models to participants without vs. with neurological/psychological disorders and compare Brain Age indices between the two groups. This means that age-prediction models from Brain Age studies focusing on neurological/psychological disorders might be under-fitted when applied to participants with neurological/psychological disorders because they were built from largely healthy participants. And thus the difference in Brain Age indices between participants without vs. with neurological/psychological disorders might be confounded by the under-fitted age-prediction models (i.e., Brain Age may predict chronological age well for the controls, but not for those with a disorder). On the contrary, our study and other Brain Age studies focusing on cognitive functioning often build age-prediction models from MRI data of largely healthy participants and apply the built age-prediction models to participants who are also largely healthy. Accordingly, the age-prediction models for explaining cognitive functioning do not suffer from being under-fitted. We consider this as a strength, not a weakness of our study.”

Reviewer 2 Public Review #3:

The second aim's results reveal a discrepancy between the accuracy of their brain-age models in estimating age and the brain-age's capacity to explain variance in cognition-fluid. The authors suggest that if the ultimate goal is to capture cognitive variance, brain-age predictive models should be optimized to predict this target variable rather than age. While this finding is important and noteworthy, additional analyses are needed to eliminate potential confounding factors, such as correlated noise between the data and cognitive outcome, overfitting, or the inclusion of non-healthy participants in the sample. Optimizing brain-age models to predict the target variable instead of age could ultimately shift the focus away from the brain-age paradigm, as it might optimize for a factor differing from age.

Response We discussed the issue regarding the discrepancy between the accuracy of their brain-age models in estimating age and the brain-age's capacity to explain variance in fluid cognition in our response to Reviewer 3 Public Review #9 (see below). This issue is found to be widespread in a recent systematic review (Jirsaraie, Gorelik, et al., 2023). We now provided several strategies to mitigate this issue to improve the utility of Brain Age in explaining other phenotypes based on our current work and others, using different MRI modalities as well as modelling techniques (Bashyam et al., 2020; Jirsaraie, Kaufmann, et al., 2023; Rokicki et al., 2021).

Regarding potential confounding factors, we are not sure what the reviewer meant by “correlated noise between the data and cognitive outcome”. The current study, for instance, used ICA-FIX (Glasser et al., 2016) to remove noise in functional MRI. It is unclear how much ‘noise’ is still left and might confound our findings. More importantly, we are not sure how to define ‘noise’ as referred to by Reviewer 2 here. As for overfitting, we used nested cross-validation to ensure that training and test sets were separate from each other (see Reviewer 1 Recommendations For The Authors #2). If overfitting happened as suggested, we should see a ‘lower’ predictive performance of age-prediction and cognitive-prediction models since the models would fit well with the training set but would not generalise well to the test set. This is not what we found. The predictive performance of our age-prediction and cognitive-prediction models was high and consistent with the literature. Regarding the inclusion of non-healthy participants in the sample, we discussed this above in our response to Reviewer 2 Public Review #2).

Reviewer 2 Public Review #4:

While a primary goal in biomarker research is to obtain indices that effectively explain variance in the outcome variable of interest, thus favouring models optimized for this purpose, the authors' conclusion overlooks the potential value of 'generic/indirect' models, despite sacrificing some additional explained variance provided by ad-hoc or 'specific/direct' models. In this context, we could consider brain-age as a 'generic' index due to its robust out-of-sample validity and significant associations across various health outcome variables reported in the literature. In contrast, the brain-cognition index proposed in this study is presumed to be 'specific' as, without out-of-sample performance metrics and testing with different outcome variables (e.g., neurodegenerative disease), it remains uncertain whether the reported effect would generalize beyond predicting cognition-fluid, the same variable used to condition the brain-cognition model in this study. A 'generic' index like brain-age enables comparability across different applications based on a common benchmark (rather than numerous specific models) and can support explanatory hypotheses (e.g., "accelerated ageing") since it is grounded in its own biological hypothesis. Generic and specific indices are not mutually exclusive; instead, they may offer complementary information. Their respective utility may depend heavily on the context and research or clinical question.

Response Thank you Reviewer 2 for pointing out this important issue. Reviewer 1 (Recommendations For The Authors #4) and Reviewer 3 (Public Review #4) bought up a similar issue. We agreed with Reviewer 2 that both 'specific/direct' index and Brain Age as a 'generic/indirect' index have merit in their own right. We made a discussion about this issue in our response to Reviewer 3 Public Review #4 (please see this response below).

Briefly, in the revision, as opposed to treating Brain Cognition and Brain Age as separate biomarkers and comparing them, we treated the capability of Brain Cognition in capturing fluid cognition as the upper limit of Brain Age’s capability in capturing fluid cognition. In other words, we now examined the extent to which Brain Age missed the variation in the brain MRI that could explain fluid cognition. We also made a discussion about using our commonality approach to test for this missing variation in future work:

Discussion

“Finally, researchers should test how much Brain Age miss the variation in the brain MRI that could explain fluid cognition or other phenotypes of interest. As demonstrated here, one straightforward method is to build a prediction model using a phenotype of interest as the target (e.g., fluid cognition) and incorporate the predicted value of this model (e.g., Brain Cognition), along with Brain Age and chronological age, into a multiple regression for commonality analyses. The unique effect of this predicted value will inform the missing variation in the brain MRI from Brain Age. If this unique effect is large, then researchers might need to reconsider whether using Brain Age is appropriate for a particular phenotype of interest.”

Reviewer 2 Public Review #5:

The study's third aim was to evaluate the authors' new index, brain-cognition. The results and conclusions drawn appear similar: compared to brain-age, brain-cognition captures more variance in the outcome variable, cognition-fluid. However, greater context and discussion of limitations is required here. Given the nature of the input variables (a large proportion of models in the study were based on fMRI data using cognitive tasks), it is perhaps unsurprising that optimizing these features for cognition-fluid generates an index better at explaining variance in cognition-fluid than the same features used to predict age. In other words, it is expected that brain-cognition would outperform brain-age in explaining variance in cognition-fluid since the former was optimized for the same variable in the same sample, while brain-age was optimized for age. Consequently, it is unclear if potential overfitting issues may inflate the brain-cognition's performance. This may be more evident when the model's input features are the ones closely related to cognition, e.g., fMRI tasks. When features were less directly related to cognitive tasks, e.g., structural MRI, the effect sizes for brain-cognition were notably smaller (see 'Total Brain Volume' and 'Subcortical Volume' models in Figure 6). This observation raises an important feasibility issue that the authors do not consider. Given the low likelihood of having task-based fMRI data available in clinical settings (such as hospitals), estimating a brain-cognition index that yields the large effects discussed in the study may be challenged by data scarcity.

Response Given the use of nested cross-validation, we do not consider the good predictive performance of Brain Cognition found here as overfitting. In fact, we found a similar level of predictive performance of Brain Cognition on another database with younger participants in the past (Tetereva et al., 2022). However, we agreed with Reviewer 2 that the prediction of fluid cognition might be driven by MRI modalities that are different from those that drive the prediction of chronological age. In our own work with other age groups, including young adults (Tetereva et al., 2022) and children (Pat, Wang, Anney, et al., 2022), cognitive functioning seems to be predicted well from task-based functional MRI. And Reviewer 2 is right that task-based fMRI is not commonly used in clinics, making it harder to translate our results. However, given our results, clinicians should be encouraged to use task-based fMRI if their goal is to predict cognitive functioning. Nevertheless, as suggested, we listed data scarcity as one of the limitations of our approach.

Discussion “For instance, the tasks used in task-based fMRI in HCP-A are not used widely in clinical settings (Horien et al., 2020). This might make it challenging to translate the approaches used here.”

Reviewer 2 Public Review #6:

This study is valuable and likely to be useful in two main ways. First, it can spur further research aimed at disentangling the lack of correspondence reported between the accuracy of the brain-age model and the brain-age's capacity to explain variance in fluid cognitive ability. Second, the study may serve, at least in part, as an illustration of the potential pros and cons of using indices that are specific and directly related to the outcome variable versus those that are generic and only indirectly related.

Response We are thankful for the encouragement. For the discrepancy between the predictive performance of age-prediction models and the utility of Brain Age indices as a biomarker for fluid cognition, we made a detailed discussion in our response to Reviewer 3 Public Review #9. More specifically, to ensure that readers can benefit from our findings, we made suggestions on how to ensure the utility of Brain Age indices as a biomarker for other phenotypes by drawing from our own strategy, as well as strategies used by Rokicki and colleagues (2021), Jirsaraie and colleagues (2023) and Bashyam and colleagues (2020).

As for the pros and cons between generic vs specific biomarkers, we made a detailed discussion in our response to Reviewer 3 Public Review #4. We also made some suggestions on how to make use of the difference in the ability between generic vs specific biomarkers (see Reviewer 2 Public Review #4, above).

Reviewer 2 Public Review #7:

Overall, the authors effectively present a clear design and well-structured procedure; however, their work could have been enhanced by providing more context for both the brain-age and brain-cognition indices, including a discussion of key concepts in the brain-age paradigm, which acknowledges that chronological age strongly predicts negative health outcomes, but crucially, recognizes that ageing does not affect everyone uniformly. Capturing this deviation from a healthy norm of ageing is the key brain-age index. This lack of context was mirrored in the presentation of the four brain-age indices provided, as it does not refer to how these indices are used in practice. In fact, there is no mention of a more common way in which brain-age is implemented in statistical analyses, which involves the use of brain-age delta as the variable of interest, along with linear and non-linear terms of age as covariates. The latter is used to account for the regression-to-the-mean effect. The 'corrected brain-age delta' the authors use does not include a non-linear term, which perhaps is an additional reason (besides the one provided by the authors) as to why there may be small, but non-zero, common effects of both age and brain-age in the 'corrected brain-age delta' index commonality analysis. The context for brain-cognition was even more limited, with no reference to any existing literature that has explored direct brain-cognitive markers, such as brain-cognition.

Response Regarding Brain Age and negative health outcomes, we addressed this in our response to Reviewer 1 Recommendations For The Authors #1 (see below). Briefly, we now discussed (1) the consistency between our findings on fluid cognition and other recent works on negative health outcomes, (2) the differences between Brain Age studies focusing on negative health outcomes vs. cognitive functioning and (3) suggested solutions to optimise the utility of brain age for both cognitive functioning and negative health outcomes.

Regarding how Brain Age was used in practice, we addressed this in our response to Reviewer 3 Public Review #2 (see below). Our argument resonates Butler and colleagues’ (2021) suggestion that the common practice for Brain Age analysis should be re-evaluated: “The MBAG and performance on the complex cognition tasks were not associated (r =  .01, p = 0.71). These results indicate that the association between cognition and the BAG are driven by the association between age and cognitive performance. As such, it is critical that readers of past literature note whether or not age was controlled for when testing for effects on the BAG, as this has not always been common practice (e.g., Beheshti et al., 2018; Cole, Underwood, et al., 2017; Franke et al., 2015; Gaser et al., 2013; Liem et al., 2017; Nenadi c et al., 2017; Steffener et al., 2016). (p. 4097).”

Importantly, we also implemented “brain-age delta as the variable of interest, along with linear and non-linear terms of age as covariates” in our additional analyses along with other implementations (see Reviewer 2 Recommendations For The Authors #3). Of particular note, we found that adding a non-linear term (i.e., a quadratic term for chronological age) barely changed the results of commonality analyses.

We now wrote this paragraph to recommend how future research should implement Brain Age:

Discussion

“First, they have to be aware of the overlap in variation between Brain Age and chronological age and should focus on the contribution of Brain Age over and above chronological age. Using Brain Age Gap will not fix this. Butler and colleagues (2021) recently highlighted this point, “These results indicate that the association between cognition and the BAG are driven by the association between age and cognitive performance. As such, it is critical that readers of past literature note whether or not age was controlled for when testing for effects on the BAG, as this has not always been common practice (p. 4097).” Similar to their recommendation (Butler et al., 2021), we suggest future work focus on Corrected Brain Age Gap or, better, unique effects of Brain Age indices after controlling for chronological age in multiple regressions. In the case of fluid cognition, the unique effects might be too small to be clinically meaningful as shown here and previously (Butler et al., 2021; Jirsaraie, Kaufmann, et al., 2023). “

Regarding brain cognition, we now expanded our explanation about Brain Cognition on how it might be relevant to Brain Age and on Brain Cognition’s predictive performance found previously.

Introduction

“Third and finally, certain variation in the brain MRI is related to fluid cognition, but to what extent does Brain Age not capture this variation? To estimate the variation in the brain MRI that is related to fluid cognition, we could build prediction models that directly predict fluid cognition (i.e., as opposed to chronological age) from brain MRI data. Previous studies found reasonable predictive performances of these cognition-prediction models, built from certain MRI modalities (Dubois et al., 2018; Pat, Wang, Anney, et al., 2022; Rasero et al., 2021; Sripada et al., 2020; Tetereva et al., 2022; for review, see Vieira et al., 2022). Analogous to Brain Age, we called the predicted values from these cognition-prediction models, Brain Cognition. The strength of an out-of-sample relationship between Brain Cognition and fluid cognition reflects variation in the brain MRI that is related to fluid cognition and, therefore, indicates the upper limit of Brain Age’s capability in capturing fluid cognition. Consequently, the unique effects of Brain Cognition that explain fluid cognition beyond Brain Age and chronological age indicate what is missing from Brain Age -- the amount of co-variation between brain MRI and fluid cognition that cannot be captured by Brain Age.”

Discussion

“Third, by introducing Brain Cognition, we showed the extent to which Brain Age indices were not able to capture the variation of brain MRI that is related to fluid cognition. Brain Cognition, from certain cognition-prediction models such as the stacked models, has relatively good predictive performance, consistent with previous studies (Dubois et al., 2018; Pat, Wang, Anney, et al., 2022; Rasero et al., 2021; Sripada et al., 2020; Tetereva et al., 2022; for review, see Vieira et al., 2022).”

Reviewer 2 Public Review #8:

While this paper delivers intriguing and thought-provoking results, it would benefit from recognizing the value that both approaches--brain-age indices and more direct, specific markers like brain-cognition--can contribute to the field.

Response Thank you so much for recognising the value of our work. As we mentioned above in our response to Reviewer 2 Public Review #4 and #6, we made some suggestions on how to make use of the difference in the ability between generic vs specific biomarkers.

Reviewer 3 (Public Review):

Reviewer 3 Public Review Overall:

The main question of this article is as follows: "To what extent does having information on brain-age improve our ability to capture declines in fluid cognition beyond knowing a person's chronological age?" While this question is worthwhile, considering that there is considerable confusion in the field about the nature of brain-age, the authors are currently missing an opportunity to convey the inevitability of their results, given how brain-age and the brain-age gap are calculated. They also argue that brain-cognition is somehow superior to brain-age, but insufficient evidence is provided in support of this claim.

Response We addressed the concerns below. The inevitability of our results is not obvious to many researchers who might be interested in Brain Age. We hope our findings might make many issues surrounding Brain Age more obvious, and we now make many suggestions on how to address some of these issues. We no longer argue that Brain Cognition is superior to Brain Age (Reviewer 3 Public Review #4). Rather, we treated the capability of Brain Cognition in capturing fluid cognition as the upper limit of Brain Age’s capability in capturing fluid cognition. We used the unique effects of Brain Cognition that explain fluid cognition beyond Brain Age and chronological age to indicate how much Brain Age misses the variation in the brain MRI that could explain fluid cognition.

Specific comments follow:

Reviewer 3 Public Review #1:

• "There are many adjustments proposed to correct for this estimation bias" (p3). Regression to the mean is not a sign of bias. Any decent loss function will result in over-predicting the age of younger individuals and under-predicting the age of older individuals. This is a direct result of minimizing an error term (e.g., mean squared error). Therefore, it is inappropriate to refer to regression to the mean as a sign of bias. This misconception has led to a great deal of inappropriate analyses, including "correcting" the brain age gap by regressing out age.

Response: Thank you so much for raising this issue. We used the word ‘bias’ following many articles in the field. For instance,

de Lange and Cole (2020) wrote: “brain-age estimation also involves a frequently observed bias: brain age is overestimated in younger subjects and underestimated in older subjects, while brain age for participants with an age closer to the mean age (of the training dataset) are predicted more accurately (Cole, Le, Kuplicki, McKinney, Yeh, Thompson, Paulus, Investigators, et al., 2018, Liang, Zhang, Niu, 2019, Niu, Zhang, Kounios, Liang, 2019, Smith, Vidaurre, Alfaro-Almagro, Nichols, Miller, 2019).”

Cole (2020) wrote: “As recent research has highlighted a proportional bias in brain-age calculation, whereby the difference between chronological age and brain-predicted age is negatively correlated with chronological age (Le et al., 2018, Liang et al., 2019, Smith et al., 2019), an age-bias correction procedure was used. This entailed calculating the regression line between age (predictor) and brain-predicted age (outcome) in the training set, then using the slope (i.e., coefficient) and intercept of that line to adjust brain-predicted age values in the testing set (by subtracting the intercept and then dividing by the slope). After applying the age-bias correction the brain-predicted age difference (brain-PAD) was calculated; chronological age subtracted from brain-predicted age.”

Beheshiti and colleagues (2019) used bias in their title: “Bias-adjustment in neuroimaging-based brain age frameworks: a robust scheme”

More recently, Cumplido-Mayoral and colleagues (2023) wrote: “As recent research has shown that brain-age estimation involves a proportional bias (de Lange et al., 2020a; Le et al., 2018; Liang et al., 2019; Smith et al., 2019), we applied a well-established age-bias correction procedure to our data (de Lange et al., 2020a; Le et al., 2018).”

Still, we agree with Reviewer 3 that using ‘bias’ might lead to misinterpretation. As Butler and colleagues (Butler et al., 2021) pointed out, ”It is important to note that regression toward the mean is not a failure, but a feature, of regression and related methods.“ We rewrote the paragraph and clarified the “regression towards the mean” issue. We no longer used the word “bias” here:

Introduction

“Note researchers often subtract chronological age from Brain Age, creating an index known as Brain Age Gap (Franke & Gaser, 2019). A higher value of Brain Age Gap is thought to reflect accelerated/premature aging. Yet, given that Brain Age Gap is calculated based on both Brain Age and chronological age, Brain Age Gap still depends on chronological age (Butler et al., 2021). If, for instance, Brain Age was based on prediction models with poor performance and made a prediction that everyone was 50 years old, individual differences in Brain Age Gap would then depend solely on chronological age (i.e., 50 minus chronological age). Moreover, Brain Age is known to demonstrate the “regression towards the mean” phenomenon (Stigler, 1997). More specifically, because Brain Age is a predicted value of a regression model that predicts chronological age, Brain Age is usually shrunk towards the mean age of samples used for training the model (Butler et al., 2021; de Lange & Cole, 2020; Le et al., 2018). Accordingly, Brain Age predicts chronological age more accurately for individuals who are closer to the mean age while overestimating younger individuals’ chronological age and underestimating older individuals’ chronological age. There are many adjustments proposed to correct for the age dependency, but the outcomes tend to be similar to each other (Beheshti et al., 2019; de Lange & Cole, 2020; Liang et al., 2019; Smith et al., 2019). These adjustments can be applied to Brain Age and Brain Age Gap, creating Corrected Brain Age and Corrected Brain Age Gap, respectively. Corrected Brain Age Gap in particular is viewed as being able to control for age dependency (Butler et al., 2021). Here, we tested the utility of different Brain Age calculations in capturing fluid cognition, over and above chronological age.”

Reviewer 3 Public Review #2:

• "Corrected Brain Age Gap in particular is viewed as being able to control for both age dependency and estimation biases (Butler et al., 2021)" (p3). This summary is not accurate as Butler and colleagues did not use the words "corrected" and "biases" in this context. All that authors say in that paper is that regressing out age from the brain age gap - which is referred to as the modified brain age gap (MBAG) - makes it so that the modified brain age gap is not dependent on age, which is true. This metric is meaningless, though, because it is the variance left over after regressing out age from residuals from a model that was predicting age. If it were not for the fact that regression on residuals is not equivalent to multiple regression (and out of sample estimates), MBAG would be a vector of zeros. Upon reading the Methods, I noticed that the authors use a metric from Le et al. (2018) for the "Corrected Brain Age Gap". If they cite the Butler et al. (2021) paper, I highly recommend sticking with the same notation, metrics and terminology throughout. That would greatly help with the interpretability of the present manuscript, and cross-comparisons between the two.

Response: We thank Reviewer 3 for pointing out the issues surrounding our choices of wording: "corrected" and "biases". We share the same frustration with Reviewer 3 in that different brain-age articles use different terminologies, and we tried to make sure our readers understand our calculations of Brain Age indices in order to compare our results with previous work.

We commented on the word “bias” in our response to Reviewer 3 Public Review #1 above and refrained from using this word in the revised manuscript. Here we commented on the use of the word “Corrected Brain Age Gap". And by doing so, we clarified how we calculated it.

Reviewer 3 is right that we cited the work of Butler and colleagues (2021), but wasn’t accurate to say that we used “a metric from Le et al. (2018) for the "Corrected Brain Age Gap". We, instead, used a method described in de Lange and Cole’s (2020) work. We now added equations to explain this method in our Materials and Method section (see below).

It is important to note that Butler and colleagues (2021) did not come up with any adjustment methods. Instead, Butler and colleagues (2021) discussed three adjustment methods:

1) A method proposed by Beheshiti and colleagues (2019). Butler and colleagues (2021) called the result of this method, Modified Brain Age Gap (MBAG). Importantly, Butler and colleagues (2021) discouraged the use of this method due to “researchers misinterpreting the reduced variability of the MBAG as an improvement in prediction accuracy.” Accordingly in our article, we performed methods (2) and (3) below.

2) A method proposed by de Lange and Cole (2020). We used this method in our article (see below for the equations). Briefly, we first fit a regression line predicting the Brain Age from a chronological age in each training set. We then used the slope and intercept of this regression line to adjust Brain Age in the corresponding test set, resulting in an adjusted index of Brain Age. Butler and colleagues (2021) called this index, “Revised Predicted Age.”, while de Lange and Cole’s (2020) originally called this Corrected Brain Age, “Corrected Predicted Age”. Butler and colleagues (2021) then subtracted the chronological age from this index and called it, “Revised Brain Age Gap (RBAG)”. We would like to follow the original terminology, but we do not want to use the word “Predicted Age” since chronological age can be predicted by other variables beyond the brain. We then settled with the word, "Corrected Brain Age" and “Corrected Brain Age Gap". We listed the terminologies used in the past in our article (see below).

3) A method proposed by Le and colleagues (2018). Here, Butler and colleagues (2021) referred to one of the approaches done by Le and colleagues: “include age as a regressor when doing follow-up analyses.” Essentially this is what we did for the commonality analysis. Le and colleagues (2018)’ approach is the same as examining the unique effects of Brain Age in a multiple regression analysis with Chronological Age and Brain Age as regressors.

While indexes from de Lange and Cole’s (2020) and Le and colleagues’ (2018) methods show poor performance in capturing fluid cognition in the current work, we need to stress that many research groups do not believe that these methods are meaningless. In fact, de Lange and Cole’s method (2020) is one of the most commonly implemented methods that can be seen elsewhere (e.g., Cole et al., 2020; Cumplido-Mayoral et al., 2023; Denissen et al., 2022). This index just does not seem to work well in the case of fluid cognition.

Here is how we described how we calculated Brain Age indexes in the revised manuscript:

Methods

“ Brain Age calculations: Brain Age, Brain Age Gap, Corrected Brain Age and Corrected Brain Age Gap In addition to Brain Age, which is the predicted value from the models predicting chronological age in the test sets, we calculated three other indices to reflect the estimation of brain aging. First, Brain Age Gap reflects the difference between the age predicted by brain MRI and the actual, chronological age. Here we simply subtracted the chronological age from Brain Age:

Brain Age Gapi = Brain Agei - chronological agei , (2)

where i is the individual. Next, to reduce the dependency on chronological age (Butler et al., 2021; de Lange & Cole, 2020; Le et al., 2018), we applied a method described in de Lange and Cole’s (2020), which was implemented elsewhere (Cole et al., 2020; Cumplido-Mayoral et al., 2023; Denissen et al., 2022):

In each outer-fold training set: Brain Agei = 0 + 1 chronological agei + εi, (3)

Then in the corresponding outer-fold test set: Corrected Brain Agei = (Brain Agei - 0)/1, (4)

That is, we first fit a regression line predicting the Brain Age from a chronological age in each outer-fold training set. We then used the slope (1) and intercept (0) of this regression line to adjust Brain Age in the corresponding outer-fold test set, resulting in Corrected Brain Age. Note de Lange and Cole (2020) called this Corrected Brain Age, “Corrected Predicted Age”, while Butler (2021) called it “Revised Predicted Age.”

Lastly, we computed Corrected Brain Age Gap by subtracting the chronological age from the Corrected Brain Age (Butler et al., 2021; Cole et al., 2020; de Lange & Cole, 2020; Denissen et al., 2022):

Corrected Brain Age Gap = Corrected Brain Age - chronological age, (5)

Note Cole and colleagues (2020) called Corrected Brain Age Gap, “brain-predicted age difference (brain-PAD),” while Butler and colleagues (2021) called this index, “Revised Brain Age Gap”.

Reviewer 3 Public Review #3:

• "However, the improvement in predicting chronological age may not necessarily make Brain Age to be better at capturing Cognitionfluid. If, for instance, the age-prediction model had the perfect performance, Brian Age Gap would be exactly zero and would have no utility in capturing Cognitionfluid beyond chronological age" (p3). I largely agree with this statement. I would be really careful to distinguish between brain-age and the brain-age gap here, as the former is a predicted value, and the latter is the residual times -1 (i.e., predicted age - age). Therefore, together they explain all of the variance in age. Changing the first sentence to refer to the brain-age gap would be more accurate in this context. The brain-age gap will never be exactly zero, though, even with perfect prediction on the training set, because subjects in the testing set are different from the subjects in the training set.

Response: Thank you so much for pointing this out. We agree to change “Brain Age” to “Brain Age Gap” in the mentioned sentence.

Reviewer 3 Public Review #4:

• "Can we further improve our ability to capture the decline in cognitionfluid by using, not only Brain Age and chronological age, but also another biomarker, Brain Cognition?". This question is fundamentally getting at whether a predicted value of cognition can predict cognition. Assuming the brain parameters can predict cognition decently, and the original cognitive measure that you were predicting is related to your measure of fluid cognition, the answer should be yes. Upon reading the Methods, it became clear that the cognitive variable in the model predicting cognition using brain features (to get predicted cognition, or as the authors refer to it, brain-cognition) is the same as the measure of fluid cognition that you are trying to assess how well brain-cognition can predict. Assuming the brain parameters can predict fluid cognition at all, it is then inevitable that brain-cognition will predict fluid cognition. Therefore, it is inappropriate to use predicted values of a variable to predict the same variable.

Response: Thank you Reviewer 3 for pointing out this important issue. Reviewer 1 (Recommendations For The Authors #4) and Reviewer 2 (Public Review #4) bought up a similar issue. While Reviewer 3 felt that “it is inappropriate to use predicted values of a variable to predict the same variable,“ Reviewer 2 viewed Brain Cognition as a 'specific/direct' index and Brain Age as a 'generic/indirect' index. And both have merit in their own right.

Similar to Reviewer 2, we believe that the specific index is as important and has commonly been used elsewhere in the context of biomarkers. For instance, to obtain neuroimaging biomarkers for Alzheimer’s, neuroimaging researchers often build a predictive model to predict Alzheimer's diagnosis (Khojaste-Sarakhsi et al., 2022). In fact, outside of neuroimaging, polygenic risk scores (PRSs) in genomics are often used following “to use predicted values of a variable to predict the same variable” (Choi et al., 2020). For instance, a PRS of ADHD that indicates the genetic liability to develop ADHD is based on genome-wide association studies of ADHD (Demontis et al., 2019).

Still, we now agreed that it may not be fair to compare the performance of a specific index (Brain Cognition) and a generic index (Brain Age) directly (as pointed out by Reviewer 3 Public Review #6 below). Accordingly, in the revision, as opposed to treating Brain Cognition and Brain Age as separate biomarkers and comparing them, we treated the capability of Brain Cognition in capturing fluid cognition as the upper limit of Brain Age’s capability in capturing fluid cognition. In other words, the strength of an out-of-sample relationship between Brain Cognition and fluid cognition reflects variation in the brain MRI that is related to fluid cognition. And consequently, the unique effects of Brain Cognition that explain fluid cognition beyond Brain Age and chronological age indicate what is missing from Brain Age -- the amount of co-variation between brain MRI and fluid cognition that cannot be captured by Brain Age. According to Reviewer 2, a generic index (Brain Age) “sacrificed some additional explained variance provided” compared to a specific index (Brain Cognition). Here, we used the commonality analyses to quantify how much scarifying was made by Brain Age. See below for the re-conceptualisation of Brain Age vs. Brain Cognition in the revision:

Abstract

“Lastly, we tested how much Brain Age missed the variation in the brain MRI that could explain fluid cognition. To capture this variation in the brain MRI that explained fluid cognition, we computed Brain Cognition, or a predicted value based on prediction models built to directly predict fluid cognition (as opposed to chronological age) from brain MRI data. We found that Brain Cognition captured up to an additional 11% of the total variation in fluid cognition that was missing from the model with only Brain Age and chronological age, leading to around a 1/3-time improvement of the total variation explained.”

Introduction:

“Third and finally, certain variation in the brain MRI is related to fluid cognition, but to what extent does Brain Age not capture this variation? To estimate the variation in the brain MRI that is related to fluid cognition, we could build prediction models that directly predict fluid cognition (i.e., as opposed to chronological age) from brain MRI data. Previous studies found reasonable predictive performances of these cognition-prediction models, built from certain MRI modalities (Dubois et al., 2018; Pat, Wang, Anney, et al., 2022; Rasero et al., 2021; Sripada et al., 2020; Tetereva et al., 2022; for review, see Vieira et al., 2022). Analogous to Brain Age, we called the predicted values from these cognition-prediction models, Brain Cognition. The strength of an out-of-sample relationship between Brain Cognition and fluid cognition reflects variation in the brain MRI that is related to fluid cognition and, therefore, indicates the upper limit of Brain Age’s capability in capturing fluid cognition. Consequently, the unique effects of Brain Cognition that explain fluid cognition beyond Brain Age and chronological age indicate what is missing from Brain Age -- the amount of co-variation between brain MRI and fluid cognition that cannot be captured by Brain Age.”

“Finally, we investigated the extent to which Brain Age indices missed the variation in the brain MRI that could explain fluid cognition. Here, we tested Brain Cognition’s unique effects in multiple regression models with a Brain Age index, chronological age and Brain Cognition as regressors to explain fluid cognition.“

Discussion

“Third, how much does Brain Age miss the variation in the brain MRI that could explain fluid cognition? Brain Age and chronological age by themselves captured around 32% of the total variation in fluid cognition. But, around an additional 11% of the variation in fluid cognition could have been captured if we used the prediction models that directly predicted fluid cognition from brain MRI.

“Third, by introducing Brain Cognition, we showed the extent to which Brain Age indices were not able to capture the variation of brain MRI that is related to fluid cognition. Brain Cognition, from certain cognition-prediction models such as the stacked models, has relatively good predictive performance, consistent with previous studies (Dubois et al., 2018; Pat, Wang, Anney, et al., 2022; Rasero et al., 2021; Sripada et al., 2020; Tetereva et al., 2022; for review, see Vieira et al., 2022). We then examined Brain Cognition using commonality analyses (Nimon et al., 2008) in multiple regression models having a Brain Age index, chronological age and Brain Cognition as regressors to explain fluid cognition. Similar to Brain Age indices, Brain Cognition exhibited large common effects with chronological age. But more importantly, unlike Brain Age indices, Brain Cognition showed large unique effects, up to around 11%. The unique effects of Brain Cognition indicated the amount of co-variation between brain MRI and fluid cognition that was missed by a Brain Age index and chronological age. This missing amount was relatively high, considering that Brain Age and chronological age together explained around 32% of the total variation in fluid cognition. Accordingly, if a Brain Age index was used as a biomarker along with chronological age, we would have missed an opportunity to improve the performance of the model by around one-third of the variation explained.”

Reviewer 3 Public Review #5:

• "However, Brain Age Gap created from the lower-performing age-prediction models explained a higher amount of variation in Cognitionfluid. For instance, the top performing age-prediction model, "Stacked: All excluding Task Contrast", generated Brain Age and Corrected Brain Age that explained the highest amount of variation in Cognitionfluid, but, at the same time, produced Brian Age Gap that explained the least amount of variation in Cognitionfluid" (p7). This is an inevitable consequence of the following relationship between predicted values and residuals (or residuals times -1): y=(y-y ̂ )+y ̂. Let's say that age explains 60% of the variance in fluid cognition, and predicted age (y ̂) explains 40% of the variance in fluid cognition. Then the brain age gap (-(y-y ̂)) should explain 20% of the variance in fluid cognition. If by "Corrected Brain Age" you mean the modified predicted age from Butler et al (2021), the "Corrected Brain Age" result is inevitable because the modified predicted age is essentially just age with a tiny bit of noise added to it. From Figure 4, though, this does not seem to be the case, because the lower left quadrant in panel (a) should be flat and high (about as high as the predictive value of age for fluid cognition). So it is unclear how "Corrected Brain Age" is calculated. It looks like you might be regressing age out of brain-age, though from your description in the Methods section, it is not totally clear. Again, I highly recommend using the terminology and metrics of Butler et al (2021) throughout to reduce confusion. Please also clarify how you used the slope and intercept. In general, given how brain-age metrics tend to be calculated, the following conclusion is inevitable: "As before, the unique effects of Brain Age indices were all relatively small across the four Brain Age indices and across different prediction models" (p10).

Response: We agreed that the results are ‘inevitable’ due to the transformations from Brain Age to other Brain Age indices. However, the consequences of these transformations may not be very clear to readers who are not very familiar with Brain Age literature and to the community at large who think about the implications of Brain Age. This is appreciated by Reviewer 1, who mentioned “While the main message will not come as a surprise to anyone with hands-on experience of using brain-age models, I think it is nonetheless an important message to convey to the community.”

Note we made clarifications on how we calculated each of the Brain Age indices above (see<br /> Reviewer 3 Public Review #2), including how we used the slope and intercept. We chose the terminology closer to the one originally used by de Lange and Cole (2020) and now listed many terminologies others have used to refer to this transformation.

Reviewer 3 Public Review #6:

"On the contrary, the unique effects of Brain Cognition appeared much larger" (p10). This is not a fair comparison if you do not look at the unique effects above and beyond the cognitive variable you predicted in your brain-cognition model. If your outcome measure had been another metric of cognition other than fluid cognition, you would see that brain-cognition does not explain any additional variance in this outcome when you include fluid cognition in the model, just as brain-age would not when including age in the model (minus small amounts due to penalization and out-of-sample estimates). This highlights the fact that using a predicted value to predict anything is worse than using the value itself.

Response Please see our response to Reviewer 3 Public Review #4 above. Briefly, we no long made this comparison. Instead, we now viewed the unique effects of Brain Cognition as a way to test how much Brain Age missed the variation in the brain MRI that could explain fluid cognition.

Reviewer 3 Public Review #7:

"First, how much does Brain Age add to what is already captured by chronological age? The short answer is very little" (p12). This is a really important point, but the paper requires an in-depth discussion of the inevitability of this result, as discussed above.

Response We agree that the tight relationship between Brain Age and chronological age is inevitable. We mentioned this from the get-go in the introduction:

Introduction “Accordingly, by design, Brain Age is tightly close to chronological age. Because chronological age usually has a strong relationship with fluid cognition, to begin with, it is unclear how much Brain Age adds to what is already captured by chronological age.”

To make this point obvious, we quantified the overlap between Brain Age and chronological age using the commonality analysis. We hope that our effort to show the inevitability of this overlap can make people more careful when designing studies involving Brain Age.

Reviewer 3 Public Review #8:

"Third, do we have a solution that can improve our ability to capture Cognitionfluid from brain MRI? The answer is, fortunately, yes. Using Brain Cognition as a biomarker, along with chronological age, seemed to capture a higher amount of variation in Cognitionfluid than only using Brain Age" (p12). I suggest controlling for the cognitive measure you predicted in your brain-cognition model. This will show that brain-cognition is not useful above and beyond cognition, highlighting the fact that it is not a useful endeavor to be using predicted values.

Response This point is similar to Reviewer 3 Public Review #6. Again please see our response to Reviewer 3 Public Review #4 above. Briefly, we no long made this comparison and said whether Brain Cognition is ‘better’ than Brain Age. Instead, we now viewed the unique effects of Brain Cognition as a way to test how much Brain Age missed the variation in the brain MRI that could explain fluid cognition.

Reviewer 3 Public Review #9:

"Accordingly, a race to improve the performance of age-prediction models (Baecker et al., 2021) does not necessarily enhance the utility of Brain Age indices as a biomarker for Cognitionfluid. This calls for a new paradigm. Future research should aim to build prediction models for Brian Age indices that are not necessarily good at predicting age, but at capturing phenotypes of interest, such as Cognitionfluid and beyond" (p13). I whole-heartedly agree with the first two sentences, but strongly disagree with the last. Certainly your results, and the underlying reason as to why you found these results, calls for a new paradigm (or, one might argue, a pre-brain-age paradigm). As of now, your results do not suggest that researchers should keep going down the brain-age path. While it is difficult to prove that there is no transformation of brain-age or the brain-age gap that will be useful, I am nearly sure this is true from the research I have done. If you would like to suggest that the field should continue down this path, I suggest presenting a very good case to support this view.

Response Thank you for your comments on this issue.

Since the submission of our manuscript, other researchers also made a similar observation regarding the disagreement between the predictive performance of age-prediction models and the utility of Brain Age. For instance, in their systematic review, Jirasarie and colleagues (2023, p7) wrote this statement, “Despite mounting evidence, there is a persisting assumption across several studies that the most accurate brain age models will have the most potential for detecting differences in a given phenotype of interest. As a point of illustration, seven of the twenty studies in this review only evaluated the utility of their most accurate model, which in all cases was trained using multimodal features. This approach has also led to researchers to exclusively use T1-weighted and diffusion-weighted MRI scans when developing brain age models36 since such modalities have been shown to have the largest contribution to a model’s predictive power.2,67 However, our review suggests that model accuracy does not necessarily provide meaningful insight about clinical utility (e.g., detection of age-related pathology). Taken with prior studies,16,17 it appears that the most accurate models tend to not be the most useful.”

We now discussed the disagreement between the predictive performance of age-prediction models and the utility of Brain Age, not only in the context of cognitive functioning (Jirsaraie, Kaufmann, et al., 2023) but also in the context of neurological/psychological disorders (Bashyam et al., 2020; Rokicki et al., 2021). Following Reviewer 3’s suggestion, we also added several possible strategies to mitigate this problem of Brain Age, used by us and other groups. Please see below.

Discussion:

“This discrepancy between the predictive performance of age-prediction models and the utility of Brain Age indices as a biomarker is consistent with recent findings (for review, see Jirsaraie, Gorelik, et al., 2023), both in the context of cognitive functioning (Jirsaraie, Kaufmann, et al., 2023) and neurological/psychological disorders (Bashyam et al., 2020; Rokicki et al., 2021). For instance, combining different MRI modalities into the prediction models, similar to our stacked models, often lead to the highest performance of age-prediction models, but does not likely explain the highest variance across different phenotypes, including cognitive functioning and beyond (Jirsaraie, Gorelik, et al., 2023).”

“Next, researchers should not select age-prediction models based solely on age-prediction performance. Instead, researchers could select age-prediction models that explained phenotypes of interest the best. Here we selected age-prediction models based on a set of features (i.e., modalities) of brain MRI. This strategy was found effective not only for fluid cognition as we demonstrated here, but also for neurological and psychological disorders as shown elsewhere (Jirsaraie, Gorelik, et al., 2023; Rokicki et al., 2021). Rokicki and colleagues (2021), for instance, found that, while integrating across MRI modalities led to age-prediction models with the highest age-prediction performance, using only T1 structural MRI gave age-prediction models that were better at classifying Alzheimer’s disease. Similarly, using only cerebral blood flow gave age-prediction models that were better at classifying mild/subjective cognitive impairment, schizophrenia and bipolar disorder.

As opposed to selecting age-prediction models based on a set of features, researchers could also select age-prediction models based on modelling methods. For instance, Jirsaraie and colleagues (2023) compared gradient tree boosting (GTB) and deep-learning brain network (DBN) algorithms in building age-prediction models. They found GTB to have higher age-prediction performance but DBN to have better utility in explaining cognitive functioning. In this case, an algorithm with better utility (e.g., DBN) should be used for explaining a phenotype of interest. Similarly, Bashyam and colleagues (2020) built different DBN-based age-prediction models, varying in age-prediction performance. The DBN models with a higher number of epochs corresponded to higher age-prediction performance. However, DBN-based age-prediction models with a moderate (as opposed to higher or lower) number of epochs were better at classifying Alzheimer’s disease, mild cognitive impairment and schizophrenia. In this case, a model from the same algorithm with better utility (e.g., those DBN with a moderate epoch number) should be used for explaining a phenotype of interest. Accordingly, this calls for a change in research practice, as recently pointed out by Jirasarie and colleagues (2023, p7), “Despite mounting evidence, there is a persisting assumption across several studies that the most accurate brain age models will have the most potential for detecting differences in a given phenotype of interest”. Future neuroimaging research should aim to build age-prediction models that are not necessarily good at predicting age, but at capturing phenotypes of interest.”

Reviewer #1 (Recommendations For The Authors):

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline using the HCP aging dataset by performing a commonality analysis in a downstream regression. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain-cognition') as an alternative that explains more unique variance in the downstream regression.

This is a reasonably good paper and the use of a commonality analysis is a nice contribution to understanding variance partitioning across different covariates. While the main message will not come as a surprise to anyone with hands-on experience of using brain-age models, I think it is nonetheless an important message to convey to the community. With that said, I have some comments that I believe the authors ought to address before publication.

Reviewer 1 Recommendations For The Authors #1:

First, from a conceptual point of view, the authors focus exclusively on cognition as a downstream outcome. This is undeniably important, but is only one application area for brain age models. They are also used for example to provide biomarkers for many brain disorders. What would the results presented here have to say about these application areas? Further, I think that since brain-age models by construction confound relevant biological variation with the accuracy of the regression models used to estimate them, my own opinion about the limits of interpretation of (e.g.) the brain-age gap is as a dimensionless biomarker. This has also been discussed elsewhere (see e.g. https://academic.oup.com/brain/article/143/7/2312/5863667). I would suggest the authors nuance their discussion to provide considerations on these issues.

Response Thank you Reviewer 1 for pointing out two important issues.

The first issue was about applications for brain disorders. We now made a detailed discussion about this, which also addressed Reviewer 3 Public Review #9. Briefly, we now bought up

1) the consistency between our findings on fluid cognition and other recent works on brain disorders,

2) under-fitted age-prediction models from Brain Age studies focusing on neurological/psychological disorders when applied to participants with neurological/psychological disorders because the age-prediction models were built from largely healthy participants,

and 3) suggested solutions we and others made to optimise the utility of Brain Age for both cognitive functioning and brain disorders.

Discussion:

“This discrepancy between the predictive performance of age-prediction models and the utility of Brain Age indices as a biomarker is consistent with recent findings (for review, see Jirsaraie, Gorelik, et al., 2023), both in the context of cognitive functioning (Jirsaraie, Kaufmann, et al., 2023) and neurological/psychological disorders (Bashyam et al., 2020; Rokicki et al., 2021). For instance, combining different MRI modalities into the prediction models, similar to our stacked models, often lead to the highest performance of age-prediction models, but does not likely explain the highest variance across different phenotypes, including cognitive functioning and beyond (Jirsaraie, Gorelik, et al., 2023).”

“There is a notable difference between studies investigating the utility of Brain Age in explaining cognitive functioning, including ours and others (e.g., Butler et al., 2021; Cole, 2020, 2020; Jirsaraie, Kaufmann, et al., 2023) and those explaining neurological/psychological disorders (e.g., Bashyam et al., 2020; Rokicki et al., 2021). That is, those Brain Age studies focusing on neurological/psychological disorders often build age-prediction models from MRI data of largely healthy participants (e.g., controls in a case-control design or large samples in a population-based design), apply the built age-prediction models to participants without vs. with neurological/psychological disorders and compare Brain Age indices between the two groups. This means that age-prediction models from Brain Age studies focusing on neurological/psychological disorders might be under-fitted when applied to participants with neurological/psychological disorders because they were built from largely healthy participants. And thus, the difference in Brain Age indices between participants without vs. with neurological/psychological disorders might be confounded by the under-fitted age-prediction models (i.e., Brain Age may predict chronological age well for the controls, but not for those with a disorder). On the contrary, our study and other Brain Age studies focusing on cognitive functioning often build age-prediction models from MRI data of largely healthy participants and apply the built age-prediction models to participants who are also largely healthy. Accordingly, the age-prediction models for explaining cognitive functioning do not suffer from being under-fitted. We consider this as a strength, not a weakness of our study.”

“Next, researchers should not select age-prediction models based solely on age-prediction performance. Instead, researchers could select age-prediction models that explained phenotypes of interest the best. Here we selected age-prediction models based on a set of features (i.e., modalities) of brain MRI. This strategy was found effective not only for fluid cognition as we demonstrated here, but also for neurological and psychological disorders as shown elsewhere (Jirsaraie, Gorelik, et al., 2023; Rokicki et al., 2021). Rokicki and colleagues (2021), for instance, found that, while integrating across MRI modalities led to age-prediction models with the highest age-prediction performance, using only T1 structural MRI gave age-prediction models that were better at classifying Alzheimer’s disease. Similarly, using only cerebral blood flow gave age-prediction models that were better at classifying mild/subjective cognitive impairment, schizophrenia and bipolar disorder. As opposed to selecting age-prediction models based on a set of features, researchers could also select age-prediction models based on modelling methods. For instance, Jirsaraie and colleagues (2023) compared gradient tree boosting (GTB) and deep-learning brain network (DBN) algorithms in building age-prediction models. They found GTB to have higher age-prediction performance but DBN to have better utility in explaining cognitive functioning. In this case, an algorithm with better utility (e.g., DBN) should be used for explaining a phenotype of interest. Similarly, Bashyam and colleagues (2020) built different DBN-based age-prediction models, varying in age-prediction performance. The DBN models with a higher number of epochs corresponded to higher age-prediction performance. However, DBN-based age-prediction models with a moderate (as opposed to higher or lower) number of epochs were better at classifying Alzheimer’s disease, mild cognitive impairment and schizophrenia. In this case, a model from the same algorithm with better utility (e.g., those DBN with a moderate epoch number) should be used for explaining a phenotype of interest. Accordingly, this calls for a change in research practice, as recently pointed out by Jirasarie and colleagues (2023, p7), “Despite mounting evidence, there is a persisting assumption across several studies that the most accurate brain age models will have the most potential for detecting differences in a given phenotype of interest”. Future neuroimaging research should aim to build age-prediction models that are not necessarily good at predicting age, but at capturing phenotypes of interest.”

The second issue was about “the brain-age gap as a dimensionless biomarker.” We are not so clear on what the reviewer meant by “the dimensionless biomarker.” One possible meaning of the “dimensionless biomarker” is the fact that Brain Age from the same algorithm and same modality can be computed, such that Brain Age can be tightly fit or loosely fit with chronological age. This is what Bashyam and colleagues (2020) did in the article Reviewer 1 referred to. We now wrote about this strategy in the above paragraph in the Discussion.

Alternatively, “the dimensionless biomarker” might be something closer to what Reviewer 2 viewed Brain Age as a “generic/indirect” index (as opposed to a 'specific/direct' index in the case of Brain Cognition) (see Reviewer 2 Public Review #4). We discussed this in our response to Reviewer 3 Public Review #4.

Reviewer 1 Recommendations For The Authors #2:

Second, from a methods perspective, I am quite suspicious of the stacked regression models the authors are using to combine regression models and I suspect they may be overfit. In my experience, stacked models are very prone to overfitting when combined with cross-validation. This is because the predictions from the first level models (i,e. the features that are provided to the second-level 'stacked' models) contain information about the training set and the test set. If cross-validation is not done very carefully (e.g. using multiple hold-out sets), information leakage can easily occur at the second level. Unfortunately, there is not sufficient explanation of the methodological procedures in the current manuscript to fully understand what was done. First, please provide more information to enable the reader to better understand the stacked regression models and if the authors are not using an approach that fully preserves training and test separability, please do so.

Response: We would like to thank Reviewer 1 for the suggestion. We now made it clearer in texts and new figure (see below) that we used nested cross-validation to ensure no information leakage between training and test sets. Regarding the stacked models more specifically, the hyperparameters of the stacked models were tuned in the same inner-fold CV as the non-stacked model (see Figure 7 below). That is, training models for both non-stacked and stacked models did not involve the test set, ensuring that there was no data leakage between training and test sets.

Methods:

“To compute Brain Age and Brain Cognition, we ran two separate prediction models. These prediction models either had chronological age or fluid cognition as the target and standardised brain MRI as the features (Denissen et al., 2022). We used nested cross-validation (CV) to build these models (see Figure 7). We first split the data into five outer folds. We used five outer folds so that each outer fold had around 100 participants. This is to ensure the stability of the test performance across folds. In each outer-fold CV, one of the outer folds was treated as a test set, and the rest was treated as a training set, which was further divided into five inner folds. In each inner-fold CV, one of the inner folds was treated as a validation set and the rest was treated as a training set. We used the inner-fold CV to tune for hyperparameters of the models and the outer-fold CV to evaluate the predictive performance of the models.

In addition to using each of the 18 sets of features in separate prediction models, we drew information across these sets via stacking. Specifically, we computed predicted values from each of the 18 sets of features in the training sets. We then treated different combinations of these predicted values as features to predict the targets in separate “stacked” models. The hyperparameters of the stacked models were tuned in the same inner-fold CV as the non-stacked model (see Figure 7). That is, training models for both non-stacked and stacked models did not involve the test set, ensuring that there was no data leakage between training and test sets. We specified eight stacked models: “All” (i.e., including all 18 sets of features), “All excluding Task FC”, “All excluding Task Contrast”, “Non-Task” (i.e., including only Rest FC and sMRI), “Resting and Task FC”, “Task Contrast and FC”, “Task Contrast” and “Task FC”. Accordingly, in total, there were 26 prediction models for Brain Age and Brain Cognition.

Reviewer 1 Recommendations For The Authors #3:

Third, the authors standardize the elastic net regression coefficients post-hoc. Why did the authors not perform the more standard approach of standardizing the covariates and responses, prior to model estimation, which would yield standardized regression coefficients (in the classical sense) by construction? Please also provide an indication of the different regression strengths that were estimated across the different models and cross-validation splits. Also, how stable were the weights across splits?

Response For model fitting, we did not “standardize the elastic net regression coefficients post-hoc.” Instead, we did all of the standardisation steps prior to model fitting (see Methods below). For regression strengths across different models and cross-validation splits, we now provided predictive performance at each of the five outer-fold test sets in Figure 1 (below). As you may have seen, the predictive performance was quite stable across the cross-validation splits.

For visualising feature importance, We originally only standardised the elastic net regression coefficients post-hoc, so that feature importance plots were in the same scale across folds. However, as mentioned by Reviewer 3 (Recommendations for the Authors #7, below), this might make it difficult to interpret the directionality of the coefficients. In the revised manuscript, we refitted the Elastic Net model to the full dataset without splitting them into five folds and visualised the coefficients on brain images (see below).

Methods

“We controlled for the potential influences of biological sex on the brain features by first residualising biological sex from brain features in each outer-fold training set. We then applied the regression of this residualisation to the corresponding test set. We also standardised the brain features in each outer-fold training set and then used the mean and standard deviation of this outer-fold training set to standardise the test set. All of the standardisation was done prior to fitting the prediction models.”

“To understand how Elastic Net made a prediction based on different brain features, we examined the coefficients of the tuned model. Elastic Net coefficients can be considered as feature importance, such that more positive Elastic Net coefficients lead to more positive predicted values and, similarly, more negative Elastic Net coefficients lead to more negative predicted values (Molnar, 2019; Pat, Wang, Bartonicek, et al., 2022). While the magnitude of Elastic Net coefficients is regularised (thus making it difficult for us to interpret the magnitude itself directly), we could still indicate that a brain feature with a higher magnitude weights relatively stronger in making a prediction. Another benefit of Elastic Net as a penalised regression is that the coefficients are less susceptible to collinearity among features as they have already been regularised (Dormann et al., 2013; Pat, Wang, Bartonicek, et al., 2022).

Given that we used five-fold nested cross validation, different outer folds may have different degrees of ‘’ and ‘l_1 ratio’, making the final coefficients from different folds to be different. For instance, for certain sets of features, penalisation may not play a big part (i.e., higher or lower ‘’ leads to similar predictive performance), resulting in different ‘’ for different folds. To remedy this in the visualisation of Elastic Net feature importance, we refitted the Elastic Net model to the full dataset without splitting them into five folds and visualised the coefficients on brain images using Brainspace (Vos De Wael et al., 2020) and Nilern (Abraham et al., 2014) packages. Note, unlike other sets of features, Task FC and Rest FC were modelled after data reduction via PCA. Thus, for Task FC and Rest FC, we, first, multiplied the absolute PCA scores (extracted from the ‘components_’ attribute of ‘sklearn.decomposition.PCA’) with Elastic Net coefficients and, then, summed the multiplied values across the 75 components, leaving 71,631 ROI-pair indices.”

Reviewer 1 Recommendations For The Authors #4:

I do not really find it surprising that the level of unique explained variance provided by a brain-cognition model is higher than a brain-age model, given that the latter is considerably more accurate (also, in view of the comment above). As such I would recommend to tone down the claims about the utility of this method, also because it is only really applicable to one application area for brain age.

Response Thank you for bringing this issue to our attention. We have now toned down the claims about the utility of Brain Cognition and importantly treated the capability of Brain Cognition in capturing fluid cognition as the upper limit of Brain Age’s capability in capturing fluid cognition. Please see Reviewer 3 Public Review #4 above for a detailed discussion about this issue.

Reviewer 1 Recommendations For The Authors #5:

Please provide more details about the task designs and MRI processing procedures that were employed on this sample so that the reader is not forced to dig through the publications from the consortia contributing the data samples used. For example, comments such as "Here we focused on the pre-processed task fMRI files with a suffix "_PA_Atlas_MSMAll_hp0_clean.dtseries.nii." are not particularly helpful to readers not already familiar with this dataset.

Response Thank you so much for pointing out this important point on the clarity of the description of our MRI methodology. We now added additional details about the data processing done by the HCP-A and by us. We, for instance, explained the meaning of the HCP-A suffix “"_PA_Atlas_MSMAll_hp0_clean.dtseries.nii”. Please see below.

Methods

“HCP-A provides details of parameters for brain MRI elsewhere (Bookheimer et al., 2019; Harms et al., 2018). Here we used MRI data that were pre-processed by the HCP-A with recommended methods, including the MSMALL alignment (Glasser et al., 2016; Robinson et al., 2018) and ICA-FIX (Glasser et al., 2016) for functional MRI. We used multiple brain MRI modalities, covering task functional MRI (task fMRI), resting-state functional MRI (rsfMRI) and structural MRI (sMRI), and organised them into 19 sets of features.

Sets of Features 1-10: Task fMRI contrast (Task Contrast)

Task contrasts reflect fMRI activation relevant to events in each task. Bookheimer and colleagues (2019) provided detailed information about the fMRI in HCP-A. Here we focused on the pre-processed task fMRI Connectivity Informatics Technology Initiative (CIFTI) files with a suffix, “_PA_Atlas_MSMAll_hp0_clean.dtseries.nii.” These CIFTI files encompassed both the cortical mesh surface and subcortical volume (Glasser et al., 2013). Collected using the posterior-to-anterior (PA) phase, these files were aligned using MSMALL (Glasser et al., 2016; Robinson et al., 2018), linear detrended (see https://groups.google.com/a/humanconnectome.org/g/hcp-users/c/ZLJc092h980/m/GiihzQAUAwAJ) and cleaned from potential artifacts using ICA-FIX (Glasser et al., 2016).

To extract Task Contrasts, we regressed the fMRI time series on the convolved task events using a double-gamma canonical hemodynamic response function via FMRIB Software Library (FSL)’s FMRI Expert Analysis Tool (FEAT) (Woolrich et al., 2001). We kept FSL’s default high pass cutoff at 200s (i.e., .005 Hz). We then parcellated the contrast ‘cope’ files, using the Glasser atlas (Gordon et al., 2016) for cortical surface regions and the Freesurfer’s automatic segmentation (aseg) (Fischl et al., 2002) for subcortical regions. This resulted in 379 regions, whose number was, in turn, the number of features for each Task Contrast set of features.

HCP-A collected fMRI data from three tasks: Face Name (Sperling et al., 2001), Conditioned Approach Response Inhibition Task (CARIT) (Somerville et al., 2018) and VISual MOTOR (VISMOTOR) (Ances et al., 2009). First, the Face Name task (Sperling et al., 2001) taps into episodic memory. The task had three blocks. In the encoding block [Encoding], participants were asked to memorise the names of faces shown. These faces were then shown again in the recall block [Recall] when the participants were asked if they could remember the names of the previously shown faces. There was also the distractor block [Distractor] occurring between the encoding and recall blocks. Here participants were distracted by a Go/NoGo task. We computed six contrasts for this Face Name task: [Encode], [Recall], [Distractor], [Encode vs. Distractor], [Recall vs. Distractor] and [Encode vs. Recall].

Second, the CARIT task (Somerville et al., 2018) was adapted from the classic Go/NoGo task and taps into inhibitory control. Participants were asked to press a button to all [Go] but not to two [NoGo] shapes. We computed three contrasts for the CARIT task: [NoGo], [Go] and [NoGo vs. Go].

Third, the VISMOTOR task (Ances et al., 2009) was designed to test simple activation of the motor and visual cortices. Participants saw a checkerboard with a red square either on the left or right. They needed to press a corresponding key to indicate the location of the red square. We computed just one contrast for the VISMOTOR task: [Vismotor], which indicates the presence of the checkerboard vs. baseline.

Sets of Features 11-13: Task fMRI functional connectivity (Task FC)

Task FC reflects functional connectivity (FC ) among the brain regions during each task, which is considered an important source of individual differences (Elliott et al., 2019; Fair et al., 2007; Gratton et al., 2018). We used the same CIFTI file “_PA_Atlas_MSMAll_hp0_clean.dtseries.nii.” as the task contrasts. Unlike Task Contrasts, here we treated the double-gamma, convolved task events as regressors of no interest and focused on the residuals of the regression from each task (Fair et al., 2007). We computed these regressors on FSL, and regressed them in nilearn (Abraham et al., 2014). Following previous work on task FC (Elliott et al., 2019), we applied a highpass at .008 Hz. For parcellation, we used the same atlases as Task Contrast (Fischl et al., 2002; Glasser et al., 2016). We computed Pearson’s correlations of each pair of 379 regions, resulting in a table of 71,631 non-overlapping FC indices for each task. We then applied r-to-z transformation and principal component analysis (PCA) of 75 components (Rasero et al., 2021; Sripada et al., 2019, 2020). Note to avoid data leakage, we conducted the PCA on each training set and applied its definition to the corresponding test set. Accordingly, there were three sets of 75 features for Task FC, one for each task. “

Reviewer 1 Recommendations For The Authors #6:

Similarly, please be more specific about the regression methods used. There are several different parameterisations of the elastic net, please provide equations to describe the method used here so that readers can easily determine how the regularisation parameters should be interpreted. The same goes for the methods used for correcting bias, e.g. what is "de Lange and Cole's (2020) 5th equation"?

Response Thank you. We now made a detailed description of Elastic Net including its equation (see below). We also added more specific details about the methods used for correcting bias in Brain Age indices (see our response to Reviewer 3 Public Review #2 above).

Methods:

“For the machine learning algorithm, we used Elastic Net (Zou & Hastie, 2005). Elastic Net is a general form of penalised regressions (including Lasso and Ridge regression), allowing us to simultaneously draw information across different brain indices to predict one target variable. Penalised regressions are commonly used for building age-prediction models (Jirsaraie, Gorelik, et al., 2023). Previously we showed that the performance of Elastic Net in predicting cognitive abilities is on par, if not better than, many non-linear and more-complicated algorithms (Pat, Wang, Bartonicek, et al., 2022; Tetereva et al., 2022). Moreover, Elastic Net coefficients are readily explainable, allowing us the ability to explain how our age-prediction and cognition-prediction models made the prediction from each brain feature (Molnar, 2019; Pat, Wang, Bartonicek, et al., 2022) (see below).

Elastic Net simultaneously minimises the weighted sum of the features’ coefficients. The degree of penalty to the sum of the feature’s coefficients is determined by a shrinkage hyperparameter ‘’: the greater the , the more the coefficients shrink, and the more regularised the model becomes. Elastic Net also includes another hyperparameter, ‘l_1 ratio’, which determines the degree to which the sum of either the squared (known as ‘Ridge’; l_1 ratio=0) or absolute (known as ‘Lasso’; l_1 ratio=1) coefficients is penalised (Zou & Hastie, 2005). The objective function of Elastic Net as implemented by sklearn (Pedregosa et al., 2011) is defined as: argmin_ ((|(|y-X|)|_2^2)/(2×n_samples )+α×l_1 _ratio×|(||)|_1+0.5×α×(1-l_1 _ratio)×|(|w|)|_2^2 ), (1) where X is the features, y is the target, and  is the coefficient. In our grid search, we tuned two Elastic Net hyperparameters:  using 70 numbers in log space, ranging from .1 and 100, and l_1-ratio using 25 numbers in linear space, ranging from 0 and 1.”

Additional minor points:

Reviewer 1 Recommendations For The Authors #7:

• Please provide more descriptive figure legends, especially for Figs 5 and 6. For example, what do the boldface numbers reflect? What do the asterisks reflect?

Response Thank you for the suggestion. We made changes to the figure legends to make it clearer what the numbers and asterisks reflect.

Reviewer 1 Recommendations For The Authors #8:

• Perhaps this is personal thing, but I find the nomenclature cognition_{fluid} to be quite awkward. Why not just define FC as an acronym?

Response Thank you for the suggestion. We now used the word ‘fluid cognition’ throughout the manuscript.

Reviewer #2 (Recommendations For The Authors):

Suggestions for improved or additional experiments, data or analyses.

Reviewer 2 Recommendations For The Authors #1:

• Since the study did not provide external validation for the indices, it is unclear how well the models would perform and generalize to other samples. Therefore, it is recommended to conduct out-of-sample testing of the models.

Response Thank you for the suggestion. We now added discussions about how consistency between our results and several recent studies that investigated similar issues with Brain Age in different populations, e.g., large samples of older adults in Uk Biobank (Cole, 2020) and younger populations (Butler et al., 2021; Jirsaraie, Kaufmann, et al., 2023), and in a broader context, extending to neurological and psychological disorders (for review, see Jirsaraie, Gorelik, et al., 2023). Please see below.

Please also noted that all of the analyses done were out-of-sample. We used nested cross-validation to evaluate the predictive performance of age- and cognition-prediction models on the outer-fold test sets, which are out-of-sample from the training sets (please see Reviewer 1 Recommendations For The Authors #2). Similarly, we also conducted all of the commonality analyses on the outer-fold test sets.

Discussion

“The small effects of the Corrected Brain Age Gap in explaining fluid cognition of aging individuals found here are consistent with studies in older adults (Cole, 2020) and younger populations (Butler et al., 2021; Jirsaraie, Kaufmann, et al., 2023). Cole (2020) studied the utility of Brain Age on cognitive functioning of large samples (n>17,000) of older adults, aged 45-80 years, from the UK Biobank (Sudlow et al., 2015). He constructed age-prediction models using LASSO, a similar penalised regression to ours and applied the same age-dependency adjustment to ours. Cole (2020) then conducted a multiple regression explaining cognitive functioning from Corrected Brain Age Gap while controlling for chronological age and other potential confounds. He found Corrected Brain Age Gap to be significantly related to performance in four out of six cognitive measures, and among those significant relationships, the effect sizes were small with a maximum of partial eta-squared at .0059. Similarly, Jirsaraie and colleagues (2023) studied the utility of Brain Age on cognitive functioning of youths aged 8-22 years old from the Human Connectome Project in Development (Somerville et al., 2018) and Preschool Depression Study (Luby, 2010). They built age-prediction models using gradient tree boosting (GTB) and deep-learning brain network (DBN) and adjusted the age dependency of Brain Age Gap using Smith and colleagues’ (2019) method. Using multiple regressions, Jirsaraie and colleagues (2023) found weak effects of the adjusted Brain Age Gap on cognitive functioning across five cognitive tasks, five age-prediction models and the two datasets (mean of standardised regression coefficient = -0.09, see their Table S7). Next, Butler and colleagues (2021) studied the utility of Brain Age on cognitive functioning of another group of youths aged 8-22 years old from the Philadelphia Neurodevelopmental Cohort (PNC) (Satterthwaite et al., 2016). Here they used Elastic Net to build age-prediction models and applied another age-dependency adjustment method, proposed by Beheshti and colleagues (2019). Similar to the aforementioned results, Butler and colleagues (2021) found a weak, statistically non-significant correlation between the adjusted Brain Age Gap and cognitive functioning at r=-.01, p=.71. Accordingly, the utility of Brain Age in explaining cognitive functioning beyond chronological age appears to be weak across age groups, different predictive modelling algorithms and age-dependency adjustments.“

“This discrepancy between the predictive performance of age-prediction models and the utility of Brain Age indices as a biomarker is consistent with recent findings (for review, see Jirsaraie, Gorelik, et al., 2023), both in the context of cognitive functioning (Jirsaraie, Kaufmann, et al., 2023) and neurological/psychological disorders (Bashyam et al., 2020; Rokicki et al., 2021). For instance, combining different MRI modalities into the prediction models, similar to our stacked models, often lead to the highest performance of age-prediction models, but does not likely explain the highest variance across different phenotypes, including cognitive functioning and beyond (Jirsaraie, Gorelik, et al., 2023). “

“Third, by introducing Brain Cognition, we showed the extent to which Brain Age indices were not able to capture the variation of brain MRI that is related to fluid cognition. Brain Cognition, from certain cognition-prediction models such as the stacked models, has relatively good predictive performance, consistent with previous studies (Dubois et al., 2018; Pat, Wang, Anney, et al., 2022; Rasero et al., 2021; Sripada et al., 2020; Tetereva et al., 2022; for review, see Vieira et al., 2022). We then examined Brain Cognition using commonality analyses (Nimon et al., 2008) in multiple regression models having a Brain Age index, chronological age and Brain Cognition as regressors to explain fluid cognition. Similar to Brain Age indices, Brain Cognition exhibited large common effects with chronological age. But more importantly, unlike Brain Age indices, Brain Cognition showed large unique effects, up to around 11%. The unique effects of Brain Cognition indicated the amount of co-variation between brain MRI and fluid cognition that was missed by a Brain Age index and chronological age. This missing amount was relatively high, considering that Brain Age and chronological age together explained around 32% of the total variation in fluid cognition. Accordingly, if a Brain Age index was used as a biomarker along with chronological age, we would have missed an opportunity to improve the performance of the model by around one-third of the variation explained. “

“There is a notable difference between studies investigating the utility of Brain Age in explaining cognitive functioning, including ours and others (e.g., Butler et al., 2021; Cole, 2020, 2020; Jirsaraie, Kaufmann, et al., 2023) and those explaining neurological/psychological disorders (e.g., Bashyam et al., 2020; Rokicki et al., 2021). That is, those Brain Age studies focusing on neurological/psychological disorders often build age-prediction models from MRI data of largely healthy participants (e.g., controls in a case-control design or large samples in a population-based design), apply the built age-prediction models to participants without vs. with neurological/psychological disorders and compare Brain Age indices between the two groups. This means that age-prediction models from Brain Age studies focusing on neurological/psychological disorders might be under-fitted when applied to participants with neurological/psychological disorders because they were built from largely healthy participants. And thus, the difference in Brain Age indices between participants without vs. with neurological/psychological disorders might be confounded by the under-fitted age-prediction models (i.e., Brain Age may predict chronological age well for the controls, but not for those with a disorder). On the contrary, our study and other Brain Age studies focusing on cognitive functioning often build age-prediction models from MRI data of largely healthy participants and apply the built age-prediction models to participants who are also largely healthy. Accordingly, the age-prediction models for explaining cognitive functioning do not suffer from being under-fitted. We consider this as a strength, not a weakness of our study.”

Reviewer 2 Recommendations For The Authors #2:

• Employ Variance Inflation Factor (VIF) to empirically test for multicollinearity.

Response Given high common effects between many of the regressors in the models (e.g., between Brain Age and chronological age), VIF will be high, but this is not a concern for the commonality analysis. We showed now that applying the commonality analysis to multiple regressions allowed us to have robust results against multicollinearity, as demonstrated elsewhere (Ray-Mukherjee et al., 2014, Using commonality analysis in multiple regressions: A tool to decompose regression effects in the face of multicollinearity). Specifically, using the multiple regressions by themselves without the commonality analysis, researchers have to rely on beta estimates, which are strongly affected by multicollinearity (e.g., a phenomenon known as the Suppression Effect). However, by applying the commonality analysis on top of multiple regressions, researchers can then rely on R2 estimates, which are less affected by multicollinearity. This can be seen in our case (Figure 5 and 6) where Brain Age indices had the same unique effects regardless of the level of common effects they had with chronological age (e.g., Brain Age vs. Corrected Brain Age Gap from stacked models).

To directly demonstrate the robustness of the current commonality analysis regarding multicollinearity, we applied the commonality analysis to Ridge regressions (see Supplementary Figures 3 and 5 below). Ridge regression is a method designed to deal with multicollinearity (Dormann et al., 2013). As seen below, the results from commonality analyses applied to Ridge regressions are closely matched with our original results.

Methods

“Note to ensure that the commonality analysis results were robust against multicollinearity (Ray-Mukherjee et al., 2014), we also repeated the same commonality analyses done here on Ridge regression, as opposed to multiple regression. Ridge regression is a method designed to deal with multicollinearity (Dormann et al., 2013). See Supplementary Figure 3 for the Ridge regression with chronological age and each Brain Age index as regressors and Supplementary Figure 5 for the Ridge regression with chronological age, each Brain Age and Brain Cognition index as regressors. Briefly, the results from commonality analyses applied to Ridge regressions are closely matched with our results done using multiple regression.”

Reviewer 2 Recommendations For The Authors #3:

• Incorporate non-linearities in the correction of brain-age indices, such as separate terms in the regression or statistical analyses.

Response Thank you for the suggestion. We now added a non-linear term of chronological age in our multiple-regression models explaining fluid cognition (see Supplementary Figure 4 and 6 below). Originally we did not have the quadratic term for chronological age in our model since the relationship between chronological age and fluid cognition was relatively linear (see Figure 1 above). Accordingly, as expected, adding the quadratic term for chronological age as suggested did not change the pattern of the results of the commonality analyses.

Methods

“Similarly, to ensure that we were able to capture the non-linear pattern of chronological age in explaining fluid cognition, we added a quadratic term of chronological age to our multiple-regression models in the commonality analyses. See Supplementary Figure 4 for the multiple regression with chronological age, square chronological age and each Brain Age index as regressors and Supplementary Figure 6 for the multiple regression with chronological age, square chronological age, each Brain Age index and Brain Cognition as regressors. Briefly, adding the quadratic term for chronological age did not change the pattern of the results of the commonality analyses.”

Reviewer 2 Recommendations For The Authors #4:

• It would be helpful to include the complete set of results in the appendix - for instance, the statistical significance for each component for the final commonality analysis.

Response Figures 5 and 6 (see above) already have asterisks to reflect the statistical significance of the unique effects. Because of this, we do not believe we need more figures/tables in the appendix to show statistical significance.

Recommendations for improving the writing and presentation.

Reviewer 2 Recommendations For The Authors #5:

• The authors are encouraged to refrain from using terms such as 'fortunately', 'unfortunately', and 'unsettling', as they may appear inappropriate when referring to empirical findings.

Response We agree with this suggestion and no long used those words.

Reviewer 2 Recommendations For The Authors #6:

• It would be helpful to clarify in the methods that you end up with 5 test folds.

Response We now made a clarification why we chose 5 test folds.

Methods

“We used nested cross-validation (CV) to build these models (see Figure 7). We first split the data into five outer folds. We used five outer folds so that each outer fold had around 100 participants. This is to ensure the stability of the test performance across folds.”

Minor corrections to the text and figures.

Reviewer 2 Recommendations For The Authors #7:

• Why use months, not years for chronological age? This seems inappropriate given the age range.

Response We originally used months since they were units used in our prediction modelling. However, to make the figures easier to understand, we now used years.

Reviewer 2 Recommendations For The Authors #8:

• The formatting, especially regarding the text embedded within the figures, could benefit from significant improvements.

Response Thank you for the suggestion. We made changes to the text embedded within the figures. They should be more readable now

Reviewer 2 Recommendations For The Authors #9:

• The legend for the neuroimaging feature labels is missing, and the captions are incomplete.

Response Please see Figure 2 above. We now revised by adding the letter L and R for the laterality of the brain images. We made some changes to the captions to make sure they are complete.

Reviewer 2 Recommendations For The Authors #10:

• Figure 5's caption: SD has a missing decimal point).

Response The numbers are not SD. The numbers to the left of the figure represent the unique effects of chronological age in %, the numbers in the middle of the figure represent the common effects between chronological age and Brain Age index in %, and the numbers to the right of the figure represent the unique effects of Brain Age Index in %. We now used the same one decimal point for these number

Reviewer #3 (Recommendations For The Authors):

The main question of this article is as follows: “To what extent does having information on Brain Age improve our ability to capture declines in fluid cognition beyond knowing a person’s chronological age?” While this question is worthwhile, considering most of the field is confused about the nature of brain age, the authors are currently missing an opportunity to convey the inevitability of their results given how Brain Age and the Brain Age Gap are calculated. They also misleadingly convey that Brain Cognition is somehow superior to Brain Age. If the authors work on conveying the inevitability of their results and redo (or remove) their section on Brain Cognition, I can see how their results would be enlightening to the general neuroimaging community that is interested in the concept of brain age. See below for specific critiques.

Response Please see our response to Reviewer 3 Public Review Overall. Note we no longer argue that Brain Cognition is superior to Brain Age (Reviewer 3 Public Review #4). Rather, we treated the capability of Brain Cognition in capturing fluid cognition as the upper limit of Brain Age’s capability in capturing fluid cognition. We used the unique effects of Brain Cognition that explain fluid cognition beyond Brain Age and chronological age to indicate how much Brain Age misses the variation in the brain MRI that could explain fluid cognition.

Reviewer 3 Recommendations For The Authors #1:

“There are many adjustments proposed to correct for this estimation bias” (p3) → Regression to the mean is not a sign of bias. Any decent loss function will result in over- predicting the age of younger individuals and under-predicting the age of older individuals. This is a direct result of minimizing an error term (e.g., mean squared error). Therefore, it is inappropriate to refer to regression to the mean as a sign of bias. This misconception has led to a great deal of inappropriate analyses, including “correcting” the brain age gap by regressing out age.

Response Please see our response to Reviewer 3 Public Review#1

Reviewer 3 Recommendations For The Authors #2:

“Corrected Brain Age Gap in particular is viewed as being able to control for both age dependency and estimation biases (Butler et al., 2021).” (p3) → This summary is not accurate as Butler and colleagues did not use the words "corrected" and "biases" in this context. All that authors say in that paper is that regressing out age from the brain age gap - which is referred to as the modified brain age gap (MBAG) - makes it so that the modified brain age gap is not dependent on age, which is true. This metric is meaningless, though, because it is the variance left over after regressing out age from residuals from a model that was predicting age. If it were not for the fact that regression on residuals is not equivalent to multiple regression (and out of sample estimates), MBAG would be a vector of zeros. Upon reading your Methods, I noticed that you are using a metric for Le et al. (2018) for your “Corrected Brain Age Gap”. If they cite the Butler et al. (2021) paper, I highly recommend sticking with the same notation, metrics and terminology throughout. That would greatly help with the interpretability of your paper, and cross-comparisons between the two.

Response Please see our response to Reviewer 3 Public Review #2.

Reviewer 3 Recommendations For The Authors #3:

“However, the improvement in predicting chronological age may not necessarily make Brain Age to be better at capturing Cognitionfluid. If, for instance, the age-prediction model had the perfect performance, Brian Age Gap would be exactly zero and would have no utility in capturing Cognitionfluid beyond chronological age.” (p3) → I largely agree with this statement. I would be really careful to distinguish between Brain Age and the Brain Age Gap here, as the former is a predicted value, and the latter is the residual times -1 (predicted age - age). Therefore, together they explain all of the variance in age. If you change the first sentence to refer to the Brain Age Gap, this statement makes more sense. The Brain Age Gap will never be exactly zero, though, even with perfect prediction on the training set, because subjects in the testing set are different from the subjects in the training set.

Response Please see our response to Reviewer 3 Public Review #3.

Reviewer 3 Recommendations For The Authors #4:

“Can we further improve our ability to capture the decline in cognitionfluid by using, not only Brain Age and chronological age, but also another biomarker, Brain Cognition?” → This question is fundamentally getting at whether a predicted value of cognition can predict cognition. Assuming the brain parameters can predict cognition decently, and the original cognitive measure that you were predicting is related to your measure of fluid cognition, the answer should be yes. This seems like an uninteresting question to me. Upon reading your Methods, it became clear that the cognitive variable in the model predicting cognition using brain features (to get predicted cognition, or as you refer to it, Brain Cognition) is the same as the measure of fluid cognition that you are trying to assess how well Brain Cognition can predict. Assuming the brain parameters can predict fluid cognition at all, of course Brain Cognition will predict fluid cognition. This is inevitable. You should never use predicted values of a variable to predict the same variable.

Response Please see our response to Reviewer 3 Public Review #4.

Reviewer 3 Recommendations For The Authors #5:

“We also examined if these better-performing age-prediction models improved the ability of Brain Age in explaining Cognitionfluid.” → Improved above and beyond what?

Response We referred to if better-performing age-prediction models improved the ability of Brain Age in explaining fluid cognition over and above lower-performing age-prediction models. We made changes to the Introduction to clarify this change.

Reviewer 3 Recommendations For The Authors #6:

Figure 1 b & c → It is a little difficult to read the text by the horizontal bars in your plots. Please make the text smaller so that there is more space between the words vertically, or even better, make the plots slightly bigger. Please also put the predicted values on the y-axis. This is standard practice for displaying regression results. To make more room, you can get rid of your rPearson or your R2 plot, considering the latter is simply the square of the former. If you want to make it clear that the association is positive between all of your variables, I would keep rPearson.

Response Thank you so much for the suggestions.

1) We now made sure that the text by the horizontal bars in Figure 1b and c is readable.

2) Note in prediction model/machine-learning literature, it is more common to plot observed/real values on the y-axis. Here is the logic of our practice: values in the x-axis are the predicted values based on the model, and we would like to see if the changes in the predicted values correspond to the changes in the observed/real value in the y-axis.

3) Regarding Pearson correlation vs R2, please note that we wrote ”for R2, we used the sum of squares definition (i.e., R2 = 1 – (sum of squares residuals/total sum of squares)) per a previous recommendation (Poldrack et al., 2020).” As such, R2 is NOT the square of the Pearson correlation. In fact, in Poldrack and colleages’s “Establishment of Best Practices for Evidence for Prediction” paper (2020), they discourage 1) the use of Pearson correlation by itself and 2) the use of the correlation coefficient square as R2 (as opposed to sum of squares definition):

“It is common in the literature to use the correlation between predicted and actual values as a measure of predictive performance; of the 64 studies in our literature review that performed prediction analyses on continuous outcomes, 30 reported such correlations as a measure of predictive performance. This reporting is problematic for several reasons. First, correlation is not sensitive to scaling of the data; thus, a high correlation can exist even when predicted values are discrepant from actual values. Second, correlation can sometimes be biased, particularly in the case of leave-one-out cross-validation. As demonstrated in Figure 4, the correlation between predicted and actual values can be strongly negative when no predictive information is present in the model. A further problem arises when the variance explained (R2) is incorrectly computed by squaring the correlation coefficient. Although this computation is appropriate when the model is obtained using the same data, it is not appropriate for out-of-sample testing23; instead, the amount of variance explained should be computed using the sum-of-squares formulation (as implemented in software packages such as scikit-learn).”

“A further problem arises when the variance explained (R2) is incorrectly computed by squaring the correlation coefficient. Although this computation is appropriate when the model is obtained using the same data, it is not appropriate for out-of-sample testing23; instead, the amount of variance explained should be computed using the sum-of-squares formulation (as implemented in software packages such as scikit-learn).”

Accordingly, we decided to keep both R2 and Pearson correlation (along with MAE) in our Figure 1.

Reviewer 3 Recommendations For The Authors #7:

Figure 2 “We calculated feature importance by, first, standardizing Elastic Net weights across brain features of each set of features from each test fold.” → What do you mean by “standardize” here? Rescale to be mean 0, variance 1? If so, this seems like a misleading transformation, because it gives the impression that the relationships are negative, when they are not necessarily. Also, why did you choose to use elastic net weights in any form as measures of effect size (or importance)? The raw values are inherently penalized, which means they are under-estimates of the true effect size. It would be more meaningful (and less biased) to plot the raw correlations.

Response For the first question regarding standardisation, we addressed this issue in our response to Reviewer 1 Recommendations For The Authors #3. Briefly, we agreed with Reviewer 3 that standardisation (with mean = 0, SD = 1) might make it difficult to interpret the directionality of the coefficients. For visualising feature importance in the revised manuscript, we refitted the Elastic Net model to the full dataset without splitting them into five folds and visualised the coefficients on brain images (see below).

For the second question regarding why using Elastic Net coefficients as feature importance (as opposed to correlations), we need to mention the goal of feature importance: to understand how the model makes a prediction based on different brain features (Molnar, 2019). Correlations between a target and each brain feature do not achieve this. Instead, they will show univariate/marginal relationships between a target and a brain feature. What we want to visualise is how the model made a prediction, which in the case of Elastic Net, the prediction is based on the sum of the features’ coefficients. In other words, the multivariate models (including Elastic Net) focus on marginal relationships that take into account all brain features within each set of features.

Elastic Net coefficients can be considered as feature importance, such that more positive Elastic Net coefficients lead to more positive predicted values and, similarly, more negative Elastic Net coefficients lead to more negative predicted values (Molnar, 2019; Pat, Wang, Bartonicek, et al., 2022). While the magnitude of Elastic Net coefficients is regularised (thus making it difficult for us to interpret the magnitude itself directly), we could still indicate that a brain feature with a higher magnitude weights relatively stronger in making a prediction. Another benefit of Elastic Net as a penalised regression is that the coefficients are less susceptible to collinearity among features as they have already been regularised (Dormann et al., 2013; Pat, Wang, Bartonicek, et al., 2022).

Reviewer 3 Recommendations For The Authors #8:

Figure 3 → Again, what exactly do you mean by “standardised” here?

Response It means mean subtraction followed by the division by an SD. Though we no longer applies standardisation for feature importance. See our response to Reviewer 1 Recommendations For The Authors #3 and Reviewer 3 Recommendations For The Authors #7.

Reviewer 3 Recommendations For The Authors #9:

“However, Brain Age Gap created from the lower-performing age-prediction models explained a higher amount of variation in Cognitionfluid. For instance, the top performing age-prediction model, “Stacked: All excluding Task Contrast”, generated Brain Age and Corrected Brain Age that explained the highest amount of variation in Cognitionfluid, but, at the same time, produced Brian Age Gap that explained the least amount of variation in Cognitionfluid.” (p7) → Yes, but you did not need to run any models to show this, considering it is an inevitable consequence of the following relationship between predicted values and residuals (or residuals times -1): 𝑦 = (𝑦 − 𝑦% ) + 𝑦% . Let’s say that age explains 60% of the variance in fluid cognition, and predicted age ( 𝑦% ) explains 40% of the variance in fluid cognition. Then the brain age gap (−(𝑦 − 𝑦% )) should explain 20% of the variance in fluid cognition. If by “Corrected Brain Age” you mean the modified predicted age from the Butler paper, the “Corrected Brain Age” result is inevitable because the modified predicted age is essentially just age with a tiny bit of noise added to it. From Figure 4, though, this does not seem to be the case, because the lower left quadrant in panel a should be flat and high (about as high as the predictive value of age for fluid cognition). So how are you calculating “Corrected Brain Age”? It looks like you might be regressing age out of Brain Age, though from your description the Methods (How exactly do you use the slope and intercept? You need equation of you are going to stick with this terminology), it is not totally clear. I highly recommend using terminology and metrics from the Butler et al. (2021) paper throughout to reduce confusion.

Response Please see our response to Reviewer 3 Public Review #5

Reviewer 3 Recommendations For The Authors #10:

“On the contrary, an amount of variation in Cognitionfluid explained by Corrected Brain Age Gap was relatively small (maximum R2 = .041) across age-prediction models and did not relate to the predictive performance of the age-prediction models.” (p7) → If by “Corrected Brain Age Gap” you mean MBAG from The Butler paper, yes, this is also inevitable, considering MBAG would be a vector of zeros if it were not for regression on residuals (and out of sample estimates), as I mentioned earlier. Also, it is not clear why you used “on the contrary” as a transition here.

Response Please see our response to Reviewer 3 Public Review #2 for the ‘MBAG’ term. Briefly, we didn’t use Butler and colleagues' (2021) MBAG, but rather we used the method described in de Lange and Cole’s (2020), which was called RBAG by Butler and colleagues.

de Lange and Cole’s (2020) method, was commonly implemented elsewhere (Cole et al., 2020; Cumplido-Mayoral et al., 2023; Denissen et al., 2022). Accordingly, researchers who use Brain Age do not usually view this method as capturing a meaningless biomarker. Yet, the small effects of the Corrected Brain Age Gap in explaining fluid cognition of aging individuals found here are consistent with studies in older adults (Cole, 2020) and younger populations (Butler et al., 2021; Jirsaraie, Kaufmann, et al., 2023) (see our response to Reviewer 2 Recommendations For The Authors #1).

“On the contrary” refers to the fact that the other three Brain Age indices (i.e., those that did not account for the relationship between Brain Age and chronological age) showed a much higher amount of variation in fluid cognition explained. As mentioned above (our response to Reviewer 2 Public Review #7), our argument resonates Butler and colleagues’ (2021) suggestion (p. 4097): “As such, it is critical that readers of past literature note whether or not age was controlled for when testing for effects on the BAG, as this has not always been common practice (e.g., Beheshti et al., 2018; Cole, Underwood, et al., 2017; Franke et al., 2015; Gaser et al., 2013; Liem et al., 2017; Nenadi c et al., 2017; Steffener et al., 2016)”.

Reviewer 3 Recommendations For The Authors #11:

“As before, the unique effects of Brain Age indices were all relatively small across the four Brain Age indices and across different prediction models.” (p10) → Yes, again, this is inevitable considering how they are calculated. You can show these analyses to demonstrate your results in data, if you want, but ignoring the inevitability given how these variables are calculated is misleading.

Response Accounting for the relationship between Brain Age and chronological age when examining the utility of Brain Age is not misleading. Similar to previous recommendations (Butler et al., 2021; Le et al., 2018), we believe that not doing so is misleading. That is, without accounting for the relationship between Brain Age and chronological age, Brain Age will likely explain the same variation of the phenotype of interest as chronological age. Please see our response to Reviewer 3 Recommendations For The Authors #18 below.

Reviewer 3 Recommendations For The Authors #12:

“On the contrary, the unique effects of Brain Cognition appeared much larger.” (p10) → This is not a fair comparison if you don’t look at the unique effects above and beyond the cognitive variable you predicted (fluid cognition) in your Brain Cognition model. When you do this, you will see that Brain Cognition is useless when you include fluid cognition in the model, just as Brain Age would be in predicting age when you include age in the model. This highlights the fact that using predicted values of a metric to predict that metric is a pointless path to take, and that using a predicted value to predict anything is worse than using the value itself.

Response Please see our response to Reviewer 3 Public Review #6.

Reviewer 3 Recommendations For The Authors #13:

“First, how much does Brain Age add to what is already captured by chronological age? The short answer is very little.” (p12) → This is a really important point, but your paper requires an in-depth discussion of the inevitability of this result, which I have discussed previously in this review.

Response Please see our response to Reviewer 3 Public Review #7.

Reviewer 3 Recommendations For The Authors #14:

“Second, do better-performing age-prediction models improve the ability of Brain Age to capture Cognitionfluid? Unfortunately, the answer is no.” (p12) → You need to be clear that you are talking about above and beyond age here.

Response Thank you so much for your suggestion. We now made the change to this sentence accordingly.

Discussion

“Second, do better-performing age-prediction models improve the utility of Brain Age to capture fluid cognition above and beyond chronological age? The answer is also no.”

Reviewer 3 Recommendations For The Authors #15:

“Third, do we have a solution that can improve our ability to capture Cognitionfluid from brain MRI? The answer is, fortunately, yes. Using Brain Cognition as a biomarker, along with chronological age, seemed to capture a higher amount of variation in Cognitionfluid than only using Brain Age.” (p12) → Again, try controlling for the cognitive measure you predicted in your Brain Cognition model. This will show that Brain Cognition is not useful above and beyond cognition, highlighting the fact that it is not a useful endeavor to be using predicted values.

Response Please see our response to Reviewer 3 Public Review #8.

Reviewer 3 Recommendations For The Authors #16:

“Accordingly, a race to improve the performance of age-prediction models (Baecker et al., 2021) does not necessarily enhance the utility of Brain Age indices as a biomarker for Cognitionfluid. This calls for a new paradigm. Future research should aim to build prediction models for Brian Age indices that are not necessarily good at predicting age, but at capturing phenotypes of interest, such as Cognitionfluid and beyond.” (p13) → I whole-heartedly agree with the first two sentences, and strongly disagree with the last. Certainly your results, and the underlying reason as to why you found these results, calls for a new paradigm (or, one might argue, a pre-brain age paradigm). They do not, however, suggest that we should keep going down the Brain Age path. In fact, I think it should be abandoned all together. While it is difficult to prove that there is no transformation of Brain Age or the Brain Age Gap that will be useful, I am nearly sure this is true from the research I have done. Therefore, if you would like to suggest that the field should continue down this path, you need to present a very good case to support this view.

Response Please see our response to Reviewer 3 Public Review #9.

Reviewer 3 Recommendations For The Authors #17:

“Perhaps this is because the estimation of the influences of chronological age was done in the training set.” (p13) → I believe this is the case, and it is testable. Try re-running your analyses where parameters are estimated and performance is evaluated on the same data.

Response Yes, we agreed with this. Based on the equations we used, this is inevitable.

Reviewer 3 Recommendations For The Authors #18:

“Similar to a previous recommendation (Butler et al., 2021), we suggest focusing on Corrected Brain Age Gap.” (p13) → To be clear, the authors did not use the term “Corrected” because it is very misleading. The authors also did not suggest that we proceed with any brain age metric; rather they mentioned that the modified brain age gap is independent of age. Note the following passage: “Further, the interpretability of the modified brain age gap (MBAG) itself is limited by the fact that it is a prediction error from a regression to remove the effects of age from a residual obtained through a regression to predict age. By virtue of these limitations, we suggest that the modified version may not provide useful information about precocity or delay in brain development. In light of this, as well as the complexities associated with interpretations of the BAG and its dependence on age, we suggest that further methodological and theoretical work is warranted.” I recognize that that this statement is hedged, as is often required in the publication process, but I am all but certain that MBAG/BAG/modified predicted age are useless constructs. Therefore, if you are going to suggest that people continue to use them, opposed to suggesting that further methodological or theoretical work is warranted, you need to make a strong case, which you did not try to make here. If anything, your results support abandoning the age- prediction endeavor altogether.

Response Please see our response to Reviewer 3 Public Review #2 for the term. Briefly, we didn’t use Butler and colleagues’ (2021) MBAG, but rather RBAG. This index was originally described in de Lange and Cole’s (2020), and has now been implemented elsewhere (Cole et al., 2020; Cumplido-Mayoral et al., 2023; Denissen et al., 2022).

We do not intend to encourage people to abandon the Brain Age endeavour altogether. However, we made main three suggestions for future research on Brain Age to ensure its utility. First, they should account for the relationship between Brain Age and chronological age either using Corrected Brain Age Gap (or other similar adjustments) or, better, examining the unique effects of Brain Age indices after controlling for chronological age through commonality analyses (see below). This is similar to the suggestion made by Le and colleagues (2018) and later rephased by Butler and colleagues (2021). More specifically, Le and colleagues (2018) mentioned (p. 10): “Based on our observations in both real and simulated data, we recommend that the relationship between chronological age and BrainAGE should be accounted for. The two methods proposed in this study are either: (1) regress age on BrainAGE, producing BrainAGER, which is centered on 0 regardless of a participant's actual age or (2) include age as a regressor when doing follow-up analyses.”

Second, we suggested that researchers should not select age-prediction models based solely on age-prediction performance (see our response to Reviewer 1 Recommendations For The Authors #1).

Third, we suggested that researchers should test how much Brain Age miss the variation in the brain MRI that could explain fluid cognition or other phenotypes of interest (see our response to Reviewer 2 Public Review #4).

Discussion

“What does it mean then for researchers/clinicians who would like to use Brain Age as a biomarker? First, they have to be aware of the overlap in variation between Brain Age and chronological age and should focus on the contribution of Brain Age over and above chronological age. Using Brain Age Gap will not fix this. Butler and colleagues (2021) recently highlighted this point, “These results indicate that the association between cognition and the BAG are driven by the association between age and cognitive performance. As such, it is critical that readers of past literature note whether or not age was controlled for when testing for effects on the BAG, as this has not always been common practice (p. 4097).” Similar to previous recommendations (Butler et al., 2021; Le et al., 2018), we suggest future work should account for the relationship between Brain Age and chronological age, either using Corrected Brain Age Gap (or other similar adjustments) or, better, examining unique effects of Brain Age indices after controlling for chronological age through commonality analyses. Note we prefer using unique effects over beta estimates from multiple regressions, given that unique effects do not change as a function of collinearity among regressors (Ray-Mukherjee et al., 2014). In our case, Brain Age indices had the same unique effects regardless of the level of common effects they had with chronological age (e.g., Brain Age vs. Corrected Brain Age Gap from stacked models). In the case of fluid cognition, the unique effects might be too small to be clinically meaningful as shown here and previously (Butler et al., 2021; Cole, 2020; Jirsaraie, Kaufmann, et al., 2023).”

Reviewer 3 Recommendations For The Authors #19:

“To compute Brain Age and Brain Cognition, we ran two separate prediction models. These prediction models either had chronological age or Cognitionfluid as the target.” (p16) → You should make it clear in the main text of your paper that the cognition variable in your Brain Cognition models is the same as what you refer to as Cognitionfluid. Some of your analyses would have been much more reasonable if you had two different measures of cognition.

Response Thank you so much for the suggestion. We believe, given the re-conceptualisation of Brain Cognition as the main text

Introduction

“certain variation in the brain MRI is related to fluid cognition, but to what extent does Brain Age not capture this variation? To estimate the variation in the brain MRI that is related to fluid cognition, we could build prediction models that directly predict fluid cognition (i.e., as opposed to chronological age) from brain MRI data.”

Reviewer 3 Recommendations For The Authors #20:

“We controlled for the potential influences of biological sex on the brain features by first residualizing biological sex from brain features in the training set.” (p16) → Why? Your question is about prediction, not causal inference.

Response While the question is about prediction, we still would like to, as much as possible, be confident about what kind of information we drew from. Here we focused on brain data and controlled for other variables that might not be neuronal. For instance, we controlled for movement and physiological noise using ICA-FIX (Glasser et al., 2016). Following conventional practices in brain-based predictive modelling, we also treated biological sex as another sort of noise (Vieira et al., 2022). The difference between movement/physiological noise and biological sex is that the former varies across TRs, and the latter varies across individuals. Thus we controlled for movement and physiological noise within each participant and controlled for biological sex within a group of participants who belonged to the same training set.

Reviewer 3 Recommendations For The Authors #20:

“Lastly, we computer Corrected Brain Age Gap by subtracting the chronological age from the Corrected Brain Age (Butler et al., 2021; Le et al., 2018).” (p17) → The modified brain age gap in that paper is the residuals from regressing BAG on age (see equation 6). I highly recommend using that terminology and notation throughout to provide consistency and interpretability across papers.

Response Please see our response to Reviewer 3 Public Review #2 for the term.

Reviewer 3 Recommendations For The Authors #21: Equations (pgs 17-19) → Please use statistical notation instead of pseudo-R code.

Response We rewrote all of the equations using statistical notations.

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Nem todas as mudanças que acontecem necessariamente devem ser para o bem ou para o mal, elas podem ser apenas mudanças.

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DOI: 10.1016/j.molcel.2023.09.010

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What is this?

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DOI: 10.1016/j.stem.2023.08.013

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DOI: 10.1016/j.isci.2023.107990

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What is this?