funções importantes para a manutenção e sobrevivência dos seres vivos, como: - Manter a temperatura média da Terra, evitando grandes amplitudes térmicas entre o dia e a noite - Fornecer oxigénio para a respiração - Proteger a Terra contra o impacto de meteoros - Proteger a Terra dos raios ultravioletas, nocivos aos seres vivos - Permitir que ocorra o efeito estufa, que mantém as temperaturas do planeta estáveis

O termo metaverso está ligado a vários equipamentos e dispositivos que o tornam mais imersivo, podendo ser entendido como um universo dentro da tecnologia. Ressalta que esse universo é “constituído como um ambiente dinâmico e navegável a partir de uma dimensão tridimensional, que se modifica à medida que os sujeitos interagem com ele” (Maria, 2012, p. 71).

O metaverso é um ecossistema complexo que envolve várias tecnologias para criar ambientes virtuais imersivos, interativos e persistentes. Algumas das principais tecnologias que sustentam o metaverso, são: * Realidade Virtual (VR): Headsets VR, Sensores de Movimento; * Realidade Aumentada (AR): Dispositivos e aplicativos de AR; * Inteligência Artificial (IA): NPCs (Personagens Não Jogáveis), Assistentes Virtuais; * Blockchain: Criptomoedas, Tokens Não Fungíveis (NFTs); * Computação em Nuvem: Serviços de Nuvem; * Redes 5G: Conectividade de Alta Velocidade; * Modelação e Animação 3D: Ferramentas de Design 3D, Captura de Movimento; * Internet das Coisas (IoT): Dispositivos Conectados; * Interface Cérebro-Computador (BCI): Leitura de Ondas Cerebrais; * Geolocalização e Sensores: Rastreamento de Posição.

Referência Bibliográfica: Ball, M. (2022). The Metaverse: And How It Will Revolutionize Everything. Liveright.

https://www.youtube.com/watch?v=AbFuNKI4o2M&t=170s&ab_channel=AIXR

No entanto, concomitante com as possibilidades advindas do metaverso, surgem também os desafios. Marcato (2022) alerta que o maior desafio tecnológico a ser enfrentado na próxima década será desenvolver o metaverso.

Alguns dos principais desafios do metaverso na educação incluem o desconforto físico dos alunos com o uso de óculos de RV, o alto custo dos equipamentos avançados necessários para uma imersão completa, e preocupações com o impacto no desenvolvimento cognitivo e questões legais e éticas sobre os limites do metaverso.

Assim como o trabalho, a economia, a cultura, o entretenimento, a educação também estão diretamente ligadas às possibilidades que o metaverso proporciona, como a criação de avatares e os métodos interativos e imersivos.

O metaverso está a transformar a educação ao oferecer métodos interativos e imersivos, como a criação de avatares e ambientes virtuais que simulam situações reais ou fantásticas. Isto torna a aprendizagem mais envolvente e facilita a compreensão de conceitos complexos, além de permitir a colaboração global em tempo real, promovendo uma educação mais inclusiva e acessível.

“ conexão da internet (Carmen, 2021). Outros aspectosque envolvem o bem-estar físico do aluno, também podem ser dificultadores, por exemplo, os óculos de RV costumam ser pesados, os olhos podem ficar secos, além dequestões relacionadas à ergonomia e o excesso de estímulos no espaço virtual. Tudo isso precisa ser considerado para se alcançar o aprendizado esperado”

Desafios: Estando a iniciar um piloto numa escola, verifico que desde com os quest 3 e comprando as fitas de contra-peso , o mesmo fica equilibrado. Sendo que as dinâmicas educativas, nas que utilizo, em matemática, ou num filmes de RV, são de 10minutos.

Desafios: * Sim, a conectividade é um desafio. * A capacidade de numa sala de aulas todos acederem e a net aguentar * adequar o recurso pedagógico ao espaço: no nosso caso, o recurso que usamos para os exercícios de matemáticas, os meninos podem estar sentados.Assim teremos 20 meninos sentados com os óculos, não havendo desafios de baterem uns nos outros. Os meninos fazem parte das cenas que estão a viver como personagens : ex: podem ser um soldado na batalha de aljubarrota, conhecer a padeira, conhecer mais sobre a envolvência, as vestimentas etc etc. * RGPD menores de 13 anos para login colocam um numero e não os seus dados. * preço dos óculos com o recurso instalado para leitura de vídeos e outras funcionabilidades à utilização do programa /recurso que vamos usar. O MAIOR DESAFIO: * A resistência dos professores a experimentarem algo que não conhecem. O MAIOR DESAFIO DE TODOS (na minha ótica): * ir demorar a podermos ter exercícios/jogos/imersões para vários pontos dos vários programas escolares e a haver orçamentos nas escolas para equipamentos + formação de professores

“ No contexto educacional, os alunosde diferentes lugares do mundo conseguem interagir entre eles, por meio da linguagem oral, gráfica e gestual, pois os avatares possibilitam manifestações de gestos e emoções”

Eu gosto de interagir no engage, uma plataforma profissional onde os avatares têm um dress code entre o casual e o executivo, Neste ambiente eu tenho formações interactivas, com exercícios um a um, com workshops , de tal forma imerso os que me esqueço que sou em casa. Temos a possibilidade de estar com pessoas de vários países, sem custos.

Uso, para trabalho a plataforma virtual speech para treino de entrevistas de embuscada, para treino em palco para plateias, enfim, nas áreas do public speaking e é impressionante o resultado de, após uma formação fora d emetaverso, os nossos clientes podem treinar no metaverso num role play imersivo como se na situação real se encontrassem.

“ esse termo é de origem hindu, que significa uma manifestação corporal de um ser imortal ou de um ser pertencente a um mundo paralelo”

Meta: prefixo de origem grega que significa "além" ou "transcendente." Verso: Derivado de "universo," implica um espaço ou um ambiente abrangente. Metaverso: a palavra combina esses elementos para significar um universo além do físico, ou um universo paralelo digital.

Também em algumas crenças espirituais se diz que aqui na Terra tudo é uma ilsuão, uma matriz de representação para cumprirmos a nossa aprendizagem e voltarmos a cãs , noutro plano. Sendo esta uma realidade paralela.

No filme matrix , com semelhanças a snow crash, "Matrix" é um filme inovador que combina ação emocionante com profundos questionamentos filosóficos e sociais. O seu sucesso teve um forte impacto cultural e influenciou a forma como pensamos a tecnologia, a realidade e a liberdade. A história do Neo e da sua luta contra a Matrix é uma alegoria poderosa sobre a procura pelo conhecimento e a libertação da nossa mente.

Metarverso na educação, oportunidadades.

O metaverso oferece grandes oportunidades para a educação, proporcionando métodos interativos e imersivos, como avatares e Realidade Virtual (RV). A RV facilita a aprendizagem significativa, superando os limites da sala de aula física e aproximando o mundo físico do virtual em tempo real, atraindo especialmente alunos familiarizados com a tecnologia digital.

Para ser eficaz, o uso da RV deve ser planeadoo com objetivos claros, evitando o uso indiscriminado. O metaverso permite experiências educativas ricas, utilizando diversas linguagens combinadas através de plataformas como Eduverse, Second Life, e Roblox. Na medicina, por exemplo, simula cirurgias com bonecos ultrarrealistas, melhorando o aprendizado prático.

Desafios na Educação

Desafios incluem a necessidade de aprimorar equipamentos de RV, que são pesados e caros, e enfrentar questões de bem-estar físico, acessibilidade financeira, além de dilemas cognitivos, legais e éticos. Apesar desses desafios, o metaverso tem um potencial transformador na educação se utilizado de maneira planeada e consciente.

O metaverso é um conceito de um universo virtual coletivo, compartilhado, que é criado pela convergência da realidade física e digital aumentada. Ele proporciona um ambiente virtual onde os utilizadores podem interagir uns com os outros e com o ambiente digital de maneira imersiva, utilizando avatares em três dimensões. Esta ideia abrange uma vasta gama de tecnologias, incluindo realidade virtual (VR), realidade aumentada (AR), redes sociais, jogos online e criptomoedas, criando uma experiência digital contínua e interconectada.

Na afirmação anotada podemos pensar na combinação de diversos espaços de aprendizagem que formou o Future ClassRoom Lab (FCL), criada pela European Schoolnet, em 2012, representando um ambiente de ensino e aprendizagem inovador, com o recurso a tecnologias digitais, composto por diversos espaços flexíveis e reconfiguráveis, que permitem aos professores a utilização de novas abordagens pedagógicas envolvendo a pedagogia, a tecnologia e a configuração espacial das salas de aula.

Em Portugal, foi adotado o Ambiente Educativo Inovador (AEI), baseado no FCL, já existente em algumas escolas este é dividido em seis cenários de inovação pedagógica, nomeadamente: Criar, Interagir, Apresentar, Investigar, Partilhar e Desenvolver, que permitem a adoção de metodologias promotoras de aprendizagens significativas. Um AEI não só contribui para motivação das aprendizagens dos alunos mas também para o desenvolvimento de competências tais como auto-estima, autonomia, competências digitais, bem como outras competências que estão no Perfil do Aluno à Saída da Escolaridade Obrigatória (Martins et al., 2017), por exemplo capacidade de resolver problemas, comunicar, colaborar e aprender ao longo da vida (Moreira & Horta, 2020, p. 11).

Imagem extraída de (Moreira & Horta, 2020, p. 9).

Bibliografia

Martins, G., Gomes, C., Brocardo, J., Pedroso, J., Carrilo, J., Silva, L., Encarnação, M., Horta, M., Calçada, M., Nery, R., & Rodrigues, S. (2017). Perfil dos Alunos à Saída da Escolaridade Obrigatória. ME,DGE.

Moreira, J. A., & Horta, M. J. (2020). Educação e ambientes híbridos de aprendizagem. Um processo de inovação sustentada. Revista UFG, 20. https://doi.org/10.5216/revufg.v20.66027

Esta inovação disruptiva tenderá a introduzir novos conceitos menos testados de uma forma mais agressiva mas mais baratos, mais simples e supostamente mais práticos, visando uma inovação que acaba por ser um risco no sentido que pode não conseguir singrar mas que por outro lado pode ser genial.

É importante ressaltar mais uma vez que o caminho para o hibridismo, não perde a essência do modelo tradicional. Ao contrário, acaba por fazer junção entre os dois modelos. Aqui mais uma vez está exposto o modelo da pedagogia digital. Ao pensarmos no modelo híbrido chegamos a conclusão que este sim é de grande importância e crescerá ao longo do tempo. Devemos ter a noção que o modelo tradicional seguirá coexistindo. Vemos que o hibridismo já é aplicado no ensino superior e devemos ser capazes de fazer uso na educação básica.

Embora não trabalhe com alunos do primeiro ciclo neste momento, considero que os alunos mais novos não possuem um grau de independência e destreza ao nível da sua capacidade de organização do seu percurso de aprendizagem que lhes permita aproveitar todas as potencialidades dos modelos mais disruptivos. Além disso, a presença de um professor é, sem dúvida, uma mais-valia para o seu desenvolvimento harmonioso.

O livro "Blended: Using Disruptive Innovation to Improve Schools" de Michael B. Horn, Heather Staker e Clayton M. Christensen: Este livro explora como a inovação disruptiva, conceito desenvolvido por Christensen, pode ser aplicada à educação através do ensino híbrido ou blended learning. Os autores argumentam que o modelo tradicional de sala de aula está ultrapassado e não atende às necessidades dos alunos no século XXI. Eles propõem o ensino híbrido, que combina a instrução online com o ensino presencial, como uma forma de transformar a educação de maneira disruptiva. São descritos quatro modelos principais de ensino híbrido: rotação, flex, à la carte e virtual enriquecido. Cada modelo utiliza a tecnologia de forma diferente, permitindo uma aprendizagem mais personalizada, centrada no aluno. O livro apresenta casos de escolas que implementaram com sucesso o ensino híbrido, destacando os desafios e estratégias para superar as resistências à mudança. Também aborda questões como formação de professores, currículos, avaliação, equidade e financiamento. Os autores defendem que o ensino híbrido permite às escolas tornarem-se organizações centradas na aprendizagem, em vez de centradas na distribuição de conteúdos. Isso pode melhorar os resultados dos alunos, aumentar a motivação e preparar melhor os estudantes para o mundo moderno. Em suma, a obra convida educadores e líderes a repensar profundamente a forma como a educação é ministrada, aproveitando a inovação disruptiva do ensino híbrido para criar um sistema mais eficaz e relevante. Referência: -Horn, M. B., Staker, H., & Christensen, C. M. (2017). Blended : using disruptive innovation to improve schools. Jossey-Bass.

Em (Staker & Horn, 2012) foi apresentado uma taxonomia com os quatro modelos principais para o ensino híbrido: o Modelo de Rotação; o Modelo Flex o Modelo Self-Blend (A La Carte) e o Modelo Virtual Enriquecido.

Os modelos de Rotação por Estações, Laboratório Rotacional e Sala de Aula Invertida (Flipped Classroom) seguem o modelo de inovações híbridas sustentadas, juntando as características da sala de aula tradicional e o ensino online. Os modelos Flex, A La Carte (Self-Blend Model), Virtual Enriquecido e de Rotação Individual, apresentam uma disrupção relativamente a sala de aula tradicional.

A seguinte imagem ilustra a classificação destes modelos de acordo o tipo de inovação, tendo como base a figura disponível em (Christensen et al., 2013, p. 28) .

Bibliografia

Staker, H., & Horn, M. B. (2012). Classifying K-12 Blended Learning. Innosight Institute, May.

Christensen, C. M., Horn, M. B. H., & Staker, H. (2013). Ensino Híbrido: uma Inovação Disruptiva? Uma introdução à teoria dos híbridos. https://www.christenseninstitute.org/publications/ensino-hibrido/

Olhando para trás, para a Microsoft e Apple, contra o golias IBM. Parece um bom exemplo do conceito de disrupção, do risco que se corre, mas também do potencial e novos horizontes que se abrem.

Cumps, Pedro Augusto

Evolução do eco-sistema hibrido, onde a tecnologia, autores humanos e não-humanos, espaços e tempos, interagem numa simbiose evolucionária ... Nesta fase de transição, a tecnologia existe, mas o humano apresenta resistência à sua integração plena (não basta estar a jogar no tlm para se considerar integrado) , e corremos o risco de uma estagnação na evolução, por os atores humanos ficarem "acomodados".

Cumps, Pedro Augusto

Esses produtos ou serviços disruptivos, ao se tornarem mais sofisticados podem transformar e, muitas vezes, substituir os produtos ou serviços dominantes. Esse processo pode redefinir a o processo educativo que temos hoje em dia. Nunca deixando abandonando a essência do analógico por completo, devemos complementar através do uso das tecnologias. Além que a execução de inovações disruptivas devem ser bem concebida com uma pedagogia bem delineada.

Vai ao encontro da minha opinião, a de criar uma base sólida através duma trajetória sustentada, e ao termos as condições adequadas (recursos tecnológicos e humanos) "darmos o salto" e optar por uma trajetória disruptiva que nos levará a novos "horizontes", que antes não se deslumbravam.

Saliento o "parecem" , julgo que a resistência que existe em colegas, e também alunos, dificulta em muito a "transição" que vivemos hoje.

A rápida evolução e disseminação das tecnologias digitais e cognitivas têm levado os educadores a repensar os modelos pedagógicos tradicionais e a adotar novos paradigmas educacionais. O surgimento de ambientes de aprendizagem inovadores, combinando ensino presencial e online, requer uma abordagem colaborativa e em rede, utilizando tecnologias digitais para enriquecer a experiência educativa. Os modelos pedagógicos para ambientes híbridos permitem aos alunos aceder a recursos educativos, participar em atividades interativas e comunicar com colegas e professores tanto presencialmente quanto online. Eles promovem a personalização da aprendizagem, adaptando o conteúdo às necessidades individuais dos alunos e facilitando a interação e o envolvimento através de diferentes formatos de aprendizagem.

-Ensino Híbrido: uma Inovação Disruptiva?Uma introdução à teoria dos híbridos. (n.d.). Christensen Institute. https://www.christenseninstitute.org/publications/ensino-hibrido/

Julgo, que devemos adoptar sempre formas hibridas na educação, "doseando" a componente analógica e a digital, conforme as necessidades e conforme a "receita" que aplicamos a cada turma, para a obtenção do maior efeito potenciador da aprendizagem.

Este artigo é o primeiro a analisar a educação híbrida utilizando a teoria da inovação disruptiva, ajudando as pessoas a antecipar e planear os potenciais efeitos da educação híbrida nas salas de aula de hoje e de amanhã. O artigo discute a introdução de inovações sustentáveis e disruptivas, que seguem caminhos diferentes e levam a resultados diferentes. As inovações sustentáveis ajudam as organizações a criar melhores produtos ou serviços, muitas vezes vendidos com maiores lucros para os seus clientes. As inovações disruptivas, por outro lado, oferecem uma nova definição do que é bom, oferecendo produtos mais simples, convenientes e mais baratos que atraem novos ou menos exigentes clientes.

Durante este estágio híbrido, as empresas podem estar a testar diferentes estratégias, a experimentar novos processos ou produtos, e a tentar encontrar um equilíbrio entre a familiaridade do que já conhecem e a necessidade de se adaptarem às exigências do ambiente em mudança. Este período de transição pode ser desafiante, mas também oferece oportunidades para o crescimento e a inovação, à medida que as empresas procuram tornar-se mais ágeis e competitivas. A integração e combinação do analógico com o tecnológico no sistema de ensino assemelha-se a este período de estágio nas empresas.

Em (Moreira & Horta, 2020, p. 7) é referido que a educação híbrida é vista como uma estratégia dinâmica que envolve diferentes ambientes de aprendizagem, abordagens pedagógicas, recursos tecnológicos e um processo de comunicação com alguma complexidade entre interações entre agentes humanos (AH) e agentes não-humanos (AH).

Bibliografia

Moreira, J. A., & Horta, M. J. (2020). Educação e ambientes híbridos de aprendizagem. Um processo de inovação sustentada. Revista UFG, 20. https://doi.org/10.5216/revufg.v20.66027

A inovação sustentada é vista como um processo contínuo de melhoria e adaptação. Um exemplo disso é o conceito de educação híbrida, que combina métodos tradicionais com tecnologias digitais para criar ambientes de aprendizagem mais eficazes. Este tipo de inovação não pretende substituir completamente os sistemas existentes, mas melhorá-los de maneira substancial.

A inovação sustentada refere-se a melhorias contínuas nos processos, métodos e tecnologias existentes, que podem passar, no contexto escolar, por melhorias curriculares e atualizações periódicas no currículo para refletir novos conhecimentos e habilidades como programação, pensamento crítico e habilidades socioemocionais. Muitas vezes para ir ao encontro destes melhoramentos passa-se também pela modernização das salas de aula e pela incorporação de tecnologia no espaço “físico” escolar, o que conduz a uma maior necessidade de formação para professores, que pretendem implementar novas pedagogias que incluam o digital. A inovação sustentada é crucial porque mantém a escola atualizada e eficiente, garantindo que os alunos recebam uma educação relevante e de qualidade, que permite uma adaptação gradual às novas exigências do século XXI, sem causar grandes ruturas no sistema educativo. Sobre estes processos de inovação sustentada gostei particularmente de ler o artigo de Moreira e Horta(2020), Educação e Ambientes Híbridos de Aprendizagem. Um Processo de Inovação Sustentada onde se lê a respeito da implementação de modelos de aprendizagem híbridos, já explicados neste fórum pelo colega Pedro Augusto: “ Saliente-se que os modelos apresentados podem ser utilizados através de múltiplas combinações, criando programas personalizados de aprendizagem. As categorias não são mutuamente exclusivas.Existem muitas possibilidades de os articular e combinar”. Aqui, os autores não equacionam a disripção, mas sim a modelagem dos diferentes modelos a fim de se encontrar uma fórmula válida para cada realidade de aprendizagem específica. Na minha opinião faz mais sentido assim do que pensarmos em modelos disruptivos ou sustentados estanques.

A inovação sustentada e a inovação disruptiva são igualmente importantes para as escolas do século XXI, embora, a meu ver, evolução e mudança devam assumir sempre uma forma espiral, recuperando conceitos já analisados e aprofundado-os e alterando-os de forma a daem resposta às necessidades atuais. Talvez a exploração destas duas formas de inovação, em fases diferentes do processo, consoante os objetivos a alcançar e o seu público-alvo possam garantir que a educação acompanhe as mudanças rápidas da sociedade prepare os alunos para serem cidadãos críticos, criativos e adaptáveis. A combinação destas duas abordagens poderá transformar a educação, tornando-a mais inclusiva, personalizada e eficaz.

Aqui temos novamente a questão já tão debatida por nós da equidade, e que surge mencionada no recurso de apoio 3, como o princípio da inclusão digital; Também Salmon inclui este patamar nas etapas 2 e 3 do seu modelo Five-Stage(socialização e partilha de informação). Joana

A escola do século XXI muda e cresce trabalhando sobre o presente, tendo em conta as realidades globais e locais; para que cada um dos alunos aprenda a viver, com sentido crítico, que lhes permite descubrir o mundo e atuar sobre ele, apropriando-se dos seus múltiplos sentidos e transformando-o.<br /> Neste processo de desenvolvimento individual, as comunidades de alunos vão evoluindo para comunidades de aprendizagem que levam, progressivamente, à inovação que nasce da pesquisa e se manifesta em pequenas ações quotidianas. O primeiro passo de toda inovação começa com uma pessoa que atua e que se comunica com outra; depois, um grupo; depois, uma escola; depois, um movimento; depois, uma comunidade que tem ação social; muitas vezes essa ação social adota traços vinculativos que norteiam a sociedade. O mundo vai aprendendo a partir das melhores experiências e atuando com ações concretas. Trata-se de gestos quotidianos protagonizados por professores e alunos em comunidade. Joana

Os ecossistemas híbridos da educação, estão aqui para ficar, e o passar desta fase de "transição" para a fase de "naturalização" é que pode demorar mais, ou menos, tempo conforme os recursos disponíveis, a formação dos professores e a resistência à mudança ! Onde uma postura "Just Do It" é essencial, na nossa geração, para que a educação seja "naturalmente" ONLIfe.

O Covid19 foi um factor disruptivo, que fez acelerar esta transição, mas esta já havia começado `já algum tempo, num ambiente de inovações sustentáveis.

Nunca devemos abandonar o analógico por completo, devemos o complementar através do uso, potenciador da aprendizagem, das tecnologias / atores digitais.

A aplicação de inovações disruptivas devem ser muito bem planeadas com uma pedagogia bem definida, e sempre com sentido critico do seu sucesso ... um grande erro que se comete é que "o que é novo é bom" e nem se analisa se o realmente é !!!

Pedro Augusto

fweflwefwfn

how to accomplish progressive overload: 1. increased intensity (more weight) 2. increased volume (more amount of work i.e. reps / sets) 3. increased density (same work, shorter period of time) 4. increased movement difficulty

DOI: 10.1161/CIRCRESAHA.123.323655

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @abever99

SciCrunch record: RRID:SCR_002798

What is this?

Para o ? ahuahuahau ta falando mineiro ? proncovô?

eu continuo confusa com a frase: O deputado, o conselheiro e o delegado são 3 pessoas diferentes? nesse caso botaria artigo em todo mundo; ou o deputado é conselheiro? nesse caso está faltando uma vírgula

migo, quando a gente se refer a algo que já foi dito, acredito que o demonstrativo é "essa", além disso eu não colocaria essa vírugla, acho que eu trocaria por "ela", dúvida.

eu tiraria a virgula

Esta me parece ser a pergunta precisa a partir da qual a reflexão deve se desdobrar. Parabéns pelo texto! Ótimo artigo para se trabalhar com os alunos.

Reviewer #2 (Public Review):

Xu et al. introduce a cellular automaton model to investigate the spatiotemporal spreading of viral infection. In this study, the author first analyzes the single-cell RNA sequencing data from experiments and identifies four clusters of cells at 48 hours post-viral infection, including susceptible cells (O), infected cells (V), IFN-secreting cells (N), and antiviral cells (A). Next, a cellular automaton model (NOVAa model) is introduced by assuming the existence of a transient pre-antiviral state (a). The model consists of an LxL lattice; each site represents one cell. The cells change their state following the rules depending on the interaction of neighboring cells. The model introduces a key parameter, p_a, representing the fraction of pre-antiviral state cells. Cell apoptosis is omitted in the model. Model simulations show a threshold-like behavior of the final attack rate of the virus when p_a changes continuously. There is a critical value p_c, so that when p_a < p_c, infections typically spread to the entire system, while at a higher p_a > p_c, the propagation of the infected state is inhibited. Moreover, the radius R that quantifies the diffusion range of N cells may affect the critical value p_c; a larger R yields a smaller value of the critical value p_c. The authors further examine the result with stochastic version dynamics, and the main findings are unchanged upon stochastic dynamics. The structure of clusters is different for different values of R; greater R leads to a different microscopic structure with fewer A and N cells in the final state. Compared with the single-cell RNA seq data, which implies a low fraction of IFN-positive cells of around 1.7%, the model simulation suggests R=5. The authors also explored a simplified version of the model, the OVA model, with only three states. The OVA model also has an outbreak size. The OVA model shows dynamics similar to the NOVAa model. However, the change in microstructure as a function of the IFN range R observed in the NOVAa model is not observed in the OVA model.

Reviewer #1 (Public Review):

This paper can be seen as an extension of a recent study by two of the same authors [1]. In the previous paper, the authors considered two variants of the Moran process, labelled Model A and Model B, and examined differences between the evolutionary dynamics of these two models. They further described the site frequency spectra, expected allele counts, and expected singleton counts of these models, building on analytical results from prior studies, and used numerical simulations to investigate the models' evolutionary dynamics. Finally, they compared the site frequency spectra of the two models (using numerical simulations) to spectra derived from a small breast cancer data set (two sets of three samples).

In the new paper, the authors consider the same two Moran process variants (Model A and Model B) and some related branching processes. As before, they compare the site frequency spectra and various summary statistics of these models, but here they present only numerical simulations (except that some prior analytical results are summarized in Appendix A, which are never referred to in the main text and seem unconnected to the study). They then compare the site frequency spectra of these models (again using numerical simulations) to those derived from the same breast cancer samples as before and thus infer some evolutionary parameters.

The first main conclusion is that the critical branching process and the Moran process models behave similarly and generate similar site frequency spectra. This finding is unsurprising (indeed, the authors acknowledge that the result "has been expected"). For a reasonably large population size, the population size in the critical branching process has been shown to vary relatively little over time and the model is thus essentially a continuous time Moran process (see, for example, Equation 8.55 in ref 2). Nor is it surprising that the authors see stronger similarities when they select only the subset of branching process replicates in which the final population size is particularly close to the initial population size (this is because, in these replicates, the population size likely varies even less than usual).

The second main conclusion is that, although "the mutational SFS alone is not adequate" to quantify the strength of selection, "All fitted values for the selective disadvantage of passenger mutations are nonzero, supporting the view that they exert deleterious selection during tumorigenesis". Although the question of whether mildly deleterious mutations play an important role in cancer evolution is of considerable interest, it's debatable whether the results presented here help resolve the issue.

Many prominent researchers have called into question whether cancer evolutionary parameters can be reliably inferred from site frequency spectra (e.g., [3-7]), even using sophisticated statistical methods. The statistical approach used here (though not named as such in the paper) is a crude kind of approximate Bayesian computation. To improve the accuracy of the results, it would have been better to have set reasonably vague priors for the uncertain mutation rates, rather than fixing them arbitrarily. It would also have been better to have chosen a likelihood function explicitly based on an analysis of the sampling and error distributions, rather than just summing the absolute logged deviations. It is well known that "Checking the model is crucial to statistical analysis" and "A good Bayesian analysis, therefore, should include at least some check of the adequacy of the fit of the model to the data and the plausibility of the model for the purposes for which the model will be used" [8]. The authors' failure to describe any attempt to validate or check their model, using simulated data or otherwise, casts doubt on the reliability of their inferences.

Putting aside the potential biassing effects of sampling error, measurement error, and the limitations of the authors' statistical method, it is well established that both population growth and spatial structure profoundly alter the shape of site frequency spectra in ways that can mimic the effects of selection (e.g. [9-11]). Indeed, Figures 3, 4 and 5 show that the critical and super-critical branching processes generate markedly different site frequency spectra. It follows that if the population dynamics and spatial structure of the mathematical model used for inference don't match those of the biological process that produced the data then any inferred evolutionary parameter values will be unreliable. Breast cancer has two indisputable ecological features that shape its evolutionary dynamics: the cell population expands by many orders of magnitude from a single cell, and the population is spatially structured. In the authors' mathematical model, the population size is initially 100 cells and either remains constant or varies little, and there is no spatial structure. These profound mismatches between model and data cast further doubt on what is supposed to be the paper's most important biological finding.

In this paper the authors offer no justification for their decision to model breast cancer as a non-growing, non-spatial cell population. Nor do they engage with the extensive recent literature on the challenges of inferring evolutionary parameters from cancer site frequency spectra (they cite none of the many relevant papers listed at https://www.sottorivalab.org/neutral-evolution.html). Their 2022 paper [1] claims that, "it sometimes makes sense to consider cancer growth in the framework of constant-population models. Our models correspond to the situation in which a constant population of N "healthy" stem cells is gradually replaced by a growing clone of transformed cells with increasing fitness." No evidence was presented to support this hypothesis regarding breast cancer progression. On the other hand, a wealth of evidence supports the consensus view that, in breast cancer and other human solid tumours, the number of cells with unlimited proliferative potential is several orders of magnitude greater than 100 and grows over time (e.g. [12]).

Analytic expressions for the site frequency spectra with neutral mutations are already known. It is well known that the site frequency spectrum of an exponentially growing population has a tail following a power law S_k ~ k^(-2) [13, 14]. Similarly, it is known that for the critical branching process or the Moran process, the site frequency spectrum at equilibrium is S_k ~ k^(-1) [13, 15]. Especially noteworthy yet uncited studies that use those results about site frequency spectra to make inferences based on sequencing data include ref 16, in which selection is inferred, and ref 17, in which evolutionary parameters of constant populations (healthy cell populations) are inferred.

Although the paper is well written, the figures are ineffective in communicating the results. As others have put it, "A figure is meant to express an idea or introduce some facts or a result that would be too long (or nearly impossible) to explain only with words" and "If your figure is able to convey a striking message at first glance, chances are increased that your article will draw more attention from the community" [18]. On the contrary, Figures 3, 4, 5 and 6 are bewilderingly complicated, crowded, and repetitive. These figures comprise no fewer than fifty-six plots, each containing numerous curves or histograms, spread across four pages. To compare the results of different scenarios, the reader is presumably expected to put these figures side by side and try to spot the differences, hampered by inconsistent axis ranges, absence of axis labels, absence of titles, absence of legends, and unreliable captions ("cyan" seems to refer to pale blue, and "orange" to something closer to red). For example, the only notable difference between Figures 3 and 4 is in the shape of a single green curve in panel I. In the main text of a published paper, one would expect fewer, more carefully curated figures drawing attention to salient features, so that the reader can infer the main results with minimal effort. The rest can be put in supplementary figures.

In summary, this paper adds somewhat to our understanding of some standard mathematical models; whether it tells us anything new about cancer is open to debate.

References<br /> (1) Kurpas, Monika K., and Marek Kimmel. "Modes of selection in tumors as reflected by two mathematical models and site frequency spectra." Frontiers in Ecology and Evolution 10 (2022): 889438.<br /> (2) Bailey, Norman TJ. The elements of stochastic processes with applications to the natural sciences. John Wiley & Sons, 1964.<br /> (3) Tarabichi, Maxime, et al. "Neutral tumor evolution?." Nature Genetics 50.12 (2018): 1630-1633.<br /> (4) McDonald, Thomas O., Shaon Chakrabarti, and Franziska Michor. "Currently available bulk sequencing data do not necessarily support a model of neutral tumor evolution." Nature Genetics 50.12 (2018): 1620-1623.<br /> (5) Balaparya, Abdul, and Subhajyoti De. "Revisiting signatures of neutral tumor evolution in the light of complexity of cancer genomic data." Nature Genetics 50.12 (2018): 1626-1628.<br /> (6) Noorbakhsh, Javad, and Jeffrey H. Chuang. "Uncertainties in tumor allele frequencies limit power to infer evolutionary pressures." Nature Genetics 49.9 (2017): 1288-1289.<br /> (7) Bozic, Ivana, Chay Paterson, and Bartlomiej Waclaw. "On measuring selection in cancer from subclonal mutation frequencies." PLoS Computational Biology 15.9 (2019): e1007368.<br /> (8) Neher, Richard A., and Oskar Hallatschek. "Genealogies of rapidly adapting populations." Proceedings of the National Academy of Sciences 110.2 (2013): 437-442.<br /> (9) Gelman, Andrew, et al. Bayesian data analysis (Third Edition). Chapman and Hall/CRC, 2014.<br /> (10) Fusco, Diana, et al. "Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments." Nature Communications 7.1 (2016): 12760.<br /> (11) Noble, Robert, et al. "Spatial structure governs the mode of tumour evolution." Nature Ecology & Evolution 6.2 (2022): 207-217.<br /> (12) Lawson, Devon A., et al. "Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells." Nature 526.7571 (2015): 131-135.<br /> (13) Gunnarsson, Einar B., Leder, Kevin, and Foo Jasmine. "Exact site frequency spectra of neutrally evolving tumors: A transition between power laws reveals a signature of cell viability" Theoretical Population Biology 142 (2021) 67-90<br /> (14) Durrett, Richard "Branching Process Models of Cancer" Springer (2015)<br /> (15) Durrett, Richard "Probability Models for DNA Sequence Evolution" Springer Science & Business media (2008)<br /> (16) Williams, Mark J. et al. "Quantification of subclonal selection in cancer from bulk sequencing data." Nature Genetics 50 (6). 895-903 (2018)<br /> (17) Moeller, Marius E. et al. "Measures of genetic diversification in somatic tissues at bulk and single-cell resolution" eLife (2024) 12:RP89780<br /> (18) Rougier, Nicolas P., Michael Droettboom, and Philip E. Bourne. "Ten simple rules for better figures." PLoS Computational Biology 10.9 (2014): e1003833.

decía todo el adjetivo que se utiliza en la página de guion es un adjetivo que tiene que ser comprobable por la cámara o el sonido. Porque hay muchos guiones muy malos que dicen juan entra en la habitación claramente abrumado por su pasado del que no puede escapar, no mames vamos a filmar cuando entrando el cuarto, tu pasado lo dejé en la casa, estás abrumado lo que puedes hacer es entonces adjetivizar ciertas acciones cuando entra a la habitación no suelte el picaporte mira para atrás no sé cómo lo irás a puntualizar pero lo haces así, esto está lo por ejemplo pongo elisa mira al linea línea al anfibio flotando en el cilidro indica que va a haber plano contraplano reverso una y le da el ritmo a la página que efectivamente permite que hagas un minuto por página porque si dices él el edificio se incendia se derrumba y pocos escapan con vida son dos minutos de pantalla tres minutos de pantalla entonces tratas de describir eso.

/- Guillermo del Toro

DOI: 10.1016/j.celrep.2024.114214

Resource: (Bethyl Cat# A300-065A, RRID:AB_2218477)

Curator: @scibot

SciCrunch record: RRID:AB_2218477

What is this?

DOI: 10.1016/j.immuni.2024.04.024

Resource: (Cell Signaling Technology Cat# 12741, RRID:AB_2617131)

Curator: @scibot

SciCrunch record: RRID:AB_2617131

What is this?

DOI: 10.1016/j.cell.2024.04.027

Resource: (BioLegend Cat# 144609, RRID:AB_2562978)

Curator: @scibot

SciCrunch record: RRID:AB_2562978

What is this?

La conciencia varia en su significado, este, va a depender de su contexto. Conciencia: responsabilidad, tmb' como sabiduría. Filosofía= Mente en general. A su vez, puede significar, actividad introspectiva. Etimología= con-ciencia / con-conocimiento. La conciencia como la capacidad de conocer, de diferenciar o discriminar un estímulo de otro. EJEMPLO: Un perro distingue a su dueño de otro, un ave distingue su nido de los otros, y las personas, diferencian si tiene frio o calor.

objetivo

como o gêmeo digital pode contribuir com o BaaS

O BaaS envolve uma mudança no conceito de serviços: os usuários tornam-se destinatários dos serviços gerados pelo edifício, e não apenas dos serviços prestados no edifício

Falta de alinhamento de dados entre o ativo construído integrando com as instalações, pessoas e processos principais é um fator para baixa eficiência e eficácia da indústria da construção.

ignorância digital da indústria da contrução. Ainda se encontra no taylorismo clássico.

Una de las cosas que encuentro interesante es la manera en que los autores definen lo que serían las digital storytelling: como cortometrajes o videoclips testimoniales, pues les da el poder y control de la narrativa a los migrantes deportados y no ejercen una injerencia siquiera en su producción, por lo que ellos deciden su propia historia, lenguaje y forma discursiva. Lo cual tiene una repercusión en la manera de mostrar el contexto que están tratando de hacer evidente: una desviación de una norma y el lugar precario en el que quedan los deportados y sus familias; además del otro lado de experimentación de los "nativos" norteamericanos al ver una inmigración controlada. Lo mismo que lo que sucedía después, la llegada nuevamente a México y el encontrarse como un outsider. La técnica de darles el control de la narrativa, parece dar una mayor posibilidad de contar todos estos aspectos. Además, como lo evidencian los autores, hay una humanización de la deportación, más allá de las cifras, hay vidas humanas que vale la pena ser contadas, ahí es donde este acercamiento me parece valioso y así lo reflejan al retomar el concepto de gnosis fornteriza de Mignolo, en el que hay víctimas de discriminación de ambos lados de la frontera.

This part is saying that people may need relationship management. And that there are methods o get better that relationships and emotions

Joint Public Review:

Summary

This manuscript explores the transcriptomic identities of olfactory ensheathing cells (OECs), glial cells that support life-long axonal growth in olfactory neurons, as they relate to spinal cord injury repair. The authors show that transplantation of cultured, immunopurified rodent OECs at a spinal cord injury site can promote injury-bridging axonal regrowth. They then characterize these OECs using single-cell RNA sequencing, identifying five subtypes and proposing functional roles that include regeneration, wound healing, and cell-cell communication. They identify one progenitor OEC subpopulation and also report several other functionally relevant findings, notably, that OEC marker genes contain mixtures of other glial cell type markers (such as for Schwann cells and astrocytes), and that these cultured OECs produce and secrete Reelin, a regrowth-promoting protein that has been disputed as a gene product of OECs.

This manuscript offers an extensive, cell-level characterization of OECs, supporting their potential therapeutic value for spinal cord injury and suggesting potential underlying repair mechanisms. The authors use various approaches to validate their findings, providing interesting images that show the overlap between sprouting axons and transplanted OECs, and showing that OEC marker genes identified using single-cell RNA sequencing are present in vivo, in both olfactory bulb tissue and spinal cord after OEC transplantation.

Despite the breadth of information presented, however, further quantification of results and explanation of experimental approaches would be needed to support some of the authors' claims. Additionally, a more thorough discussion is needed to contextualize their findings relative to previous work.

(1) Important quantification is lacking for the data presented. For example, multiple figures include immunohistochemistry or immunocytochemistry data (Figures 1, 5, 6), but they are presented without accompanying measures like fractions of cells labeled or comparisons against controls. As a result, for axons projecting via OEC bridges in Figure 1, it is unclear how common these bridges are in the presence or absence of OECs. For Figure 6., it is unclear whether cells having an alternative OEC morphology coincide with progenitor OEC subtype marker genes to a statistically significant degree. Similar quantification is missing in other types of data such as Western blot images (Fig. 9) and OEC marker gene data (for which p-values are not reported; Table S2).

The addition of quantitative measures and, where appropriate, statistical comparisons with p-values or other significance measures, would be important for supporting the authors' claims and more rigorously conveying the results.

(2) Some aspects of the experimental design that are relevant to the interpretation of the results are not explained. For example, OECs appear to be collected from only female rats, but the potential implications of this factor are not discussed.

Additionally, it is unclear from the manuscript to what degree immunopurified cells are OECs as opposed to other cell types. The antibody used to retain OECs, nerve growth factor receptor p75 (Ngfr-p75), can also be expressed by non-OEC olfactory bulb cell types including astrocytes [1-3]. The possible inclusion of Ngfr-p75-positive but non-OEC cell types in the OEC culture is not sufficiently addressed. Such non-OEC cell types are also not distinguished in the analysis of single-cell RNA sequencing data (only microglia, fibroblasts, and OECs are identified; Figure 2). Thus, it is currently unclear whether results related to the OEC subtype may have been impacted by these experimental factors.

(3) The introduction, while well written, does not discuss studies showing no significant effect of OEC implantation after spinal cord injury. The discussion also fails to sufficiently acknowledge this variability in the efficacy of OEC implantation. This omission amplifies bias in the text, suggesting that OECs have significant effects that are not fully reflected in the literature. The introduction would need to be expanded to properly address the nuance suggested by the literature regarding the benefits of OECs after spinal cord injury. Additionally, in the discussion, relating the current study to previous work would help clarify how varying observations may relate to experimental or biological factors.

(a) Cragnolini, A.B. et al., Glia, (2009), doi: 10.1002/glia.20857.<br /> (b) Vickland H. et al., Brain Res., (1991), doi: 10.1016/0006-8993(91)91659-O.<br /> (c) Ung K. et al., Nat Commun., (2021), doi: 10.1038/s41467-021-25444-3.

Moverse-con estas voces nos permite sentir que las relaciones afectivas que nos (con)mueves son inseparables de nuestros cuerpos-mentes. De esta manera, mi práctica investigación apunta a la (re)valoración del conocimiento corporal.

Al sumergirnos en estas voces, podemos experimentar empatía, compasión, dolor o alegría a medida que nos conectamos con las emociones y los relatos de los demás, y estas experiencias resonarán dentro de nosotros de manera tangible y visceral.

En este sentido, la práctica de investigación desafía las jerarquías tradicionales de conocimiento y presenta una perspectiva más holística e inclusiva que reconoce la interconexión entre el cuerpo, la mente y las emociones en la producción y comprensión del conocimiento.

La emoción es una sensación, experimentada en el cuerpo, a la cual se le da un nombre para entenderla en el lenguaje (discurso).

Las plataformas digitales ofrecen un espacio de expresión de emociones que son compartidas y entendidas de maneras nuevas:

Primero, las narrativas digitales tienen la capacidad de transmitir emociones a través de una variedad de medios, como texto, imágenes, videos y sonidos. Estos elementos pueden combinarse para crear experiencias inmersivas que evocan emociones intensas en el espectador o lector. Por ejemplo, un video que muestra testimonios de personas que han pasado por experiencias difíciles puede provocar empatía y compasión en el espectador, mientras que una serie de imágenes evocadoras puede despertar nostalgia o melancolía.

En el ámbito de la investigación y el análisis de datos, las narrativas digitales también pueden utilizarse para explorar y comprender las emociones de las personas en un contexto más amplio. Mediante el análisis de grandes cantidades de datos, como publicaciones en redes sociales o comentarios en blogs, los investigadores pueden identificar patrones y tendencias en las emociones de las personas en respuesta a eventos o temas específicos.

Responsabilidad relacional.

En primer lugar, implica respetar la autonomía y dignidad de los individuos cuyas historias están siendo compartidas o representadas en narrativas digitales. Esto significa obtener consentimiento informado y asegurarse de que las personas comprendan cómo se utilizarán sus historias y cómo se presentarán en el entorno digital.

Además, implica una consideración cuidadosa de las posibles repercusiones que pueden tener las narrativas en las personas involucradas, especialmente en términos de privacidad, seguridad y bienestar emocional. Los investigadores y creadores de contenido tienen la responsabilidad de proteger la confidencialidad de la información personal y de evitar cualquier forma de explotación o daño a los participantes.

La responsabilidad relacional también implica un compromiso con la justicia y la equidad en la representación de las voces y experiencias de las personas en el entorno digital. Esto puede implicar dar voz a aquellos que tradicionalmente han sido marginados o subrepresentados en los medios de comunicación y garantizar que se aborden las desigualdades estructurales que pueden influir en quién tiene acceso y quién controla la narrativa.

Algunas ideas adicionales que podrían contextualizar esta afirmación:

Explicación mecánica de los fenómenos: Kropotkin sugiere que el anarquismo se basa en una comprensión de la realidad que busca explicar los fenómenos sociales, políticos, económicos y naturales utilizando principios y conceptos que reflejan un enfoque mecánico o materialista. Esto implica ver el mundo como un sistema en el que las relaciones y procesos están determinados por causas y efectos objetivos, en lugar de ser atribuidos a fuerzas sobrenaturales o abstractas.

Rechazo de la arbitrariedad y la autoridad: Kropotkin sugiere que el anarquismo rechaza la arbitrariedad y la autoridad como bases para la organización social. En cambio, aboga por comprender y abordar los problemas sociales y políticos desde una perspectiva racional y basada en la observación empírica.

Materialismo y determinismo: Al reconocer que los fenómenos sociales están determinados por causas materiales y condiciones objetivas, el anarquismo busca identificar y cambiar las estructuras de poder y dominación que perpetúan la opresión y la injusticia.

Visión holística: La frase sugiere que el anarquismo no se limita a ser simplemente una teoría política, sino que constituye una concepción del mundo integral que abarca aspectos sociales, económicos, políticos y filosóficos. Esto implica una comprensión amplia y profunda de la realidad y una búsqueda de transformación social radical en todos los ámbitos de la vida.

Los anarquistas profesionales defienden el derecho de cada individuo a desarrollarse libremente, es decir, a explorar y expresar su identidad, intereses y potencial sin interferencias externas. Esto implica la autonomía individual y el respeto por la diversidad de experiencias y elecciones de vida.

DOI: 10.1038/s41598-024-61295-w

Resource: RRID:Addgene_49386

Curator: @scibot

SciCrunch record: RRID:Addgene_49386

What is this?

1. O Direito de ler não importa o quê- Cada pessoa deve ter a liberdade de ler qualquer tipo de livro que deseje, sem restrições ou censuras. Pois a leitura é uma atividade pessoal e privada e ninguém deve ser julgado ou impedido de ler um conteúdo que lhe interesse. Contribuindo para a liberdade de expressão, desenvolvimento intelectual e cultural de uma pessoa.
2. O Direito de amar os "Heróis" dos romances- As pessoas devem ser livres para admirar e apaixonar-se pelos personagens dos livros que leem. Amar esses personagens faz parte da experiência de leitura e permite que os leitores explorem emoções e a imaginação sem julgamentos ou limitações.

O Direito de Reler

Este direito permite ao leitor reviver momentos de prazer que teve na primeira leitura de um livro que gostou muito.

1. O direito de não acabar um livro Este direito é importante porque respeita a liberdade pessoal do leitor, evitando o desperdício de tempo com leituras não prazerosas e reconhece a diversidade de gostos literários.

2. O direito de ler não importa o quê Este direito é importante porque garante a liberdade de expressão, promove o acesso ao conhecimento, e encoraja a diversidade de perspetivas. Além disso, estimula o prazer pela leitura, contribuindo para o desenvolvimento pessoal e a sua identidade.

O Direito de Ler em Voz Alta

Na minha opinião, ler em voz alta é muito importante, porque podemos “saborear” as palavras de uma forma diferente do que se estivéssemos a ler “para dentro”.

O Direito de Ler em Voz Alta

Na minha opinião, ler em voz alta é muito importante, porque podemos “saborear” as palavras de uma forma diferente do que se estivéssemos a ler “para dentro”.

O Direito de Não Acabar um Livro

Na minha opinião, este direito do leitor é um dos mais importantes, pois, se não gostamos de um livro, devemos poder "abandoná-lo", para selecionarmos um de que verdadeiramente gostamos, não acham? E vocês, quais são os dois direitos que consideram fundamentais. Selecionem-nos e justifiquem as vossas opções.

Cada leitor tem o direito de falar ou não acerca do que leu. Nem todos nos sentimos confortáveis a falara sobre o que lemos, pois pode ter sido um tema muito pessoal e sensível, ou simplesmente não nos sentimos à vontade para o compartilhar.

Ler em voz alta, permite que as palavras "ganhem vida" e, permite também, que os leitores criem uma conexão mais intensa com as histórias. Esta prática pode ser especialmente gratificante para todos os leitores, desde que não incomode quem se encontra no mesmo ambiente.

Com este capítulo, o autor mostra aos leitores, que estes tem o direito de "saltar" para outro livro, uma vez que o que estão a ler não os esteja a cativar ou agradar. Os leitores devem seguir e descobrir o seu próprio caminho no mundo das leituras.

Cada leitor tem o direito de escolher um local para ler o seu livro, "não importa onde" pois, podemos começar ou continuar as nossas leituras em qualquer lugar, desde que nos sintamos confortáveis com isso.

Neste capítulo, percebemos que cada leitor tem a liberdade de se poder identificar com os protagonistas das histórias, muitas vezes pelas características ou experiencias em comum. Na maioria das vezes, são essas características que nos cativam e nos fazem ler o livro até ao fim.

Com este capítulo, temos a consciência que cada um tem o direito de ler aquilo que prefere, ou seja, devemos respeitar cada gosto individual de cada um, sem qualquer tipo de julgamento, pois nas bibliotecas e nos locais de leitura encontramos todos os tipos de livros para cada gosto.

Reler um livro, permite-nos revisitar o passado e muitas vezes dar ênfase a detalhes que anteriormente podem ter passado despercebidos. Cada leitura é única e voltar a ler o mesmo livro é bastante enriquecedor.

"Não há vergonha em não acabar um livro", com esta frase do terceiro capítulo, percebemos que nem toda a gente consegue terminar um livro, por diversas razões, mas que não existe nada de errado, pois cada um pode decidir fazer com o seu livro o que melhor entender. Nem sempre as histórias são o que era esperado pelo leitor, e ninguém se deve sentir culpado por "abandonar" o seu livro.

No segundo capítulo somos confrontados com o tema de "saltar páginas". Apesar de cada livro ter páginas e enumerá-lo, com este capítulo, percebemos que cada leitor tem autonomia para ler apenas aquilo que ele acha que é importante, ou benéfico para si.

No primeiro capítulo é nos apresentado o tema da não leitura. Nos dias de hoje, os indivíduos sentem cada vez menos necessidade de ler pois tem outro tipo de ferramentas e atividades que ocupam os seus dias. No entanto, e apesar da leitura ser algo benéfico para cada pessoa, pois com a leitura, adquirimos conhecimento e novas formas de vocabulário, o autor critica a leitura nas instituições educacionais, pois são impostas aos alunos. Devemos perceber que cada um deve ter o direito de escolha, ou seja, se lê ou não, pois a leitura não é a única forma de adquirir conhecimento e cada indivíduo pode aprender por outro meio, sem ser o da leitura.

Para o autor, a leitura é uma jornada individual e íntima, uma presença que constrói uma conexão entre a vida e o leitor.

A prática de ler em voz alta é retratada como uma ação que anima as palavras e permite uma melhor compreensão.

No capítulo 8, enfatiza-se a relevância de reservarmos pequenos intervalos para a leitura em qualquer obra de nossa coleção, reconhecendo a liberdade de escolha e a chance de imergir em diversos mundos literários.

Os leitores têm o privilégio de ler em qualquer lugar. Cada indivíduo possui o direito de escolher onde ler desde que se sinta confortável para o fazer.

Este capítulo oferece uma reflexão sobre reconhecer e honrar os momentos em que os leitores se identificam profundamente com os protagonistas das histórias. Isso é visto como uma parte essencial do desenvolvimento tanto literário quanto pessoal.

O quinto capítulo enfatiza a diferença entre livros considerados "de qualidade" e aqueles tidos como menos satisfatórios. Nesta parte, o autor salienta que é importante os indivíduos lerem o que realmente gostam sem terem medo de críticas ou julgamentos.

O quarto capítulo comemora a liberdade e a felicidade de reler obras que foram anteriormente descartadas.

O texto enfatiza a relevância da independência relativamente à leitura, encorajando os leitores a confiarem nas suas preferências e vivências pessoais. Isso pode incluir a decisão de abandonar a leitura de certos livros.

Neste segundo capítulo, o autor defende que os leitores têm o direito de escolher o que desejam ler e de que maneira desejam fazê-lo, incluindo a liberdade de saltar partes menos cativantes. O autor fala sobre a importância crucial da autonomia individual na abordagem à leitura.

Neste capítulo, investiga-se a interligação entre a leitura e a autonomia pessoal. O autor reconhece os benefícios inegáveis da leitura, porém adverte contra a imposição dela como uma norma moral. Ele salienta a necessidade de respeitar a decisão de não ler, destacando que essa escolha não deprecia o indivíduo. É uma reflexão acerca da liberdade de escolha e da importância de evitar julgamentos sobre os hábitos literários dos indivíduos.

O leitor tem o direto de falar ou não sobre o que leu. Às vezes o livro significou tanto para o leitor a nível pessoal que prefere manter apenas para si mesmo. "Ínfimas e secretas conivências que falam da paradoxal alegria de viver, mesmo quando referem o trágico absurdo da vida. "

Ler em voz alta é como se o leitor estivesse a dar vida às palavras e não existe nenhuma razão para não o fazermos a não ser que estejamos e incomodar quem está à nossa volta.

O leitor tem o direito de saltar para outro livro se este não o cativa, é uma questão de liberdade. Se eu estou a ler um livro e não estou a gostar tenho o direito de o tirar da minha "prateleira" e passar para outro assim como também ler vários ao mesmo tempo e ler conforme o que desejamos.

Este texto aborda o "bovarismo" na leitura, enfatizando a importância de entender e respeitar a intensa identificação dos leitores com personagens literários como parte do seu crescimento pessoal e literário.

Este texto distingue a diferença entre "bons" de "maus" romances, criticando a literatura de massa por usar estereótipos e simplificar conteúdos, e destaca uma preferência por obras mais complexas e substanciais.

O direito de ler não importa onde, significa que todos temos a oportunidade de ler em qualquer sítio físico ou geográfico pois não existe sítios específicos nem impróprios para iniciar as nossas leituras. A leitura pode acontecer a qualquer momento e em qualquer situação "Sim, posso sem mentir — senta-te, pedagogo— Afirmar ter lido todo o meu Gogol nas latrinas.*"

Cada leitor tem o direito e a liberdade de amar as personagens do livro que está a ler. Muitas vezes, os heróis com os quais nos identificamos apresentam características e qualidades que nos cativam e aumentam ainda mais a nossa vontade de ler aquele livro.

Este capítulo, salienta como é importante ler sem nenhuma restrição, ou seja, ler aquilo que realmente gostamos, que temos interesse e mais nos cativa. As prateleiras das bibliotecas estão cheias, temos uma diversidade de cultura literária portanto todos temos o direito de ler o que mais é adequado para nós. O importante é que cada livro seja capaz de nos enriquecer e nos ensinar algo novo.

Este texto celebra a alegria de reler livros que antes foram rejeitados, equiparando essa experiência à vontade infantil de repetição e descoberta, enfatizando a procura por crescimento e permanência.

Este texto enfatiza a importância da liberdade e maturidade na leitura, incentivando os leitores a seguir os seus próprios gostos e experiências, mesmo que isso signifique deixar de ler ou adiar certos livros.

Reler um livro é considerado algo especial, memorável e uma grande oportunidade para novas experiências. Reler um livro é muito mais do que simplesmente reler a história ou me lembrar daquilo que foi ali escrito, é relembrar-me de personagens com as quais me identifiquei, reviver emoções e sentimentos e absorver mais detalhas que na primeira leitura não foi capaz de o fazer. Cada página lida novamente é um reencontro com aquilo que nós já fomos ou vivemos, "As nossas releituras de adultos assemelham-se a esse desejo: encontrarmo-nos com uma permanência, e encontrá-la cada vez mais rica de novos deslumbramentos."

Este texto salienta a liberdade na leitura, usando "Guerra e Paz" como exemplo. Defende o direito de escolher e personalizar a leitura, incluindo saltar partes, e alerta contra o uso de resumos simplificados que podem limitar a experiência.

Este texto aborda a relação entre ler e ser livre, destacando que ler é benéfico, mas não deve ser imposto como obrigação moral. Enfatiza a importância de respeitar quem escolhe não ler.

Existe várias razões para deixarmos um livro a meio e o capítulo 3 reflete isso mesmo, o leitor tem a liberdade de escolher as suas leituras como também abandoná-las. Às vezes não nos apetece acabar um livro por inúmeras razões, a história não foi aquilo que esperávamos, não está interessante ou até porque estou a gostar imenso do livro e não quero que acabe. "Mas, ao contrário de alguns vinhos, os bons livros não envelhecem. Continuam à nossa espera nas estantes, e nós é que envelhecemos."

No capítulo "O Direito de Saltar Páginas" significa a liberdade do leitor em ler apenas aquilo que faz sentido para o próprio. Os livros estão com as páginas todas numeradas mas não nos proibi de as saltar, o que realmente importa é que aquilo que lemos faça sentido para nós ou então que nos deixe uma mensagem. Isto demonstra a liberdade e a autonomia que todos os leitores possuem, não somos obrigados a ler aquilo que não queremos nem tão pouco de seguir uma ordem.

O primeiro capítulo do livro " Os Direitos Inalienáveis do Leitor" transfere qualquer indivíduo para um turbilhão de pensamentos em relação à leitura, então devo ou não devo ler livros diariamente? Será que deveria ler mais? A grande realidade é que estamos divididos em dois grandes grupos, uns não têm a leitura como um foco principal do seu dia a dia e outros têm os livros como companheiros de vida. E não está errado o que lê demais nem o que simplesmente não lê. O que devemos entender é o que significa a leitura para cada um de nós, para o sujeito x a leitura pode ser apenas a visualização das imagens e e esperar que as próprias lhe transmitam alguma mensagem, já para o sujeito y a leitura é apenas esfolhar o livro até que este lhe desperte algum interesse ou então a troca de mensagens já conta como uma grande leitura realizada no seu dia. Se formos a analisar a leitura no século em que estamos a viver, esta não está a ficar escassa, está é a ser trocada pelo mundo digital. A leitura de livros é facilmente trocada por horas e horas nas redes sociais ou então pela visualização de uma série, mas não estamos a ler? Esta é a nossa realidade, mas não nos devemos esquecer que "Ser excluído dos livros — mesmo daqueles que não fazem falta —, é uma enorme tristeza, uma solidão dentro da solidão." Não ler livros ou então não termos o hábito de ler traz-nos enormes desvantagens como a falta de conhecimento, de imaginação e de criatividade, desvantagens profissionais e limitações dos pontos de vista. Mas, temos "O direito de não ler".

O direito de não falar do que se leu ou do que o livro trata, constituí um direito do leitor. Cada leitor reage e define a sua interpretação da maneira que a vê e sente, e por isso não querer ou se sentir confortável para partilhar o que lê.

Neste tópico, aborda-se uma temática interessante de contextualizar, uma vez que, quando o leitor realiza uma leitura em voz alta melhora a sua comunicação e compreensão à cerca do livro, permitindo assim, uma leitura mais interessante.

Neste capítulo, o que o autor pretende transmitir é que o leitor tem a liberdade de "saltar" de livro em livro, uma vez que, enriquece as suas experiências e o seu gosto pela leitura.

10-O Direito de Não Falar do Que se Leu Para o autor, a leitura é uma jornada pessoal e íntima, uma presença que tece uma ligação cúmplice entre a vida e o leitor, revelando a estranha e paradoxal alegria de existir.

A leitura, para o autor, é uma viagem pessoal e íntima, uma companhia que tece uma rede de conivências entre a vida e o leitor, revelando a estranha e paradoxal alegria de viver.

9-O Direito de Ler em Voz Alta Ler em voz alta é retratado como uma prática que dá vida ao texto, colocando o leitor sob a atenção total dos ouvintes, tanto visual quanto auditivamente.

A leitura em voz alta é descrita como um ato que dá vida às palavras, expondo o leitor por completo aos olhos e ouvidos dos que o escutam.

8-O Direito de Saltar de Livro em Livro

Neste excerto, enfatiza-se a relevância de reservar breves intervalos para a leitura em qualquer livro, destacando a liberdade de escolha e a chance de escapar para diversos mundos literários, mesmo que por apenas alguns minutos.

7-O Direito de Ler não Importa Onde Neste texto, o soldado Fulano descobre conforto nas obras de Nicolau Gogol, convertendo o entediante trabalho nas latrinas em preciosos momentos de leitura e escapismo do ambiente militar.

Neste texto, o soldado Fulano encontra consolo nas obras de Nicolau Gogol, transformando a tediosa tarefa das latrinas em momentos valiosos de leitura e fuga ao ambiente militar.

6-O Direito de Amar os "Heroís" dos Romances Este texto aborda de maneira sensível o fenômeno do "bovarismo" na leitura, enfatizando a necessidade de reconhecer e valorizar as fases em que os leitores se identificam intensamente com os heróis dos romances. Isso é visto como parte essencial do crescimento tanto literário quanto pessoal.

Este texto reflete de forma sensível sobre o "bovarismo" na leitura, destacando a importância de compreender e respeitar as fases de identificação intensa dos leitores com os heróis dos romances, como parte do processo de crescimento literário e pessoal.

5-O Direito de Ler e não Importa o Quê Neste texto, ressalta-se a diferença entre romances considerados "bons" e "maus", criticando a "literatura industrial" por perpetuar estereótipos e simplificações. Também enfatiza a mudança dos padrões de preferência literária em direção a obras mais profundas e complexas.

4- O Direito de Reler Este texto celebra a oportunidade e o prazer de reler obras que foram inicialmente descartadas, comparando essa experiência à nostalgia infantil pela repetição e descoberta. Destaca também a procura pela permanência e crescimento através dessa prática.

3- O Direiito de Não Acabar um Livro Este texto ressalta a importância da liberdade e da maturidade ao abordarmos a leitura, encorajando os leitores a confiarem em seus gostos e experiências pessoais. Isso pode significar abandonar ou adiar a leitura de certos livros, e tudo bem.

**2- O Direito de Saltar Páginas ** Este texto destaca a liberdade na leitura, defendendo que os leitores têm o direito de escolher o que ler e como ler. Adverte contra resumos simplificados de livros, que podem privar os leitores de experiências valiosas. Em resumo, enfatiza a importância da liberdade individual na abordagem à leitura.

1- O Direito de Não Ler Este texto aborda a conexão entre ler e a liberdade pessoal. Embora reconheça os benefícios da leitura, o autor adverte contra a imposição desta como uma obrigação moral. É enfatizada a importância de respeitar a decisão de não ler, ressaltando que isso não diminui o valor de alguém. A reflexão destaca a liberdade de escolha e a necessidade de evitar julgamentos sobre os hábitos de leitura das pessoas.

Este texto celebra o direito e a alegria de reler obras previamente rejeitadas, equiparando a experiência de releitura ao desejo infantil de repetição e descoberta, enquanto realça a busca por permanência e enriquecimento.

Cada leitor tem o direito de escolher onde fazer a sua leitura, para que com isso, se sintam confortáveis e livres nas suas escolhas.

Este texto realça a liberdade na leitura, usando o exemplo do autor com "Guerra e Paz". Defende que os leitores têm o direito de escolher o que ler e como ler, até mesmo pulando partes menos interessantes. Adverte contra resumos simplificados de livros, que podem privar os leitores de experiências valiosas. Em resumo, destaca a importância da liberdade individual na abordagem à leitura.

Este texto explora a relação entre a leitura e a liberdade individual. O autor reconhece os benefícios da leitura, mas alerta para não impor sua prática como uma obrigação moral. Destaca a importância de respeitar a escolha de não ler, enfatizando que isso não diminui o valor de alguém. É uma reflexão sobre liberdade de escolha e evitar julgamentos sobre os hábitos de leitura das pessoas.

É importante gostar dos "Heróis" de ficção, uma vez que isso permite uma melhor experiência na leitura e posteriormente, criar assim uma admiração pelas personagens de ficção, ou também uma ensinamento visto por outras perspetivas.

Neste capítulo encontramo-nos perante uma perspetiva importante, visto que, para um leitor é importante que o livro seja cativante, pois assim faz com que o mesmo desenvolva o seu pensamento e o gosto pela leitura de livros que vão ao interesse do mesmo.

O direito de reler um livro permite assim uma felicidade e uma alegria pelo prazer de repetição de um livro que tanto significado teve.

As características das personagens de um livro podem torná-lo, do nosso ponto de vista pessoal, bom ou mau. Neste capítulo, é destacada a importância de, criticamente, julgarmos essas personagens e tomá-las como exemplo, positivo ou negativo, para a nossa vida.

Segundo variados relatos de pessoas conhecidas, o livro "O Principezinho" é um excelente exemplo do direito de reler. Dizem que a nossa perspectiva e a forma como o interpretamos a história muda consoante a nossa maturidade e, por isso, devemos reler os nossos livros.

Existem variados motivos para um leitor não terminar a leitura de um livro. Se a história deixa de interessar ou se não superou as expectativas, a continuação da leitura não deve ser uma obrigação. Por outro lado, posso relatar uma história interessante: tenho uma amiga que não tem coragem de ler um dos livros do Harry Potter (não me recordo exatamente qual), pois soube, através dos filmes, que a sua personagem favorita morre. Sendo assim, enquanto ela não ler o livro, a personagem continua viva. Ou seja, o facto de estarmos tão envolvidos na história pode fazer com que não a queiramos acabar.

Neste primeiro capítulo somos confrontados com o direito de não ler. Como é referido, e como podemos comprovar pelas nossas experiências pessoais, os indivíduos sentem, cada vez menos, a necessidade de ler, pois são assoberbados com outras formas bastante mais apelativas de entretenimento. No entanto, está também a perder-se o incentivo à leitura e, também por isso, a falta de necessidade de leitura sentida - é cada vez menos dada a oportunidade aos indivíduos de se envolverem na leitura e saberem se querem ou não (ler é um direito, não um dever) continuar a ler. Por outro lado, acho importante contrapor a expressão "não estamos permanentemente a ler". A verdade é que, apesar desta leitura poder não ser de livros, somos frequentemente (e até inconscientemente) chamados à leitura: seja um jornal ou uma revista digital, uma publicação no Instagram/Facebook, um outdoor à margem da estrada, etc.. Todas estas ferramentas, que estão já incutidas nosso cotidiano, apesar de não serem, maioritariamente, educativas, estimulam também a leitura (e, na verdade, nem todos os livros são educativos).

O direito de não ler é algo que está à diposição de todos, por isso, torna-se muitas vezes uma ação que passa para segundo plano, muito rapidamente, seja por falta de tempo para o realizar, porque não consideram interessante ou até mesmo porque preferem outras atividades que não englobam a leitura. Neste contexto, é relevante salientar, que as pessoas não têm todas de gostar ou sentir vontade de ler,contudo, é muito importante criar ou ter o hábito de ler e especialmente, incutir esta vontade nas crianças.

O direito de não falar do que se leu, é um direito que cada leitor define. Cada um têm a sua interpretação e a sua reação em relação à leitura de um livro, por isso é muito importante respeitarmos que a pessoa não partilhe aquilo que leu. Os leitores quando decidem ler um livro têm as suas razões para o fazer " Por isso as razões que temos para ler são tão estranhas como as que temos para viver".

O facto de o leitor realizar uma leitura em voz alta permite não só uma melhor compressão por parte do leitor como também do ouvinte " O homem que lê em voz alta expõe-se totalmente aos olhos que o escutam ". Além disso, o leitor ao ler em voz alta vai captar com muito mais facilidade quem o está a ouvir e permite que se torne uma leitura mais atrativa e interessante.

Um leitor ao ter o direito de saltar de livro em livro, consegue retirar uma maior satifação daquilo que lê, pois não se foca apenas num estilo de livro. Ao ir mudando de livro consegue perceber qual o gênero de livro que prefere e que se sente disposto para o ler. Por vezes, os leitores ao vizualizarem um livro sentem a necessidade de parar a leitura do livro que estam a ler e optam por ler um outro " de o abrir onde nos apetecer e de mergulharmos nele por um instante, porque só dispomos desse instante"

Os leitores ao terem o direito de ler não importa onde, permite que quando estão a realizar a leitura se sintam concentrados, confortáveis e predispostos para o fazer. Cada pessoa têm o direito de poder escolher em que local prefere realizar a sua leitura, tudo depende das características de cada leitor.

Todos temos o direito de amar os heróis da ficção, o facto o leitor se identificar com alguma parte da história ou com uma personagem permite uma leitura muito mais significante, pois desta forma, o leitor vai sentir diversos sentimentos, uma maior curiosidade e para além disso, vai sentir melhor experiência na realização da leitura. O que não significa, que deva cumprir e acreditar-se em tudo o que é feito nos livros.

Um leitor tem o direito de não querer acabar o livro que está a ler. De facto, a escolha de um livro nem sempre é a mais adequada e com isso, surge a vontade de não ler mais, assim como é referido pelo autor, " Há outros que ainda estão à minha espera, e desses provavelmente alguns continuarão para sempre à minha espera".

O direito de reler um livro permite reviver uma leitura ou voltar a tentar ler um livro, que não conseguimos fazer no momento. Reler permite reavivar a memória dos acontecimentos de um livro, repensar e também confirmar o que já tinhamos lido. Posto isto, um leitor ao ter o direito de reler um livro, promove que o mesmo consiga usufruir novamante de uma leitura, mas vista com outros olhos, com uma outra reação e por fim, com uma nova perspetiva da história.

Por vezes os leitores optam por não terminar a leitura de um livro, sendo um ato que não têm de ser posto em causa. Nem sempre as histórias que são contadas nos livros, vão de encontro ao que o leitor esperava ou apenas não correponde às suas especatativas, por isso têm o direito de não acabar de ler, o que não significa que futuramente não sinta vontade de o ler,tal como é referido pelo autor " Talvez voltaremos a tentar, talvez não".

Neste parágrafo, o autor reforça a ideia de que apesar da "ordem" de um livro, o leitor não necessita de a seguir porque o mesmo formula a sua própria opinião, fazendo com que o "Saltar Páginas" expresse uma vontade maior de continuar a ler o livro e dedicar-se melhor ao mesmo.

O direito de não ler é algo que diariamente está presente na vida de cada individuo fazendo com o que o mesmo, se orgulhe de expressar isso mesmo. Ler, hoje em dia, é uma atividade que é desvalorizada, sendo oprimida por outras mesmo atividades, tal como demonstra neste primeiro tópico. Este parágrafo serve para salientar a importância da consciencialização do ato da leitura, principalmente nas crianças, visto que, noutras faixas etárias o gosto pela leitura tem vindo a diminuir.

O direito de não ler é algo que, hoje em dia, está muito presente na vida de cada individuo, fazendo com que o gosto pela leitura seja desvalorizado e fazendo com que, assim os indivíduos se orgulhem disso mesmo. A leitura acaba por ser oprimida por outras atividades, acabando assim por ser cada vez menos comum a valorização de um simples livro. Com isto, neste parágrafo, podemos observar a importância da consciencialização para a leitura, principalmente em crianças, visto que, noutras faixas etárias a mesma, tem vindo a diminuir.

Nesta parte do texto, o autor reforça a ideia do direito de saltar páginas. O facto de um livro apresentar uma determinada ordem de capítulos ou páginas, não obriga o leitor, a cumprir a mesma, tal como é referido no texto " Digamos o que dissermos, o aborrecimento teimoso que impomos a nós próprios não está relacionado com o dever, é uma categoria do nosso prazer de ler", ou seja, o leitor ao poder saltar páginas livremente, permite que o mesmo, sinta que pode apenas ler aquilo que está a disposto, além disso, proporciona a quem está a ler uma convição maior para realizar a leitura do livro e de uma forma mais prazerosa.

expor o que leu faz com que o leitor tenha de se abrir perante outras pessoas sobre as suas leituras

seja uma experiencia de melhor compreensão do texto, consiga aprofundar o tema e conexão

faz com que o leitor possa ler vários estilos de livros e seja livre em escolher, e depois perceber qual será o seu favorito

permite ao leitor ler onde quiser e sela livre

é importante amar os "heróis" pois muitas vezes são uma inspiração embora que também seja importante diferenciar da realidade e ficção

permite o desenvolvimento da exploração dos seus interesses ao longo da leitura

reler um livro, permite que explore outras partes do livro que da primeira vez não o tenha feito

por vezes, não acabamos de ler um livro não ser interessante e não existe problema com isso

o facto de saltar páginas realça o facto de por nem todos lemos de uma maneira soe sim por vezes existe liberdade em ler

todos temos o direito de não ler , noa só por não gostarmos ou não termos nenhum livro

100 Direito de Não Falar do Que se Leu

O direito à privacidade na leitura é crucial, pois permite aos leitores uma experiência íntima e pessoal, onde podem explorar os seus próprios pensamentos e sentimentos sem a pressão de conformar-se às opiniões alheias. Essa liberdade proporciona um refúgio para a reflexão solitária, onde os leitores podem se conectar mais profundamente com o texto. Ao respeitar essa privacidade, garantimos que a leitura permaneça como um espaço seguro e pessoal, onde cada indivíduo se pode envolver com as obras de forma autêntica e sem julgamento externo.

90 Direito de Ler em Voz Alta

Ler em voz alta é uma prática que enriquece significativamente a experiência de leitura. Ao recitar as palavras em voz alta, os leitores não so interiorizam o texto de maneira mais profunda, como também aprimoram a sua compreensão, pois a audição complementa a visão na absorção da informação. Além disso, a leitura em voz alta dá vida ao texto, permitindo que os leitores criem uma conexão mais íntima com a obra, à medida que exploram os ritmos e nuances da linguagem. Esta prática também oferece a oportunidade de partilhar a paixão pela leitura com os outros, promovendo a conexão e o diálogo entre os entusiastas da leitura. Ao ler em voz alta, os leitores não só partilham a história, mas também as suas interpretações e emoções, enriquecendo a experiência para todos os envolvidos.

80 Direito de Saltar de Livro em Livro

O direito de saltar de livro em livro é uma expressão da liberdade do leitor de moldar a sua própria jornada literária. Essa liberdade não só permite que os leitores escolham as obras que mais os interessam, mas dá a capacidade de abandonar livros que não os cativam. Ao ter essa liberdade, os leitores podem direcionar o seu tempo e energia para as leituras que lhes proporcionam mais prazer e satisfação, evitando assim a frustração de ficar preso em uma leitura desinteressante. A capacidade de "debicar" em diferentes livros sem o medo de decepção é essencial para uma experiência de leitura verdadeiramente enriquecedora, permitindo aos leitores explorar uma variedade de estilos, géneros e temas sem restrições. Isso não só amplia os seus horizontes literários, como enriquece a sua compreensão do mundo e de si mesmos através das múltiplas perspectivas oferecidas pela diversidade de obras disponíveis. Em última análise, o direito de saltar de livro em livro capacita os leitores a serem os mestres da sua própria jornada literária, moldando-a de acordo com suas preferências individuais e experiências únicas.

70 Direito de Ler não Importa Onde

O direito de ler em qualquer lugar não só aumenta a acessibilidade à leitura, como promove a liberdade individual e a diversidade de experiências literárias. Isso permite que os leitores explorem novas ideias em momentos oportunos e adaptem o seu ambiente de leitura às suas preferências pessoais, resultando numa experiência mais significativa e envolvente.

60 Direito de Amar os "Heróis" dos Romances

Amar os heróis fictícios é essencial, pois inspiram-nos e ajudam a compreender o mundo e geram felicidade ao identificarmo-nos com eles. Mesmo que não sejam perfeitos, as suas falhas podem ensinar importantes lições sobre a humanidade e os relacionamentos.

50 Direito de Ler não Importa o Quê

Ler sem restrições é fulcral para os leitores, permitindo explorar interesses, desenvolver o senso crítico e desfrutar da leitura. Esse direito, sem censura ou culpa, é fundamental para a vida, a liberdade de pensamento e a imaginação.

O direito de não ler

Esta parte do livro questiona a visão convencional de que a leitura é essencial para a formação completa do ser humano, destacando que a leitura não é necessariamente o único caminho para a humanização. A frase "A ideia de que a leitura humaniza o homem" sugere que há outras formas de enriquecimento pessoal além da leitura. Reconhece-se que nem todos têm o mesmo apreço pela leitura, e é importante respeitar o direito de cada indivíduo de optar por não ler, pois isso faz parte da sua liberdade individual.

DOI: 10.20944/preprints202401.1738.v1

Resource: (Santa Cruz Biotechnology Cat# sc-25726, RRID:AB_649789)

Curator: @scibot

SciCrunch record: RRID:AB_649789

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DOI: 10.1016/j.isci.2024.109785

Resource: (Proteintech Cat# HRP-66002, RRID:AB_2883834)

Curator: @scibot

SciCrunch record: RRID:AB_2883834

What is this?

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Response to Reviewer 1

__Glycosaminoglycan (GAG)-binding proteins regulating essential processes such as cell growth and migration are essential for cell homeostasis. It is reported that the GAG has the ability to bind to Herpin sulfate. As both GAGs and the LPS lipid A disaccharide core of gram-negative bacteria contain negatively charged disaccharide units, the researchers proposed that heparin-binding peptides might have cryptic antimicrobial peptide motifs. To prove the hypothesis, they have synthesized five candidates [HBP1-5], which showed a binding affinity towards heparin and LPS binding. By using various methods, they showed that these molecules have antimicrobial activity. The key finding in this study is the finding of the CPC domain, where C is a cationic amino acid and P is a polar amino acid. __

Major comments

1. __ Even though the Authors propose here that CPC' clip motif is needed for antimicrobial activity. However, various studies have demonstrated that the mere presence of cationic amino or hydrophobic amino acids does not give the activity, the location of these amino acids at the strategic position is critically needed. The major issue in this work, the authors have not presented, whether there was a single CPC motif or multiple in the 5 peptides they have synthesized. Further, they need to demonstrate how are the charged and hydrophobic amino acids distributed in the peptides. these things will clearly explain the difference in the activity as well spectrum of the peptides. The authors should make an extra figure or add information highlighting this unique characteristic for better understanding to the reader.__

We thank the reviewer for his/her comments and suggestions. We concur that the distribution of amino acids is crucial for the antimicrobial activity of the peptides and their ability to bind heparin. We also agree with the suggestion of illustrating the location of the CPC' motifs of HBPs in the context of the parental proteins and have accordingly done so in the new Supplementary Figure 1. In all cases, only one CPC' motif was identified in the antimicrobial region, as highlighted in the figure, and the inter-residue distances measured are consistent with the CPC' motif definition. Thus, we demonstrate that a CPC' motif exists in all five HBPs, which explains how they recognize and bind heparin.

To illustrate the distribution of charged and hydrophobic amino acids in HBPs, we have also prepared new Supplementary Figure 2, displaying electrostatic potentials in the predicted HBP structures, and showing how the distribution of charged residues creates hydrophobic and cationic patches on the surface of the peptides. Our analysis reveals cationic patches to be surrounded by hydrophobic residues, which may explain the ability of the peptides to disrupt membranes and exert antimicrobial activity.

__ It is strange to observe that there are quite a number of reports showing that the peptides derived from the Herprin binding proteins have antimicrobial activity, but no one has reported their efficacy in the in vivo mouse model. if possible, the authors could add their observations if in vivo studies were done. or as a future line of study.__

We thank the reviewer for his/her comment on the observation of antimicrobial activity in peptides derived from heparin-binding proteins. Indeed, a few such studies have appeared in the literature, some with moderate success [1]. It is possible that a lack of understanding on how to identify heparin-binding regions in proteins and AMPs underlies their relative paucity. In this context, we believe our results will spur further efforts, specifically by providing a rationale on how to identify CPC' motifs hence heparin-binding regions in protein sequences.

Regarding the suggestion of assessing the in vivo efficacy of HBPs, we would agree that it would be helpful for better understanding their potential therapeutic applications. However, we feel that such experiments are beyond the scope of our manuscript, which offers ample, compelling in vitro and in silico evidence of how heparin-binding proteins can be a source of AMPs. We have done this by showing that CPC' motifs embedded in such proteins can be unveiled, accurately defined in structural terms, and experimentally shown to possess antimicrobial activity. Furthermore, we have shown that heparin binding correlates with LPS binding, allowing us to propose a mechanistic explanation for how heparin binding can be related to antimicrobial activity.

Translating these results to animal models is possibly premature at this stage as, from a classical medicinal chemistry perspective, it would require previous structural elaboration in terms of, e.g., optimized serum half-life or serum protein binding, both of which can modulate activity in in vivo studies regardless of heparin affinity or bactericidal activity per se. Ongoing work in our laboratories is focused in these directions and will be reported in due time.

*Referees cross-commenting**

Minor comments

1. __ The presence of Cryptic antimicrobial domain in various heparin-binding proteins like laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitor, matrix glycoproteins thrombospondin, proline arginine-rich end leucine-rich repeat protein and fibronectin, have been reported previous. It is not clear why the authors did not refer to that work. The authors should refer to the works. (same as reviewer 3)__

We were aware of other prior studies on heparin-binding proteins and did indeed cite some of them, though not exhaustively for conciseness' sake. However, as encouraged by reviewers 1 and 3 we have cited the following studies:

Malmström E, Mörgelin M, Malmsten M, Johansson L, Norrby-Teglund A, Shannon O, Schmidtchen A, Meijers JC, Herwald H. Protein C inhibitor--a novel antimicrobial agent. PLoS Pathog. 2009 Dec;5(12):e1000698. doi: 10.1371/journal.ppat.1000698. Epub 2009 Dec 18. PMID: 20019810; PMCID: PMC2788422.

Ishihara, J., Ishihara, A., Fukunaga, K. et al. Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing. Nat Commun 9, 2163 (2018). https://doi.org/10.1038/s41467-018-04525-w

Chillakuri Chandramouli R, Jones Céline and Mardon Helen J(2010), Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading, FEBS Letters, 584, doi: 10.1016/j.febslet.2010.06.023

Papareddy P, Kasetty G, Kalle M, Bhongir RK, Mörgelin M, Schmidtchen A, Malmsten M. NLF20: an antimicrobial peptide with therapeutic potential against invasive Pseudomonas aeruginosa infection. J Antimicrob Chemother. 2016 Jan;71(1):170-80. doi: 10.1093/jac/dkv322. Epub 2015 Oct 26. PMID: 26503666.

All the earlier studies related to the antimicrobial activity of the peptides derived from the Heparin-binding protein reported a consensus Cardin and Weintraub motifs i.e, XBBBXXBX or XBBXBX, where X represents hydrophobic or uncharged amino acids, and B represents basic amino acids. However, in this work, the researchers report about the presence of the new CPC motif. So, this is unique and a novelty in the study.

We thank the reviewers for these observations. Indeed, our quest to unveil CPC' motifs in antimicrobial regions of heparin-binding proteins is the key point of our investigation, and what distinguishes it from previous studies on consensus motifs such as XBBBXXBX or XBBXBX. We believe our definition of CPC' motifs in simple, structure-based, and experimentally verifiable terms is not only a significant departure but also a step forward from earlier views, highlighting the importance of a structural perspective in defining heparin-binding regions. In point of fact, we show that our peptides, even without consensus Cardin-Weintraub motifs, bind heparin with high affinity. The presence of the CPC' motif is crucial for such binding, as well as for LPS binding, and the new experiments performed at editor/reviewer's request, where the CPC motif in HBP5 is abolished, with predictable impact, fully support our view, see new section "Insights into the CPC' motif of HBP-5 and its implication on the antibacterial mechanism" and new Table 3 in the revised manuscript.

__ Even though the researchers report on the role of the CPC motif in the antimicrobial activity and binding to the heprin, the authors did not show any data or draw the conclusions related to the CPC domain when it comes to differences in the activity. this is the weakness of the manuscript. (same as reviewer 2)__

We welcome the reviewer's observation. To address it, we made and tested three HBP-5 mutants aimed at showing how alterations in the CPC' motif might influence interaction with heparin and LPS, as well as antimicrobial properties. The first two mutants involved replacing positively charged R10 and R14 residues with glutamine, similar in size and polarity but uncharged. As shown in the new section "Insights into the CPC' motif of HBP-5 and its implication on the antibacterial mechanism" and on the new Table 3 of the revised manuscript, the changes reduced heparin binding, i.e., shorter retention times on affinity chromatography, as well as LPS binding, i.e., a decrease in EC50 in the cadaverine assay (Table 3). The modifications had a lesser impact on antimicrobial activity, most likely due to the low resolution of MIC assays.

In a further step to assess the effect of the CPC' motif on antimicrobial activity, we deleted it in full by replacing residues H9, R10 and R14 of HBP-5 by alanine. As expected, this DCPC' peptide showed a sharp reduction in both heparin and LPS binding (Table 3) and, most importantly, a significant and asymmetric change in antimicrobial activity, with substantial impact on Gram-negatives yet practically no effect on Gram-positives, suggesting that LPS plays a key role in this selective response. Altogether, these observations align with our hypothesis that heparin-binding proteins might exploit their intrinsic affinity for heparin as an opportunity to developing antimicrobial properties by leveraging structural similarities between glycosaminoglycans and LPS.

__ It is strange to observe that there are quite a number of reports showing that the peptides derived from the Herprin (sic) binding proteins have antimicrobial activity, but no one has reported their efficacy in the in vivo mouse model. if possible, the authors could add their observations if in vivo studies were done. or as a future line of study. (Same as reviewer 2)__

We would kindly direct attention to #2 in the response to reviewer 1 above.

__ There are more than 20 different AMP databases or prediction software. however, not all of them are 100 % current, their success rate varies from 30-50% only. It needs to be investigated if adding this search in the hit peptides might increase the success rate of the extra in silico-based AMPs prediction software.__

If we understand the question correctly, the reviewer wonders whether including a CPC' motif predictor would increase the accuracy of AMP search algorithms. In our view, this strategy has two main limitations to be considered: (i) locating a CPC' motif in a peptide sequence typically requires a known 3D structure. Unfortunately, this is not always the case, and for proteins lacking reliable 3D data it can be a challenging and resource-intensive process; (ii) while CPC' motifs may predispose proteins to evolve antimicrobial properties, it is unclear if this is a required feature for all AMPs. Imposing the presence of a CPC' motif may not be applicable to all AMPs, although it might help identifying peptides with specific activity against gram-negative strains.

In summary, while the query of including a CPC' motif search tool in AMP predictors is intriguing and worthy of exploration for its potential bearing on antimicrobial research, it is technically complicated and beyond the scope of our manuscript.

__Reviewer #1 (Significance (Required)): __

__All the earlier studies related to the antimicrobial activity of the peptides derived from the Heparin-binding protein reported a consensus Cardin and Weintraub motifs i.e, XBBBXXBX or XBBXBX, where X represents hydrophobic or uncharged amino acids, and B represents basic amino acids. However, in this work, the researchers report about the presence of the new CPC motif. So this is unique and a novelty in the study. __

Even though the researchers report on the role of the CPC motif in the antimicrobial activity and binding to the heparin, the authors did not show any data or draw conclusions related to the CPC domain when it comes to differences in the activity. This is the weakness of the manuscript.

We would direct reviewer's attention to #1 in the Referee's cross-commenting section above.

Response to Reviewer 2

This is a very nice paper by the Andreu and Torrent groups that report the antimicrobial and heparin-binding of several encrypted peptides. Overall, this study presents an intriguing exploration into the potential dual functionality of glycosaminoglycan (GAG)-binding proteins, specifically heparin-binding proteins (HBPs), in recognizing lipopolysaccharide (LPS) and exhibiting antimicrobial properties. The findings, particularly the identification and characterization of novel encrypted peptides, such as HBP-5, are promising and contribute to our understanding of the intricate interplay between GAG-binding proteins and immunity. The data provided and methodology are thorough and well described. In sum, this is a very nice work. Please see below my minor comments.

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Minor comments:

1. __ Fig. 1 legend does not show antimicrobial activity. Please remove from the figure legend title.__

As pointed out by the reviewer, the legend was incorrect and has been corrected accordingly and now reads "Figure 1. Structural and bioinformatics analysis of HBPs".

__ Discussion section: the authors should expand this section a bit to discuss recent work in the encrypted/cryptic peptide area. There are some recent relevant papers published in the past 3 years that should be discussed.__

We agree with the reviewer's suggestion to expand the discussion section to address recent work in the field of encrypted/cryptic peptides. We have carefully reviewed the recent literature and added several references in this topic:

Torres MDT, Melo MCR, Flowers L, Crescenzi O, Notomista E, de la Fuente-Nunez C. Mining for encrypted peptide antibiotics in the human proteome. Nat Biomed Eng. 2022 Jan;6(1):67-75. doi: 10.1038/s41551-021-00801-1. Epub 2021 Nov 4. Erratum in: Nat Biomed Eng. 2022 Dec;6(12):1451. PMID: 34737399.

• *

Santos MFDS, Freitas CS, Verissimo da Costa GC, Pereira PR, Paschoalin VMF. Identification of Antibacterial Peptide Candidates Encrypted in Stress-Related and Metabolic Saccharomyces cerevisiae Proteins. Pharmaceuticals (Basel). 2022 Jan 28;15(2):163. doi: 10.3390/ph15020163. PMID: 35215278; PMCID: PMC8877035.

• *

Boaro A, Ageitos L, Torres MT, Blasco EB, Oztekin S, de la Fuente-Nunez C. Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom. Cell Rep Phys Sci. 2023 Jul 19;4(7):101459. doi: 10.1016/j.xcrp.2023.101459. PMID: 38239869; PMCID: PMC10795512.

__ References provided are a bit outdated and do not accurately reflect the latest in the field (see comment above).__

We thank the reviewer for this comment. Older references were updated as suggested.

__ Gram should be capitalized throughout the text.__

Gram has been capitalized as suggested by the reviewer.

__ Can the authors comment on the potential translatability of HBP-5? Please also comment on the potential advantages of having peptides that 1) bind to heparin; and 2) kill bacteria.__

We appreciate the reviewer's interest in the potential of HBP-5. Indeed, we believe it has promise for clinical applications due to its unique attributes, but further studies, including in vivo experiments and pharmacokinetic assessments, are needed to fully evaluate its potential. The advantages of peptides that bind to heparin and kill bacteria include targeted delivery or localization of therapeutic agents, enhanced efficacy, and minimized off-target effects. HBP-5's ability to perturb outer membrane LPS, a crucial aspect of its antibacterial activity, makes it a promising approach to combat Gram-negative bacterial infections, which are often challenging to treat. By disrupting the outer membrane integrity, HBP-5 may also enhance the susceptibility of Gram-negative bacteria to other antimicrobial agents or host immune responses, underscoring its translational potential for treating bacterial infections.

__ More details on the computational tools and methods used to mine the peptides are needed.__

We have updated the Methods section to provide more details on the computational tools used for defining AMPs. Briefly, from the library of heparin-binding proteins obtained from previous studies [2] and AMP scanning for all these proteins was performed using the AMPA tool. The predicted antibacterial segments were located in the 3D structure of their respective proteins. Then, the CPC' motifs were searched in each segment following the criteria previously reported in [3, 4]. The motif involves two cationic residues (Arg or Lys) and a polar residue (preferentially Asn, Gln, Thr, Tyr or Ser), with fairly conserved distances between the carbons and the side chain center of gravity, defining a clip-like structure where heparin would be lodged. This structural motif is highly conserved and can be found in many proteins with reported heparin binding capacity. Finally, for all these regions, docking with a heparin disaccharide was performed using AutoDock Vina to evaluate the potential binding energy.

Response to Reviewer 3

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__Summary: This manuscript has identified and investigated antimicrobial peptides from GAG binding proteins. Authors hypothesized that due to physiochemical similarity between GAG and LPS, fragments of GAG binding proteins might exert antimicrobial activity particularly against G- bacteria. Authors have identified few such AMPs that demonstrate LPS binding and displayed antibacterial activity. They have also solved NMR structure of the potent peptide and mode of action. __

Major comments: AMPs are promising molecules that can serve as lead for the development of therapeutics against MDR bacteria. In particular, currently therapeutic options to treat MDR Gram negative pathogens are limited. The current study is interesting and provides new non-toxic AMPs. Conclusions drawn from the works are largely valid. However, authors should address following comments:

1. __ The design and characterization of the peptide YI12WF is not described. Previous studies had shown design of β-boomerang peptides (Bhattacharjya and coworkers) that target LPS.__

---

We thank the reviewer for this comment. YI12WF (YVLWKRKRFIFI-amide) has been previously reported [4, 5] and shown to bind LPS with high affinity. YI12WF also contains a CPC' motif that, if deleted, reduces heparin binding [4]. References have been added in the text.

__ Mutations or substitution of the key residues peptide 5 might improve the novelty of the work.__

We thank the reviewer for this comment and agree that targeted substitutions in HBP-5 might shed light on the importance of the CPC' motif. As this point was also raised by reviewer 1, we would direct the reviewer's attention to #2 in the *Referees cross-commenting** section above.

__ How these peptides disrupt LPS permeability is not investigated. As LPS is the major target.__

We thank the reviewer for this suggestion and have accordingly evaluated the outer membrane (OM) permeability of the peptides by the 1-N-phenyl-naphthylamine (NPN) assay, a widely used method to assess OM integrity in Gram-negative bacteria. NPN is typically unable to cross the intact outer membrane; however, when the membrane is damaged or disrupted, it can penetrate and interact with lipids and proteins inside the cell, leading to an increase in fluorescence which is directly correlated with the degree of OM permeability and serves as an indicator of membrane damage.

Our results, illustrated in the new Figure 2D, show that all peptides are able to disrupt the OM of Gram-negative bacteria comparably to the LL-37 positive control, except for HBP2. Notably, HBP-5 exhibits the highest activity against OM, consistent with findings elsewhere in the manuscript and altogether confirming the ability of HBPs to bind to and disrupt the LPS structure.

__ Are the D-enantiomers of the peptides active against bacteria?__

We tested the antibacterial activity of the D-enantiomer of HBP5 (dHBP-and 5) and found it to be even higher than that of all-L HBP-5 against both Gram-negative and -positive bacteria, probably due to increased proteolytic stability as found in many AMP studies [6, 7]. As for LPS and heparin affinity, L- and D-HBP-5 behaved similarly (Table R1). As expected, the CD signatures of L- and D-HBP-5 were mirror images (Figure R1). These results suggest that the conformation of the CPC' motif is preserved in dHBP5, in tune with all previous results.

Antibacterial Activity

ID

E. Coli

P. Aeruginosa

A. Baumannii

S. Aureus

E. Faecium

L. monocytognes

HPB-5

0.4

0.8

0.2

6.3

25

1.6

dHBP-5

0.1

0.2

0.2

1.6

0.4

0.2

Binding Affinity

LPS (EC50, µM)

Heparin (% Elution buffer)

HPB-5

0.9 {plus minus} 0.7

98.0

dHBP-5

1.1 {plus minus} 0.8

97.2

Table R1. Antimicrobial activity of HBP-5 and dHBP-5

Figure R1. CD spectra of HBP-5 (red line) and dHBP-5 (green line) in LPS (left panel) and heparin (right panel).

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__ 3D structure of peptide 5 is solved in DPC micelle which is a mimic for eukaryotic cells. Authors should attempt to determine structure in LPS as shown in several recent studies with potent AMPs thanatin, MSI etc.__

We appreciate the suggestion and have indeed attempted to obtain NMR spectra of HBP-5 in LPS micelles. However, we've been hindered by peptide precipitation and, despite considerable efforts, have not been able to obtain satisfactory results thus far. In contrast, we have succeeded in obtaining CD spectra of HBP5 in LPS micelles, showing an a-helix conformation similar to the one in SDS micelles, hence suggesting similar conformation in both environments.

Minor comments: There are examples of AMPs derived from human proteins. Authors should highlight such works.

Other studies have been cited according to the reviewers' comments:

Malmström E, Mörgelin M, Malmsten M, Johansson L, Norrby-Teglund A, Shannon O, Schmidtchen A, Meijers JC, Herwald H. Protein C inhibitor--a novel antimicrobial agent. PLoS Pathog. 2009 Dec;5(12):e1000698. doi: 10.1371/journal.ppat.1000698. Epub 2009 Dec 18. PMID: 20019810; PMCID: PMC2788422.

Ishihara, J., Ishihara, A., Fukunaga, K. et al. Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing. Nat Commun 9, 2163 (2018). https://doi.org/10.1038/s41467-018-04525-w

Chillakuri Chandramouli R, Jones Céline and Mardon Helen J(2010), Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading, FEBS Letters, 584, doi: 10.1016/j.febslet.2010.06.023

Papareddy P, Kasetty G, Kalle M, Bhongir RK, Mörgelin M, Schmidtchen A, Malmsten M. NLF20: an antimicrobial peptide with therapeutic potential against invasive Pseudomonas aeruginosa infection. J Antimicrob Chemother. 2016 Jan;71(1):170-80. doi: 10.1093/jac/dkv322. Epub 2015 Oct 26. PMID: 26503666.

References

1. Papareddy, P., et al., An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014. 1838(5): p. 1225-1234.
2. Ori, A., M.C. Wilkinson, and D.G. Fernig, A systems biology approach for the investigation of the heparin/heparan sulfate interactome. J Biol Chem, 2011. 286(22): p. 19892-904.
3. Torrent, M., et al., The "CPC Clip Motif": A Conserved Structural Signature for Heparin-Binding Proteins.PLOS ONE, 2012. 7(8): p. e42692.
4. Pulido, D., et al., Structural similarities in the CPC clip motif explain peptide-binding promiscuity between glycosaminoglycans and lipopolysaccharides. J R Soc Interface, 2017. 14(136).
5. Bhunia, A., et al., Designed beta-boomerang antiendotoxic and antimicrobial peptides: structures and activities in lipopolysaccharide. J Biol Chem, 2009. 284(33): p. 21991-22004.
6. Varponi, I., et al., Fighting Pseudomonas aeruginosa Infections: Antibacterial and Antibiofilm Activity of D-Q53 CecB, a Synthetic Analog of a Silkworm Natural Cecropin B Variant. Int J Mol Sci, 2023. 24(15).
7. Chen, Y., et al., Comparison of Biophysical and Biologic Properties of α-Helical Enantiomeric Antimicrobial Peptides. Chemical Biology & Drug Design, 2006. 67(2): p. 162-173.

Author response:

Response to Reviewer #1 (Public Review):

We thank the reviewer for their constructive criticism of our study, their proposed solutions, and for highlighting areas of the methodology and analytical pipeline where explanations were unclear or unsatisfactory. We will take the reviewer’s feedback into account to improve the clarity and readability of the revised manuscript. We acknowledge the importance of ruling out eye movements as a potential confound. We address these concerns briefly below, but a more detailed explanation (and a full breakdown of the relevant analyses, including the corrected and uncorrected results) will be provided in the revised manuscript.

First, the source of EEG activity recorded from the frontal electrodes is often unclear. Without an external reference, it is challenging to resolve the degree to which frontal EEG activity represents neural or muscular responses1. Thus, as a preventative measure against the potential contribution of eye movement activity, for all our EEG analyses, we only included activity from occipital, temporal, and parietal electrodes (the selected electrodes can be seen in the final inset of Figure 3).

Second, as suggested by the reviewer, we re-ran our analyses using the activity measured from the frontal electrodes alone. If the source of the nonlinear decoding accuracy in the AV condition was muscular activity produced by eye movements, we would expect to observe better decoding accuracy from sensors closer to the source. Instead, we found that decoding accuracy from the frontal electrodes (peak d' = 0.08) was less than half that of decoding accuracy from the more posterior electrodes (peak d' = 0.18). These results suggest that the source of neural activity containing information about stimulus position was located over occipito-parietal areas, consistent with our topographical analyses (inset of Figure 4).

Third, we compared the average eye movements between the three main sensory conditions (auditory, visual, and audiovisual). In the visual condition, there was little difference in eye movements corresponding to the five stimulus locations, likely because the visual stimuli were designed to be spatially diffuse. For the auditory and audiovisual conditions, there was more distinction between eye movements corresponding to the stimulus locations. However, these appeared to be the same between auditory and audiovisual conditions. If consistent saccades to audiovisual stimuli had been responsible for the nonlinear decoding we observed, we would expect to find a higher positive correlation between horizontal eye position and stimulus location in the audiovisual condition than in the auditory or visual conditions. Instead, we found no difference in correlation between audiovisual and auditory stimuli, indicating that eye movements were equivalent in these conditions and unlikely to explain better decoding accuracy for audiovisual stimuli.

Finally, we note that the stricter eye movement criterion acknowledged in the Discussion section of the original manuscript resulted in significantly better audiovisual d' than the MLE prediction, but this difference did not survive cluster correction. This is an important distinction to make as, when combined with the results described above, it seems to support our original interpretation that the stricter criterion combined with our conservative measure of (mass-based) cluster correction2 led to type 2 error.

References

(1) Roy, R. N., Charbonnier, S., & Bonnet, S. (2014). Eye blink characterization from frontal EEG electrodes using source separation and pattern recognition algorithms. Biomedical Signal Processing and Control, 14, 256–264.

(2) Pernet, C. R., Latinus, M., Nichols, T. E., & Rousselet, G. A. (2015). Cluster-based computational methods for mass univariate analyses of event-related brain potentials/fields: A simulation study. Journal of Neuroscience Methods, 250, 85–93.

Response to Reviewer #2 (Public Review):

We thank the reviewer for their insight and constructive feedback. As emphasized in the review, an interesting question that arises from our results is that, if the neural data exceeds the optimal statistical decision (MLE d'), why doesn’t the behavioural data? We agree with the reviewer’s suggestion that more attention should be devoted to this question, and plan to provide a deeper discussion of the relationship between behavioural and neural super-additivity in the revised manuscript. We also note that while this discrepancy remains unexplained, our results are consistent with the literature. That is, both non-linear neural responses (single-cell recordings) and behavioural responses that match MLE are reliable phenomenon in multisensory integration1,2,3,4.

One possible explanation for this puzzling discrepancy is that behavioural responses occur sometime after the initial neural response to sensory input. There are several subsequent neural processes between perception and a behavioural response5, all of which introduce additional noise that may obscure super-additive perceptual sensitivity. In particular, the mismatch between neural and behavioural accuracy may be the result of additional neural processes that translate sensory activity into a motor response to perform the behavioural task.

Our measure of neural super-additivity (exceeding optimally weighted linear summation) differs from how it is traditionally assessed (exceeding summation of single neuron responses)2. However, neither method has yet fully explained how this neural activity translates to behavioural responses, and we think that more work is needed to resolve the abovementioned discrepancy. However, our method will facilitate this work by providing a reliable method of measuring neural super-additivity in humans, using non-invasive recordings.

References

(1) Alais, D., & Burr, D. (2004). The ventriloquist effect results from near-optimal bimodal integration. Current Biology, 14(3), 257–262.

(2) Ernst, M. O., & Banks, M. S., (2002). Humans integrate visual and haptic information in a statistically optimal fashion. Nature, 415(6870), 429–433.

(3) Meredith, M. A., & Stein, B. E. (1993). Interactions among converging sensory inputs in the superior colliculus. Science, 221, 389–391.

(4) Stanford, T. R., & Stein, B. E. (2007). Superadditivity in multisensory integration: putting the computation in context. Neuroreport 18, 787–792.

(5) Heekeren, H., Marrett, S. & Ungerleider, L. (2008). The neural systems that mediate human perceptual decision making. Nature Reviews Neuroscience, 9, 467–479.

Author Response:

We appreciate the thorough comments from the reviewers. Before revising the manuscript, we would like to briefly reply to the main concerns raised:

• Is pupil size a reliable proxy of effort? A vast amount of work demonstrates that pupil size sensitively scales with fluctuations in effort: for instance, the pupil dilates when increasing load in working memory, or multiple object tracking tasks, and such pupillary effects robustly explain individual differences in cognitive ability and fluctuations in performance across trials.1–4 This extends to the planning of movements as pupil dilations are observed prior to the execution of (eye) movements.5 As reviewed previously6–12 (based on vast literature each), any increase in effort is associated with an increase in pupil size. Inadvertently, we phrased as if the link between effort and pupil size was established via shared neural correlates. However, this is not the case as the link between effort and pupil size had been established well before the underlying neural circuitry of this relationship was investigated in detail. During the revision, we plan to rewrite this section to clarify that pupil size indexes effort and to provide a clear distinction between this link and putative neural underpinnings of such effort-linked modulations.

• Is saccade latency an alternative explanation for the link between effort and saccade selection? Longer saccade latencies may imply more complex oculomotor programming (e.g. saccades with larger amplitudes require longer latencies for non-microsaccades13, and latencies increase when distractors are presented14), and latencies are indeed known to differ across directions15,16. As suggested, it is possible that saccade latencies may also predict saccade preferences. However, even if this is the case, this would not constitute an alternative explanation. As saccade latency may index oculomotor programming complexity, it can potentially be considered an alternative outcome measure of effort, albeit restricted to the context of saccades. Therefore, if saccade latencies predict saccade preferences, this would not affect our conclusion, rather it would constitute as converging evidence that supports the conclusion that effort drives saccade selection.

A related question is why one would use pupil size as a measure of effort, given the methodological care that pupillometry requires. There are a number of points that make pupil size sensible and promising in comparison with saccade latencies. In contrast to saccade latencies, pupil size allows to capture the effort of different effector systems (e.g. head or hand movements), and potentially even the effort associated with covert shifts of attention. Moreover, pupil size is a temporally rich and continuous measure that allows to isolate processes unfolding prior to (eye) movement onset (e.g. oculomotor programming). Together, this makes pupil size a powerful tool to study the costs of visual selection more broadly. In the revision, we will add analyses incorporating latencies and other other saccade metrics. We will also discuss the differences between pupil size and saccade latencies in capturing saccade costs and effort.

• Are the current results causal or correlational? Most of the currently reported results are indeed correlational in nature. In our first tasks, we correlated pupil size during saccade planning to saccade preferences in a subsequent task. Although the link between across tasks was correlational, the observed relationship clearly followed our previously specified hypothesis.17 Moreover, experiments 1 and 2 of the visual search data replicated and extended this relationship. We also directly manipulated cognitive demand in the second visual search experiment. In line with the hypothesis that effort affects saccade selection, participants executed less saccades overall when performing a (primary) auditory dual task, and even cut the costly saccades most. Whilst mostly correlational, we do not know of a more fitting and parsimonious explanation for our findings than effort predicting saccade selection. We will address causality in the discussion for transparency and point more clearly to the second visual search experiment for causal evidence.

References

(1) Alnæs, D. et al. Pupil size signals mental effort deployed during multiple object tracking and predicts brain activity in the dorsal attention network and the locus coeruleus. J. Vis. 14, 1 (2014).

(2) Koevoet, D., Strauch, C., Van der Stigchel, S., Mathôt, S. & Naber, M. Revealing visual working memory operations with pupillometry: Encoding, maintenance, and prioritization. WIREs Cogn. Sci. e1668 (2023) doi:10.1002/wcs.1668.

(3) Robison, M. K. & Unsworth, N. Pupillometry tracks fluctuations in working memory performance. Atten. Percept. Psychophys. 81, 407–419 (2019).

(4) Unsworth, N. & Miller, A. L. Individual Differences in the Intensity and Consistency of Attention. Curr. Dir. Psychol. Sci. 30, 391–400 (2021).

(5) Richer, F. & Beatty, J. Pupillary Dilations in Movement Preparation and Execution. Psychophysiology 22, 204–207 (1985).

(6) Bumke, O. Die Pupillenstörungen Bei Geistes-Und Nervenkrankheiten. (Fischer, 1911).

(7) Kahneman, D. Attention and Effort. (Prentice-Hall, 1973).

(8) van der Wel, P. & van Steenbergen, H. Pupil dilation as an index of effort in cognitive control tasks: A review. Psychon. Bull. Rev. 25, 2005–2015 (2018).

(9) Loewenfeld, I. E. Mechanisms of reflex dilatation of the pupil. Doc. Ophthalmol. 12, 185–448 (1958).

(10) Mathôt, S. Pupillometry: Psychology, Physiology, and Function. J. Cogn. 1, 16 (2018).

(11) Sirois, S. & Brisson, J. Pupillometry. WIREs Cogn. Sci. 5, 679–692 (2014).

(12) Strauch, C., Wang, C.-A., Einhäuser, W., Van der Stigchel, S. & Naber, M. Pupillometry as an integrated readout of distinct attentional networks. Trends Neurosci. 45, 635–647 (2022).

(13) Kalesnykas, R. P. & Hallett, P. E. Retinal eccentricity and the latency of eye saccades. Vision Res. 34, 517–531 (1994).

(14) Walker, R., Deubel, H., Schneider, W. X. & Findlay, J. M. Effect of Remote Distractors on Saccade Programming: Evidence for an Extended Fixation Zone. J. Neurophysiol. 78, 1108–1119 (1997).

(15) Hanning, N. M., Himmelberg, M. M. & Carrasco, M. Presaccadic attention enhances contrast sensitivity, but not at the upper vertical meridian. iScience 25, 103851 (2022).

(16) Hanning, N. M., Himmelberg, M. M. & Carrasco, M. Presaccadic Attention Depends on Eye Movement Direction and Is Related to V1 Cortical Magnification. J. Neurosci. 4

4, (2024).

(17) Koevoet, D., Strauch, C., Naber, M. & Van der Stigchel, S. The Costs of Paying Overt and Covert Attention Assessed With Pupillometry. Psychol. Sci. 34, 887–898 (2023).

Reviewer #1 (Public Review):

Abbasi et al. assess in this MEG study the directed connectivity of both cortical and subcortical regions during continuous speech production and perception. The authors observed bidirectional connectivity patterns between speech-related cortical areas as well as subcortical areas in production and perception. Interestingly, they found in speaking low-frequency connectivity from subcortical (the right cerebellum) to cortical (left superior temporal) areas, while connectivity from the cortical to subcortical areas was in the high frequencies. In listening a similar cortico-subcortical connectivity pattern was observed for the low frequencies, but the reversed connectivity in the higher frequencies was absent.

The work by Abbasi and colleagues addresses a relevant, novel topic, namely understanding the brain dynamics between speaking and listening. This is important because traditionally production and perception of speech and language are investigated in a modality-specific manner. To have a more complete understanding of the neurobiology underlying these different speech behaviors, it is key to also understand their similarities and differences. Furthermore, to do so, the authors utilize state-of-the-art directed connectivity analyses on MEG measurements, providing a quite detailed profile of cortical and subcortical interactions for the production and perception of speech. Importantly, and perhaps most interesting in my opinion, is that the authors find evidence for frequency-specific directed connectivity, which is (partially) different between speaking and listening. This could suggest that both speech behaviors rely (to some extent) on similar cortico-cortical and cortico-subcortical networks, but different frequency-specific dynamics.

These elements mentioned above (investigation of both production and perception, both cortico-cortical and cortico-subcortical connectivity is considered, and observing frequency-specific connectivity profiles within and between speech behaviors), make for important novel contributions to the field. Notwithstanding these strengths, I find that they are especially centered on methodology and functional anatomical description, but that precise theoretical contributions for neurobiological and cognitive models of speech are less transparent. This is in part because the study compares speech production and perception in general, but no psychophysical or psycholinguistic manipulations are considered. I also have some critical questions about the design which may pose some confounds in interpreting the data, especially with regard to comparing production and perception.

(1) While the cortico-cortical and cortico-subcortical connectivity profiles highlighted in this study and the depth of the analyses are impressive, what these data mean for models of speech processing remains on the surface. This is in part due, I believe, to the fact that the authors have decided to explore speaking and listening in general, without targeting specific manipulations that help elucidate which aspects of speech processing are relevant for the particular connectivity profiles they have uncovered. For example, the frequency-specific directed connectivity is it driven by low-level psychophysical attributes of the speech or by more cognitive linguistic properties? Does it relate to the monitoring of speech, timing information, and updating of sensory predictions? Without manipulations trying to target one or several of these components, as some of the referenced work has done (e.g., Floegel et al., 2020; Stockert et al., 2021; Todorović et al., 2023), it is difficult to draw concrete conclusions as to which representations and/or processes of speech are reflected by the connectivity profiles. An additional disadvantage of not having manipulations within each speech behavior is that it makes the comparison between listening and speaking harder. That is, speaking and listening have marked input-output differences which likely will dominate any comparison between them. These physically driven differences (or similarities for that matter; see below) can be strongly reduced by instead exploring the same manipulations/variables between speaking and listening. If possible (if not to consider for future work), it may be interesting to score psychophysical (e.g., acoustic properties) or psycholinguistic (e.g., lexical frequency) information of the speech and see whether and how the frequency-specific connectivity profiles are affected by it.

(2) Recent studies comparing the production and perception of language may be relevant to the current study and add some theoretical weight since their data and interpretations for the comparisons between production and perception fit quite well with the observations in the current work. These studies highlight that language processes between production and perception, specifically lexical and phonetic processing (Fairs et al., 2021), and syntactic processing (Giglio et al., 2024), may rely on the same neural representations, but are differentiated in their (temporal) dynamics upon those shared representations. This is relevant because it dispenses with the classical notion in neurobiological models of language where production and perception rely on (partially) dissociable networks (e.g., Price, 2010). Rather those data suggest shared networks where different language behaviors are dissociated in their dynamics. The speech results in this study nicely fit and extend those studies and their theoretical implications.

(3) The authors align the frequency-selective connectivity between the right cerebellum and left temporal speech areas with recent studies demonstrating a role for the right cerebellum for the internal modelling in speech production and monitoring (e.g., Stockert et al., 2021; Todorović et al., 2023). This link is indeed interesting, but it does seem relevant to point out that at a more specific scale, it does not concern the exact same regions between those studies and the current study. That is, in the current study the frequency-specific connectivity with temporal regions concerns lobule VI in the right cerebellum, while in the referenced work it concerns Crus I/II. The distinction seems relevant since Crus I/II has been linked to the internal modelling of more cognitive behavior, while lobule VI seems more motor-related and/or contextual-related (e.g., D'Mello et al., 2020; Runnqvist et al., 2021; Runnqvist, 2023).

(4) On the methodological side, my main concern is that for the listening condition, the authors have chosen to play back the speech produced by the participants in the production condition. Both the fixed order as well as hearing one's own speech as listening condition may produce confounds in data interpretation, especially with regard to the comparison between speech production and perception. Could order effects impact the observed connectivity profiles, and how would this impact the comparison between speaking and listening? In particular, I am thinking of repetition effects present in the listening condition as well as prediction, which will be much more elevated for the listening condition than the speaking condition. The fact that it also concerns their own voice furthermore adds to the possible predictability confound (e.g., Heinks-Maldonado et al., 2005). In addition, listening to one's speech which just before has been articulated may, potentially strategically even, enhance inner speech and "mouthing" in the participants, hereby thus engaging the production mechanism. Similarly, during production, the participants already hear their own voice (which serves as input in the subsequent listening condition). Taken together, both similarities or differences between speaking and listening connectivity may have been due to or influenced by these order effects, and the fact that the different speech behaviors are to some extent present in both conditions.

(5) The ability of the authors to analyze the spatiotemporal dynamics during continuous speech is a potentially important feat of this study, given that one of the reasons that speech production is much less investigated compared to perception concerns motor and movement artifacts due to articulation (e.g., Strijkers et al., 2010). Two questions did spring to mind when reading the authors' articulation artifact correction procedure: If I understood correctly, the approach comes from Abbasi et al. (2021) and is based on signal space projection (SSP) as used for eye movement corrections, which the authors successfully applied to speech production. However, in that study, it concerned the repeated production of three syllables, while here it concerns continuous speech of full words embedded in discourse. The articulation and muscular variance will be much higher in the current study compared to three syllables (or compared to eye movements which produce much more stable movement potentials compared to an entire discourse). Given this, I can imagine that corrections of the signal in the speaking condition were likely substantial and one may wonder (1) how much signal relevant to speech production behavior is lost?; (2) similar corrections are not necessary for perception, so how would this marked difference in signal processing affect the comparability between the modalities?

References:<br /> - Abbasi, O., Steingräber, N., & Gross, J. (2021). Correcting MEG artifacts caused by overt speech. Frontiers in Neuroscience, 15, 682419.<br /> - D'Mello, A. M., Gabrieli, J. D., & Nee, D. E. (2020). Evidence for hierarchical cognitive control in the human cerebellum. Current Biology, 30(10), 1881-1892.<br /> - Fairs, A., Michelas, A., Dufour, S., & Strijkers, K. (2021). The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. Cerebral Cortex Communications, 2(3), tgab040.<br /> - Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.<br /> - Giglio, L., Ostarek, M., Sharoh, D., & Hagoort, P. (2024). Diverging neural dynamics for syntactic structure building in naturalistic speaking and listening. Proceedings of the National Academy of Sciences, 121(11), e2310766121.<br /> - Heinks‐Maldonado, T. H., Mathalon, D. H., Gray, M., & Ford, J. M. (2005). Fine‐tuning of auditory cortex during speech production. Psychophysiology, 42(2), 180-190.<br /> - Price, C. J. (2010). The anatomy of language: a review of 100 fMRI studies published in 2009. Annals of the new York Academy of Sciences, 1191(1), 62-88.<br /> - Runnqvist, E., Chanoine, V., Strijkers, K., Pattamadilok, C., Bonnard, M., Nazarian, B., ... & Alario, F. X. (2021). Cerebellar and cortical correlates of internal and external speech error monitoring. Cerebral Cortex Communications, 2(2), tgab038.<br /> - Runnqvist, E. (2023). Self-monitoring: The neurocognitive basis of error monitoring in language production. In Language production (pp. 168-190). Routledge.<br /> - Stockert, A., Schwartze, M., Poeppel, D., Anwander, A., & Kotz, S. A. (2021). Temporo-cerebellar connectivity underlies timing constraints in audition. Elife, 10, e67303.<br /> - Strijkers, K., Costa, A., & Thierry, G. (2010). Tracking lexical access in speech production: electrophysiological correlates of word frequency and cognate effects. Cerebral cortex, 20(4), 912-928.<br /> - Todorović, S., Anton, J. L., Sein, J., Nazarian, B., Chanoine, V., Rauchbauer, B., ... & Runnqvist, E. (2023). Cortico-cerebellar monitoring of speech sequence production. Neurobiology of Language, 1-21.

DOI: 10.1016/j.cell.2024.04.018

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Curator: @abever99

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DOI: 10.1101/2024.05.06.592806

Resource: (BDSC Cat# 7473,RRID:BDSC_7473)

Curator: @scibot

SciCrunch record: RRID:BDSC_7473

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Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Description of the planned revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

• Again, in Figure 5, were FoxP3/CD4+ cells enumerated? Author Response: Fig 5 showed that the inflammatory score, and activation of CD4 and CD8 cells, were lower in the intestine of DSS-treated mice transplanted with Jag1Ndr/Ndr lymphocytes than in those transplanted with Jag1+/+ lymphocytes. However, in Figure 5 we had not quantified the number of FoxP3/CD4+ cells (Tregs). We agree that it would be interesting to know whether the dampened intestinal inflammation (in response to a classical inflammatory disease model (DSS-treatment)) is also mediated by excess Tregs. We will therefore now quantify Foxp3+ cells on the intestinal sections of experimental animals used for acquisition of data in Fig 5.

• *

Description of the revisions that have already been incorporated in the transferred manuscript.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Reviewer 1 comment: This is an interesting study that examines defects in the Jag1ndr/ndr mouse model of Alagille syndrome. The novel aspects of this manuscript are the comparisons, at many levels, between the mouse model and ALG patient samples, including an examination of immune profiles. The conclusions that the Jag1ndr/ndr mouse model is an accurate representation of the human ALG syndrome appear valid. However the reported differences in immune profiles, particularly in the Jag1ndr/ndr mouse model are difficult to understand. The data presented indicate a reduction in CD4+ cells in the Jag1ndr/ndr mouse at day P3 in both liver and spleen. Additionally, the authors report differences between the the Jag1ndr/ndr mouse and controls at day P30 in the relative percentages of DN, DP and SP CD4 and CD8 cells in the thymus. When examining the peripheral lymphoid system, CD4+ numbers are the same in both the Jag1ndr/ndr animals and controls however CD8+ numbers are reduced and FoxP3/CD4+ cells are increased in both the spleen and the thymus. FoxP3/CD4+ T cells are usually assumed to be regulatory T cells that dampen the inflammatory responses of T cells. Therefore, the increase in this population in an animal model of what is assumed to be an inflammatory disease is confusing and confounding. The authors do not present a clear analysis of how they feel an increase of Tregs would lead to this disease. One possibility is that this population is not functioning as conventional Tregs and rather are promoting inflammation but this conclusion would require a functional analysis of this population of cells, at the very least in an in vitro analysis of T cell suppression. From an immunologist's point of view, their data are antithetical to what one would expect to find in an inflammatory disease. Perhaps this reviewer is missing an important point but if I am missing it, then other who read this manusgcript also may be confused.

Author Response: *We thank the reviewer for carefully assessing our work, and for noting which aspects of the immune analyses should be more thoroughly explained. We apologize for any confusion, which a clearer introduction will help to avoid. *

*Alagille syndrome is not thought of as an inflammatory disorder, it is a congenital disorder affecting bile duct development (Kohut et al 2021, Semin Liver Dis). During normal bile duct development, JAG1+ portal fibroblasts signal to NOTCH2+ hepatoblasts to instruct bile duct development. In the context of low JAG1 signaling, hepatoblasts either fail to adopt a cholangiocyte fate, or fail to undergo bile duct morphogenesis, resulting in bile duct paucity and cholestasis. This cholestasis should activate inflammatory processes leading to fibrosis, which is the subject of this study. *

• *

We agree with the reviewer that Tregs would be expected to suppress inflammation, and our data are consistent with Treg suppression of inflammation. We show, for the first time, that Tregs are enriched in Jag1Ndr/Ndr mice (Fig 4) and present evidence that they suppress inflammation (Fig 5) and fibrosis (Fig 6), which could explain the atypical fibrosis seen in patients with ALGS.

• *

*To clarify that ALGS is a genetic liver disease affecting bile duct formation, we: *

1. Modified and extended the following text in the Introduction (Page 2, lines 14-17): “ALGS is mainly caused by mutations in the Notch ligand JAGGED1 (JAG1, 94%) (Mašek & Andersson, 2017; Oda et al, 1997), affecting bile duct development and morphogenesis, resulting in bile duct paucity and cholestasis. Immune dysregulation has also been described (Tilib Shamoun et al, 2015), but how this might interact with liver disease in ALGS to affect fibrosis is not known.”
2. *Introduce the disease, the animal model, and the scientific question in a schematic in new Fig 1A. *
3. * Reviewer 1 comment: Minor points that should be addressed include: • The source cells used in the transfer experiments reported in Figure 5 is unclear. Are they using total spleen cells with T, B and myeloid cells or are they using purified T cells. And if it is the latter, have they assessed the ratio of CD4+ versus FoxP3/CD4+ cells in the transferred cells?

Author Response: *Total spleen cells including all lymphocytes were transplanted, as described in Materials and Methods. The constituent T-cell populations are characterized and shown in Fig 4F. To clarify this, we: *

1. *added the text “Adoptive transfer of lymphocytes” to the schematic in Fig 5A, FigS5A, and Fig 6A, and *
2. modified the opening paragraph related to results presented in Fig.5 and FigS5 in the following way (page 8, line 209): “To investigate Jag1Ndr/Ndr T cell function, we performed adoptive transfer of the splenic lymphocytes into Rag1-/- mice, which lack mature B- and T cell populations, but provide a host environment with normal Jag1 (Mombaerts et al, 1992).”
3. *

*To acknowledge that B-cells and innate lymphoid cells might contribute to the observed results, we include a following sentence in the Discussion: *

(page 12, lines 369-371) “Finally, our experimental setup does not exclude an additional contribution of other lymphocytes (B-cells or innate lymphoid cells) to the BDL-induced fibrosis, and selective testing of the individual subpopulations would be an intriguing follow up to this study.”

Reviewer 1 comment: In the DSS experiments in Figure 5, there does not appear to be a no DSS control. What does the architecture look like without DSS?

Author Response: The intestinal architecture and phenotype of mice transplanted with Jag1+/+ or Jag1Ndr/Ndr lymphocytes, not treated with DSS, are presented in Supplementary Figure 5. In the absence of DSS, Jag1+/+- or Jag1Ndr/Ndr -transplanted mice exhibit no overt differences in survival or weight gain/loss. The intestinal inflammatory score was not different in the two conditions and was *2.29 +/-0.44 and 2.03 +/-0.92 for Jag1+/+- or Jag1Ndr/Ndr -transplanted mice, respectively. *

To compare the results with and without DSS, we added the following text to the results section, when describing the DSS results (Page 9, lines 223-226):

“As expected, histological scoring of intestinal and colonic inflammation revealed elevated inflammation in Jag1+/+→Rag1-/- mice treated with DSS (Fig. 5C,D) compared to Jag1+/+→Rag1-/- mice not treated with DSS (Fig. S5). However, there was significantly less inflammation in Jag1Ndr/Ndr→Rag1-/- mice than in Jag1+/+→Rag1-/- mice (Fig. 5C,D)."

Reviewer 1 comment: The authors noted that splenomegaly was observed in the Jag1ndr/ndr mouse model. Again this is antithetical to what one would expect when one sees an increase in FoxP3/CD4+ T regs.

Author Response: *We thank the reviewer for pointing at a possible discrepancy, related to Fig1 in which we report the presence of splenomegaly. Although there can be multiple causes of splenomegaly, it is one of the hallmarks of portal hypertension (as also corroborated by Reviewer 2), tightly connected with liver fibrosis, present in patients with ALGS and we report it as such in the manuscript. To clarify this, we added the following text sections: *

1. Results (page 2, lines 37,38) “Liver fibrosis compresses blood vessels and reduces their blood flow, leading to portal hypertension, a serious consequence of liver disease which can manifest as splenomegaly.”
2. Discussion (page 13, line 394-401): “Splenomegaly has been described as a consequence of portal hypertension in ALGS (Kamath et al, 2020), but could also be attributed to immune-related pathology. Jag1Ndr/Ndr mice exhibit splenomegaly as early as P10, and is exacerbated at P30 ( 1E,F). Patients with other liver diseases display portal hypertension and cirrhosis, with both splenomegaly and hypersplenism associated with a high CD4+/CD8+ ratio, but a low Treg+/CD4+ ratio (Nomura et al, 2014). However, Jag1Ndr/Ndr mice present with splenomegaly but not hypersplenism. An overactive spleen (hypersplenism) would remove red blood cells which are instead enriched in Jag1Ndr/Ndr mice, and Tregs were enriched in Jag1Ndr/Ndr mice, not depleted as seen in cirrhosis/hypersplenism. These data are thus consistent with portal hypertension-induced splenomegaly rather than hypersplenism.*” *

Reviewer #1 (Significance (Required)):

Reviewer 1 comment: The strengths of this paper are the careful comparisons between the mouse model and the human ALG syndrome. These comparisons are valuable and worth publication.

Author Response: We thank the reviewer for these comments.

Reviewer 1 comment: Weaknesses are stated above. Needs a clearer explanation for their immune analysis.

Author Response: *We thank the reviewers for highlighting points requiring clarification and hope the proposed text changes and additional data presented in response to the comments of all three reviewers lead to a significant clarification of the immunological aspect of our study. *

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Reviewer 2 comment:

Summary: Masek and colleagues use multi-pronged studies on the Jag1[Ndr/Ndr] mouse model of Alagille syndrome (ALGS) combined with transcriptomic analysis on livers from patients with ALGS to elucidate the potential mechanisms regulating liver fibrosis in this disease. The authors first show that Jag1[Ndr/Ndr] animals develop pericellular and perisinusoidal fibrosis and exhibit evidence for portal hypertension, similar to patients with ALGS. Single-cell RNA-sequencing indicated more hepatoblasts and less hepatocytes, relatively speaking, in Jag1[Ndr/Ndr] P3 livers, which suggested hampering of hepatoblast differentiation to hepatocytes. Deconvolution of previously generated bulk RNA-seq data from Jag1[Ndr/Ndr] P10 livers and GESA on RNAseq data from livers of these mice and patients with ALGS confirmed the P3 scRNA-seq observations and indicated mild pro-inflammatory activation of immature hepatocytes in ALGS livers. GESA also suggested an inability of Jag1[Ndr/Ndr] livers to attract T cells upon cholestatic injury. Indeed, 25-color flow cytometry on liver and spleen from mutant and control mice indicated a defect in T cell response to cholestasis in this model. The authors then examined the effects of the Ndr mutation on T-cell development and function. They found that the Ndr/Ndr thymi were significantly smaller than control thymi. Moreover, Ndr/Ndr thymi showed an increase in CD4+ T-cells and Tregs at the expense of double-positive T-cells. The authors then performed lymphocyte transplantation studies and concluded that Ndr/Ndr T-cells fail to mount an adequate response to inflammation in a DSS model of ulcerative colitis. The authors tested the contribution of Ndr/Ndr immune cells to liver fibrosis in a model of experimentally induced cholestasis (bile duct ligation; BDL). Ndr/Ndr T-cells did not show any defects in migrating into the liver upon BDL. However, the periportal fibrosis observed in BDL model was reduced in animals receiving Ndr/Ndr immune cells compared to those receiving Jag1+/+ immune cells. This was accompanied by significantly less aSMA staining in these livers. Finally, reanalysis of bulk RNAseq data from liver samples from ALGS and other liver diseases suggested that the presence of FOXP3+ T-reg cells in the liver is associated with higher liver fibrosis in non-ALGS liver diseases but lower liver fibrosis in ALGS livers. The authors have used an impressive combination of single-cell RNA-sequencing, reanalysis of previous bulk RNA-sequencing data from their group and others, 25-color FACS analysis, and adoptive immune transfer experiments in this manuscript, and systematically provide quantification and statistical analysis for their data. Overall, this is an interesting and important study. Prior studies are referenced appropriately. The text and figures are clear and accurate. I don't think any additional experiments are essential. However, the issues listed under Major comments should be discussed and clarified in the manuscript, especially the first item.

Author Response: *We sincerely thank the reviewer for the comprehensive and insightful assessment of our manuscript. We are particularly gratified to note your acknowledgment of the thoroughness of our experimental approach and the clarity of our presentation. We are pleased that no further experiments would be required, and will address the points raised under Major comments which enhance our study's quality and accessibility. *

Reviewer 2 comment:

Major comments:

• Only a small fraction of the cells in scRNA-seq experiments have been assigned to hepatocytes/hepatoblast clusters, with the majority of these cells allocated to Hepato-Ery cluster. This suggests that many hepatocytes and potentially hepatoblasts have been lost during sample preparation. The authors should discuss this issue and its potential implications on the interpretation of the cell ratios and gene expression conclusions of scRNA-seq data. Author Response: We agree with the reviewer regarding this aspect of our study. We mentioned this limitation in the supplementary methods section: ”Liver parenchymal cells constituted ~6.5% of cells at E16.5, and ~7.5% of cells at P3 and included mesenchymal cells, endothelial cells, hepatoblasts and hepatocytes (Fig. S1D), this parenchymal proportion is lower than in vivo, but consistent with ex vivo liver digest (Guilliams et al, 2022).” We recognize it may be too inaccessible there, and we thus added the following text to the Discussion section of the manuscript: (Pages 11-12, lines 330-337) “A limitation of this study is the underrepresentation of the hepatoblast/cyte parenchymal cells in the scRNA-seq dataset (Fig. 2A-D), which constituted ~6.5% of analyzed cells at E16.5, and ~7.5% of cells at P3 (Fig. S1D). This parenchymal proportion is lower than in vivo, but is consistent with scRNA seq datasets obtained with ex vivo liver digest (Guilliams et al, 2022). One risk is that cell stress as a result of dissociation could result in further loss of injured Jag1Ndr/Ndr hepatocytes, impacting the interpretation of cell type abundance. Nuclear scRNAseq can overcome cell type-dependent dissociation sensitivity bias (Guilliams et al, 2022), and could provide further insights into Jag1Ndr/Ndr livers at the single cell level. Nonetheless, both bulk RNA seq deconvolution and histological analyses confirmed that patients and Jag1Ndr/Ndr mice exhibit hepatoblast enrichment and less differentiated hepatocytes.”

Reviewer 2 comment: The Jag1[Ndr/Ndr] strain is an excellent model for various aspects of ALGS phenotypes. However, when it comes to linking the effects of this mutation to the function of a specific cell type, it is worth considering that Jag1[Ndr/Ndr] might not recapitulate the effects of loss of one copy of JAG1 observed in most patients with ALGS. This is especially important given the sensitivity of various cellular and organ-level processes to the degree of Notch pathway activation. In the context of the present manuscript, it is possible that what the authors have observed in Jag1[Ndr/Ndr] lymphocytes does not mirror how a JAG1-heterozygous human lymphocyte behaves. This is not a major concern, but it is worth considering.

Author Response: We agree and thus added the following discussion paragraph (page 11, lines 315-321) “In patients with ALGS, who have a single mutation in either JAG1 or NOTCH2, the remnant healthy allele(s) could be expected to mediate signaling. However, some JAG1 mutations exhibit dominant negative effects (Ponio et al, 2007; Xiao et al, 2013; Guan et al, 2023), which could entail further repression of JAG1/NOTCH2 signaling. In this context, it is important to note that the Jag1Ndr/Ndr mice are homozygous for the missense mutation, but retain some JAG1 activity, and it is not clear to which degree this mimics JAG1 heterozygosity in humans. It would be of interest to test whether Jag1 potency affects hepatoblast differentiation or injury-induced reversion of hepatocytes in patients as a function of their genotype.”

Reviewer 2 comment: •The basis for the opposite type of correlation between COL1A1 expression and POXP3 level in ALGS versus non-ALGS liver disease is not clear.

Author Response: We thank the reviewer for pointing out the unclear interpretation of the patient data. In patients with ALGS, the extent of fibrosis is likely to be highly multifactorial, involving (as we show) hepatocyte immaturity, dampened inflammation, and immune system dysregulation (possibly involving more than T-cells). Since human patients ARE so heterogeneous, teasing apart the relative contribution of each is currently outside the scope of our study, but will be an important area of future research. Nonetheless we thought it was important and interesting to show these patterns in supplementary Fig 6, now extended with further data, and analyses, and described in the following manner:

• *

Results section: (page 10, lines 267-275) “Liver damage in non-ALGS liver disease (using liver injury marker LGALS3BP) (Yang et al, 2021), was positively correlated with recruitment of lymphocytes (including CD8A+,and FOXP3+ populations of T cells), as well as the extent of fibrosis (COL1A1 abundance) (Fig. S6G). However, in ALGS, the extent of liver damage, lymphocyte recruitment and fibrosis were unlinked (Fig. S6G). These data are in line with the observation that liver stiffness (a proxy for fibrosis) in ALGS is independent of biomarkers of liver disease (Leung et al, 2023). While Treg infiltration in ALGS was independent of liver damage, it exhibited a tendency towards a negative correlation with fibrosis (Fig. S6G), corroborating that elevated levels of Tregs may limit fibrosis in ALGS. Altogether, these data suggest that the liver and lymphocytes may be differentially affected in different patients with ALGS, a disorder that is well known for its heterogenous presentation.”

Minor comments:

• Page 2, last paragraph of Introduction, Page 12 last sentence, and Supplementary Methods: Please use "adoptive immune transfer" instead of "adaptive immune transfer". • Pages 3 and 4: Reference is made to Figures 3E-O, which appears to be Figure 2E-O. • Figure 3 legend: "Analysis in (E) is one-way ANOVA with Dunnett's multiple comparison test". Panel E compares two means, so ANOVA is not the appropriate statistical analysis for these data. Is this sentence related to panel D? • Page 9: Please correct misspelling: "response to intestinal insult (Fig. 5). W therefore". • The Science Translation Medicine references lack page number. Author Response: *We thank the reviewer deeply for taking the time to meticulously note and convey these errors, helping us to correct these. The suggested corrections have been implemented. Science Transl Med is an online journal and does not have page numbers – we have added an issue number to facilitate retrieval of these references. *

• *

Additionally, we noticed that the image of a consecutive liver section with CYP1A2 staining from Jag1Ndr/Ndr liver in Fig 2 L was accidentally flipped along the horizontal axis, which we have now corrected. We also changed the scRNAseq cell cluster naming from Hepatoblasts/cytes, Hepato_Ery, and Kupffer cells, Kuffer cells_Ery to Hepatoblasts/cytes I, and II, and Kupffer cells I and II, respectively, to match the Neutrophil progenitors I and II naming convention. Names were subsequently also changed in Fig S1 and methods.

**Referees cross-commenting**

To my knowledge, ALGS is not considered to be an inflammatory disorder. Furthermore, the splenomagaly observed in the mouse model could be due to portal hypertension rather than a primary immune disturbance. Having said that, I agree with the other reviewers that the manuscript will benefit from further discussion and clarification on the immune-related observations.

Author Response: We thank Reviewer 2 for indicating to Reviewer 1 that ALGS is not considered an inflammatory disorder, which we agree with. It was not our intention to convey this idea. To avoid confusion, we now:

1. *Added a schematic in Fig 1A. *
2. Modified and extended the following text in the Introduction: (Page 2, lines 14-17): “ALGS is mainly caused by mutations in the Notch ligand JAGGED1 (JAG1, 94%) (Mašek & Andersson, 2017; Oda et al, 1997), affecting bile duct development and morphogenesis, resulting in bile duct paucity and cholestasis. Immune dysregulation has also been described (Tilib Shamoun et al, 2015), but how this might interact with liver disease in ALGS to affect fibrosis is not known.” *Furthermore, we have addressed or will address all comments from reviewer 1 to clarify the immune-related observations. *

Reviewer #2 (Significance (Required)):

Despite severe cholestasis, ALGS patients do not show as much fibrosis as other cholestatic diseases, including biliary atresia (BA). A previous study had suggested that this phenomenon could be due to the difference in the nature of reactive hepatobiliary cells in ALGS compared to BA (Fabris et al, 2007). Moreover, a number of studies have suggested a role for Notch pathway activation in several cell types in the liver in the development of liver fibrosis (for example, Sawitza et al, Hepatology, 2009; Chen et al, Plos One, 2012; Duan et al, Hepatology, 2018; Yu et al, Science Translational Medicine, 2021). However, although a role for Notch signaling in T-cells is well established, it was not known whether impaired T-cell development/function contributes to reduced fibrosis in ALGS liver disease. Accordingly, the current manuscript provides novel insight into the mechanism of fibrosis in this disease. Moreover, the observation that Jag1-mutant T-cells do not confer as much protection as control T-cells to immunodeficient mice subjected to DSS-induced ulcerative colitis provides strong evidence for impaired T-cell immunity in this ALGS model and might help explain other aspects of ALGS phenotypes.

The manuscript will be of interest to broad audience (Notch signaling, cholestatic liver disease, mechanisms of liver fibrosis, T-cell development).

I have expertise in Notch signaling and in using animal models of human developmental disorders.

__Author Response: __We thank the reviewer for the balanced assessment of our manuscript in light of the current knowledge, and for highlighting its importance in the context of not only Notch and ALGS, but also other cholestatic and fibrotic liver diseases.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The article entitled "Jag1 Insufficiency Disrupts Neonatal T Cell Differentiation and Impairs Hepatocyte Maturation, Leading to Altered Liver Fibrosis" by Mašek et al described the role of Notch ligand JAGGED1 (JAG1) in the T-cell differentiation contributing to liver fibrosis and immune system development in ALGS. This article is well written and has important preliminary findings that could establish Jag1 and its downstream signaling pathways as potential therapeutic targets to attenuate liver fibrosis.

Author Response: We thank the reviewer for recognizing our work and pointing out the therapeutical implications of our findings.

Reviewer 3 comment 1: Minor comments: In page 4, they mentioned that "the hepatoblast marker alpha fetoprotein (AFP) was 3.1-fold enriched (Fig. 3J,K), while the mature hepatocyte marker CYP1A2 protein was 1.7-fold less expressed (Fig. 3L-M)", the figure numbers should be changed to 2J, K, L-M etc.

Author Response:* We thank the reviewer for identifying these errors. The suggested corrections have been implemented. *

Reviewer 3 comment 2: In liver fibrosis the Th17 cells play crucial roles. Please show the level of IL17A mRNA level in the liver in the Jag1Ndr/Ndr mice compared to the Jag1+/+ mice.

Author Response: We thank the reviewer for the insightful comments. We indeed investigated the Th17 vs Treg immune response, however we detect neither Th17-expressed Il17, Il17a, Il17f, nor Il21 and Il22 mRNA in the bulk RNA data, suggesting their expression is either masked or they are not present in significant numbers within the liver tissue at P10, preventing us from drawing any conclusions about this cell population.

Reviewer ____3 comment 3: Also, please show the expression level of pro-inflammatory molecules, for example, TNFα, IL1β, MCP1 etc and the level of MMPs (especially MMP2, MMP8, MMP9) in the livers of the mice models used.

Author Response: *The expression of Il10, Il1b, Mcp1(Ccl2), was presented in the manuscript Fig. 2O, and we attach in the response to reviewers *

*a full list together with the expression levels of Mmp2/8/9, Tnfa, Ifng, Il17 receptor family and Tgfb1-3. Out of these, Mmp8 (0.9 Log2fold change = 1.9-fold), Ccl2 (2.2 Log2fold change = 4.7-fold), and Tl17rb (1.1 Log2fold change = 2.1-fold) were significantly upregulated, but do not indicate any specific leukocyte population’s response. This is in line with data in Fig S2E, demonstrating a dominance of myeloid over adaptive immune response in the GSEA of the immune KEGGs. *

*Since lymphocytes are underrepresented in the bulk transcriptomics, and individual genes might report activity of many different cell types, we chose to focus on the list of genes shown to be markers of activated hepatocytes, to avoid over interpretation of the RNA sequencing data. Instead, the immune analyses were based on flow cytometry data, which we expect should accurately report cell type abundance across organ systems. *

Reviewer 3 comment____ 4. Authors have shown significant alterations in the Treg population in their Jag1Ndr/Ndr mice of ALGS. Please also show the expression of IL10 and TGFβ in the liver and whether they are correlated with the level of Treg populations.

Author response:* IL10 and Tgfb mRNA levels in liver are shown in the heatmap in the response to reviewers, and were not significantly different between genotypes at P10. They were also not correlated with Foxp3 levels, as shown in the correlation matrices below (Pearson’s R values in top row, significance values in bottom row). *

Reviewer 3 comment 5. It would be interesting to know whether the IFNγ mRNA expression in the livers were altered in the Jag1Ndr/Ndr mice with altered populations of CD8 T cells.

Author Response: There was no significant difference in IFNγ mRNA expression levels between Jag1+/+ and Jag1Ndr/Ndr *livers at P10 (please see the heatmap in response to comment no.3, above). *

Reviewer #3 (Significance (Required)): Strength: This article is well written and has important preliminary findings that could establish Jag1 and its downstream signaling pathways as potential therapeutic targets to attenuate liver fibrosis.

Author Response: Thank you for these comments and pointing out the wider implications of our findings.

Reviewer 3____ Limitations: This study lacked the detailed molecular pathways which could explain how the Jag1 altered the T-cell recruitment, development and hepatocyte maturation in the development of liver fibrosis in the ALGS model.

Author Response: We agree that this study does not focus on molecular pathways. The intention of this study was to identify which cell populations contribute to atypical neonatal fibrosis in ALGS. Because we expected this process to be multifactorial, Jag1Ndr/Ndr mice, carrying a systemic mutation, present both advantages (Jag1 abrogation in all cells --> ALGS-like organ interactions) and limitations (inability to identify contributions of individual cell types). However, by identifying maturing hepatocytes and Tregs as dysregulated, and demonstrating that Jag1Ndr/Ndr lymphocytes behave abnormally and suppress inflammation and fibrosis in Rag1-/- mice (with normal Jag1 expression), we establish a biological framework that can now be further investigated with conditional genetic tools and in vitro systems, to elucidate specific molecular pathways, that were beyond the scope of the current study.

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Referee #2

Evidence, reproducibility and clarity

Summary:

Masek and colleagues use multi-pronged studies on the Jag1[Ndr/Ndr] mouse model of Alagille syndrome (ALGS) combined with transcriptomic analysis on livers from patients with ALGS to elucidate the potential mechanisms regulating liver fibrosis in this disease. The authors first show that Jag1[Ndr/Ndr] animals develop pericellular and perisinusoidal fibrosis and exhibit evidence for portal hypertension, similar to patients with ALGS. Single-cell RNA-sequencing indicated more hepatoblasts and less hepatocytes, relatively speaking, in Jag1[Ndr/Ndr] P3 livers, which suggested hampering of hepatoblast differentiation to hepatocytes. Deconvolution of previously generated bulk RNA-seq data from Jag1[Ndr/Ndr] P10 livers and GESA on RNAseq data from livers of these mice and patients with ALGS confirmed the P3 scRNA-seq observations and indicated mild pro-inflammatory activation of immature hepatocytes in ALGS livers. GESA also suggested an inability of Jag1[Ndr/Ndr] livers to attract T cells upon cholestatic injury. Indeed, 25-color flow cytometry on liver and spleen from mutant and control mice indicated a defect in T cell response to cholestasis in this model. The authors then examined the effects of the Ndr mutation on T-cell development and function. They found that the Ndr/Ndr thymi were significantly smaller than control thymi. Moreover, Ndr/Ndr thymi showed an increase in CD4+ T-cells and Tregs at the expense of double-positive T-cells. The authors then performed lymphocyte transplantation studies and concluded that Ndr/Ndr T-cells fail to mount an adequate response to inflammation in a DSS model of ulcerative colitis. The authors tested the contribution of Ndr/Ndr immune cells to liver fibrosis in a model of experimentally induced cholestasis (bile duct ligation; BDL). Ndr/Ndr T-cells did not show any defects in migrating into the liver upon BDL. However, the periportal fibrosis observed in BDL model was reduced in animals receiving Ndr/Ndr immune cells compared to those receiving Jag1+/+ immune cells. This was accompanied by significantly less aSMA staining in these livers. Finally, reanalysis of bulk RNAseq data from liver samples from ALGS and other liver diseases suggested that the presence of FOXP3+ T-reg cells in the liver is associated with higher liver fibrosis in non-ALGS liver diseases but lower liver fibrosis in ALGS livers. The authors have used an impressive combination of single-cell RNA-sequencing, reanalysis of previous bulk RNA-sequencing data from their group and others, 25-color FACS analysis, and adoptive immune transfer experiments in this manuscript, and systematically provide quantification and statistical analysis for their data. Overall, this is an interesting and important study. Prior studies are referenced appropriately. The text and figures are clear and accurate. I don't think any additional experiments are essential. However, the issues listed under Major comments should be discussed and clarified in the manuscript, especially the first item.

Major comments:

• Only a small fraction of the cells in scRNA-seq experiments have been assigned to hepatocytes/hepatoblast clusters, with the majority of these cells allocated to Hepato-Ery cluster. This suggests that many hepatocytes and potentially hepatoblasts have been lost during sample preparation. The authors should discuss this issue and its potential implications on the interpretation of the cell ratios and gene expression conclusions of scRNA-seq data.
• The Jag1[Ndr/Ndr] strain is an excellent model for various aspects of ALGS phenotypes. However, when it comes to linking the effects of this mutation to the function of a specific cell type, it is worth considering that Jag1[Ndr/Ndr] might not recapitulate the effects of loss of one copy of JAG1 observed in most patients with ALGS. This is especially important given the sensitivity of various cellular and organ-level processes to the degree of Notch pathway activation. In the context of the present manuscript, it is possible that what the authors have observed in Jag1[Ndr/Ndr] lymphocytes does not mirror how a JAG1-heterozygous human lymphocyte behaves. This is not a major concern, but it is worth considering.
• The basis for the opposite type of correlation between COL1A1 expression and POXP3 level in ALGS versus non-ALGS liver disease is not clear.

Minor comments:

• Page 2, last paragraph of Introduction, Page 12 last sentence, and Supplementary Methods: Please use "adoptive immune transfer" instead of "adaptive immune transfer".
• Pages 3 and 4: Reference is made to Figures 3E-O, which appears to be Figure 2E-O.
• Figure 3 legend: "Analysis in (E) is one-way ANOVA with Dunnett's multiple comparison test". Panel E compares two means, so ANOVA is not the appropriate statistical analysis for these data. Is this sentence related to panel D?
• Page 9: Please correct misspelling: "response to intestinal insult (Fig. 5). W therefore".
• The Science Translation Medicine references lack page number.

Referees cross-commenting

To my knowledge, ALGS is not considered to be an inflammatory disorder. Furthermore, the splenomagaly observed in the mouse model could be due to portal hypertension rather than a primary immune disturbance. Having said that, I agree with the other reviewers that the manuscript will benefit from further discussion and clarification on the immune-related observations.

Significance

Despite severe cholestasis, ALGS patients do not show as much fibrosis as other cholestatic diseases, including biliary atresia (BA). A previous study had suggested that this phenomenon could be due to the difference in the nature of reactive hepatobiliary cells in ALGS compared to BA (Fabris et al, 2007). Moreover, a number of studies have suggested a role for Notch pathway activation in several cell types in the liver in the development of liver fibrosis (for example, Sawitza et al, Hepatology, 2009; Chen et al, Plos One, 2012; Duan et al, Hepatology, 2018; Yu et al, Science Translational Medicine, 2021). However, although a role for Notch signaling in T-cells is well established, it was not known whether impaired T-cell development/function contributes to reduced fibrosis in ALGS liver disease. Accordingly, the current manuscript provides novel insight into the mechanism of fibrosis in this disease. Moreover, the observation that Jag1-mutant T-cells do not confer as much protection as control T-cells to immunodeficient mice subjected to DSS-induced ulcerative colitis provides strong evidence for impaired T-cell immunity in this ALGS model and might help explain other aspects of ALGS phenotypes.

The manuscript will be of interest to broad audience (Notch signaling, cholestatic liver disease, mechanisms of liver fibrosis, T-cell development).

I have expertise in Notch signaling and in using animal models of human developmental disorders.

o

Cientistas, tão elucidados!!! Como pode a gente se submeter a essas coisas?

Eu não me considero superior..... (kkkkkkkk)

One formate hash and for peer and for key

Author response

Reviewer #1 (Public Review):

Summary:

The authors aimed to modify the characteristics of the extracellular matrix (ECM) produced by immortalized mesenchymal stem cells (MSCs) by employing the CRISPR/Cas9 system to knock out specific genes. Initially, they established VEGF-KO cell lines, demonstrating that these cells retained chondrogenic and angiogenic properties. Additionally, lyophilized carriage tissues produced by these cells exhibited retained osteogenic properties.

Subsequently, the authors established RUNX2-KO cell lines, which exhibited reduced COLX expression during chondrogenic differentiation and notably diminished osteogenic properties in vitro. Transplantation of lyophilized carriage tissues produced by RUNX2-KO cell lines into osteochondral defects in rat knee joints resulted in the regeneration of articular cartilage tissues as well as bone tissues, a phenomenon not observed with tissues derived from parental cells. This suggests that gene-edited MSCs represent a valuable cell source for producing ECM with enhanced quality.

Strengths:

The enhanced cartilage regeneration observed with ECM derived from RUNX2-KO cells supports the authors' strategy of creating gene-edited MSCs capable of producing ECM with superior quality. Immortalized cell lines offer a limitless source of off-the-shelf material for tissue regeneration.

We thank the reviewer for the interest in our work. We however want to clarify that the present manuscript does not report the generation of ECM with “superior quality”, but rather of modulated composition and thus function.

Weaknesses:

Most data align with anticipated outcomes, offering limited novelty to advance scientific understanding. Methodologically, the chondrogenic differentiation properties of immortalized MSCs appeared deficient, evidenced by Safranin-O staining of 3D tissues and histological findings lacking robust evidence for endochondral differentiation. This presents a critical limitation, particularly as authors propose the implantation of cartilage tissues for in vivo experiments. Instead, the bulk of data stemmed from type I collagen scaffold with factors produced by MSCs stimulated by TGFβ.

The chondrogenic differentiation of our MSOD-B line and their capacity of undergoing endochondral ossification has been robustly demonstrated in previous studies (Pigeot et al., Advanced Materials 2021 and Grigoryan et al., Science Translational Medicine 2022). In the present manuscript, we thus compare the chondrogenic capacity of newly established VEGF-KO and RUNX-KO lines to those of MSOD-B cells. We demonstrate by qualitative (Safranin-O staining, Collagen type 2 and Collagen type X immuno-stainings) and quantitative (glycosaminoglycans assay) assays that the generated tissues consist in cartilage grafts of similar quality than the MSOD-B counterpart. Of note, the safranin-O stainings were performed on lyophilized tissues, which can alter the staining quality/intensity. We will thus provide additional stainings of generated tissues pre-lyophilization.

The rationale behind establishing VEGF-KO cell lines remains unclear. What specific outcomes did the authors anticipate from this modification?

VEGF is a known master regulator of angiogenesis and a key mediator of endochondral ossification. It has also been extensively used in bone tissue engineering studies as a supplemented factor – primarily in the form of VEGFα – to increase the vascularization and thus outcome of bone formation of engineered grafts (https://www.nature.com/articles/s42003-020-01606-9, https://www.sciencedirect.com/science/article/pii/S8756328216301752). In our study, it was thus identified as a natural candidate to demonstrate the possibility to generate VEGF-KO cartilage and subsequently assess the functional impact on both the angiogenic and osteogenic potential of resulting cartilage tissue.

Insufficient depth was given to elucidate the disparity in osteogenic properties between those observed in ectopic bone formation and those observed in transplantation into osteochondral defects. While the regeneration of articular cartilage in RUNX2-KO ECM presents intriguing results, the study lacked an exploration into underlying mechanisms, such as histological analyses at earlier time points.

Using RUNX2-KO ECM, we aimed at demonstrating the impact on cartilage remodeling and bone formation. This was performed ectopically but also in the rat osteochondral defect as a regenerative set-up of higher clinical relevance. We agree with the reviewer that additional experimental groups and time-points (not only earlier but also longer ones) would offer a better mechanistic understanding of the ECM contribution to the joint repair. However, as stated in our manuscript this is a proof-of-concept study that successfully demonstrated the influence of the cartilage ECM modification on the in vivo skeletal regeneration. A follow-up study would need to be performed to complement existing evidence and strengthen the relevance of our approach for cartilage repair.

Reviewer #2 (Public Review):

The manuscript submitted by Sujeethkumar et al. describes an alternative approach to skeletal tissue repair using extracellular matrix (ECM) deposited by genetically modified mesenchymal stromal/stem cells. Here, they generate a loss of function mutations in VEGF or RUNX2 in a BMP2-overexpressing MSC line and define the differences in the resulting tissue-engineered constructs following seeding onto a type I collagen matrix in vitro, and following lyophilization and subcutaneous and orthotopic implantation into mice and rats. Some strengths of this manuscript are the establishment of a platform by which modifications in cell-derived ECM can be evaluated both in vitro and in vivo, the demonstration that genetic modification of cells results in complexity of in vitro cell-derived ECM that elicits quantifiable results, and the admirable goal to improve endogenous cartilage repair. However, I recommend the authors clarify their conclusions and add more information regarding reproducibility, which was one limitation of primary-cell-derived ECMs.

We thank the reviewer for the positive evaluation of our work.

Overcoming the limitations of native/autologous/allogeneic ECMs such as complete decellularization and reduction of batch-to-batch variability was not specifically addressed in the data provided herein. For the maintenance of ECM organization and complexity following lyophilization, evidence of complete decellularization was not addressed, but could be easily evaluated using polarized light microscopy and quantification of human DNA for example in constructs pre and post-lyophilization.

We will clarify the experiments and characterization performed with lyophilized tissues versus those performed with decellularized ones. We will also provide evidence of DNA removal in our decellularized ECMs.

It would be ideal to see minimization of batch-to-batch variability using this approach, as mitigation of using a sole cell line is likely not sufficient (considering that the sole cell line-derived Matrigel does exhibit batch-to-batch and manufacturer-to-manufacturer variability). I recommend adding details regarding experimental design and outcomes not initially considered. Inter- and intra-experimental reproducibility was not adequately addressed. The size of in vitro-derived cartilage pellets was not quantified, and it is not clear that more than one independent 'differentiation' was performed from each gene-edited MSC line to generate in vitro replicates and constructs that were implanted in vivo.

We thank the Reviewer for the comment on variability/reproducibility concern. Using a cell line does confer higher robustness but indeed does not grant unlimited consistency of batch production. We will temper our claims in the discussion and mention the need to regularly re-characterize cell lines properties upon passages.

In our study, our grafts have been generated from various batches and tested in more than one experimental repeat. This will be further described in the revised version of our manuscript. We will also implement data on the size variability of generated tissues.

The use of descriptive language in describing conclusions may mislead the reader and should be modified accordingly throughout the manuscript. For example, although this reviewer agrees with the comparative statements made by the authors regarding parental and gene-edited MSC lines, non-quantifiable terms such as 'frank' 'superior' (example, line 242) are inappropriate and should rather be discussed in terms of significance. Another example is 'rich-collagenous matrix,' which was not substantiated by uniform immunostaining for type II collagen (line 189).

I have similar recommendations regarding conclusive statements from the rat implantation model, which was appropriately used for the purpose of evaluating the response of native skeletal cells to the different cell-derived ECMs. Interpretations of these results should be described with more accuracy. For example, increased TRAP staining does not indicate reduced active bone formation (line 237). Many would not conclude that GAGs were retained in the RUNX2-KO line graft subchondral region based on the histology. Quantification of % chondral regeneration using histology is not accurate as it is greatly influenced by the location in the defect from which the section was taken. Chondral regeneration is usually semi-quantified from gross observations of the cartilage surface immediately following excision. The statements regarding integration (example line 290) are not founded by histological evidence, which should show high magnification of the periphery of the graft adjacent to the native tissue.

We thank the Reviewer for the constructive suggestions. We will revise language accordingly throughout the manuscript.

Reviewer #3 (Public Review):

Summary:

In this study, the authors have started off using an immortalized human cell line and then gene-edited it to decrease the levels of VEGF1 (in order to influence vascularization), and the levels of Runx2 (to decrease chondro/osteogenesis). They first transplanted these cells with a collagen scaffold. The modified cells showed a decrease in vascularization when VEGF1 was decreased, and suggested an increase in cartilage formation.

In another study, the matrix generated by these cells was subsequently remodeled into a bone marrow organ. When RUNX2 was decreased, the cells did not mineralize in vitro, and their matrices expressed types I and II collagen but not type X collagen in vitro, in comparison with unedited cells. In vivo, the author claims that remodeling of the matrices into bone was somewhat inhibited. Lastly, they utilized matrices generated by RUNX2 edited cells to regenerate chondro-osteal defects. They suggest that the edited cells regenerated cartilage in comparison with unedited cells.

Strengths:

-The notion that inducing changes in the ECM by genetically editing the cells is a novel one, as it has long been thought that ECM composition influences cell activity.

-If successful, it may be possible to make off-the-shelf ECMS to carry out different types of tissue repair.

We thank the Reviewer for the critical evaluation of our work and the highlighted novelty of it.

Weaknesses:

-The authors have not generated histologically identifiable cartilage or bone in their transplants of the cells with a type I scaffold.

The chondrogenic differentiation of our MSOD-B line and their capacity of undergoing endochondral ossification has been robustly demonstrated in previous studies (Pigeot et al., Advanced Materials 2021 and Grigoryan et al., Science Translational Medicine 2022). In the present manuscript, we thus compare the chondrogenic capacity of newly established VEGF-KO and RUNX-KO lines to those of MSOD-B. We demonstrate by qualitative (Safranin-O staining, Collagen type 2 and Collagen type X immuno-stainings) and quantitative (glycosaminoglycans assay) assays that the generated tissues consist in cartilage tissue of similar quality than the MSOD-B. However, the safranin-O stainings were performed on lyophilized tissues, which can alter the staining quality/intensity. We will thus provide additional stainings of generated tissues pre-lyophilization.

On the contested formation of bone in vivo by our ECMs grafts, we have provided compelling qualitative evidence via Masson´s Trichrome stainings and quantification of mineralized volume by µCT. Both cortical bone and trabecular structures were identified ectopically. Those are standard evaluation methods in the field, we would be happy to receive additional suggestions by the Reviewer.

-In many cases, they did not generate histologically identifiable cartilage with their cell-free-edited scaffold. They did generate small amounts of bone but this is most likely due to BMPs that were synthesized by the cells and trapped in the matrix.

We now appreciate that the Reviewer agrees on the successful formation of bone induced by our engineered grafts. We however still respectfully disagree with the “small amount of bone” statement since our MSOD-B and MSOD-B VEGF KO cartilage grafts led to the full generation of a mature ectopic bone organ (that is, also composed of extensive marrow). This has been assessed qualitatively and quantitatively.

We agree with the Reviewer on the key role of BMP-2 in the remodeling process into bone and bone marrow, which we have extensively described in our previous publication (Pigeot et al., Advanced Materials 2021). We previously demonstrated that the low amount of BMP-2 (in the dozens of nanogram/tissue range) embedded in the matrix is not sufficient per se to induce ectopic endochondral ossification. It is the combined presence of GAGs in the matrix -thus cartilage- that allows the success of bone formation. Since we have already demonstrated in the present manuscript that the GAGs content is the same in MSOD-B and MSOD-B edited ECMs, we will provide additional data demonstrating the maintenance of BMP-2 content in all generated cartilage tissues.

-There is a great deal of missing detail in the manuscript.

We will provide additional information on the MSOD-B line and the overall methodology in our revised version.

-The in vivo study is underpowered, the results are not well documented pictorially, and are not convincing.

We will provide additional information and pictures related to our in vivo studies. We believe our group size supports our conclusions confirmed by statistical assessment.

-Given the fact that they have genetically modified cells, they could have done analyses of ECM components to determine what was different between the lines, both at the transcriptome and the protein level. Consequently, the study is purely descriptive and does not provide any mechanistic understanding of what mixture of matrix components and growth factors works best for cartilage or bone. But this presupposes that they actually induced the formation of bona fide cartilage, at least.

We thank the Reviewer for the suggestion. However, our study did not aim at understanding what ECM graft composition work best for cartilage nor bone regeneration respectively. Instead, we propose the exploitation of our cellular tools to interrogate the function of key ECM constituents and their impact in skeletal regeneration. We once more confirm that we generated lyophilized cartilage grafts which will be more evidently supported by histological assessment before lyophilization.

DOI: 10.1016/j.isci.2024.109691

Resource: RZ6 Multi-I/O Processor (RRID:SCR_018153)

Curator: @scibot

SciCrunch record: RRID:SCR_018153

What is this?

DOI: 10.1083/jcb.202309015

Resource: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)

Curator: @scibot

SciCrunch record: RRID:CVCL_0063

What is this?

The British Lawnmower Museum is a museum dedicated to the history of the lawnmowers in Southport, Merseyside, northern England.

https://en.wikipedia.org/wiki/British_Lawnmower_Museum

The nation's foremost garden machinery collection, multi-award winning unique museum of over 250 restored machines.

https://www.tripadvisor.co.uk/Attraction_Review-g191255-d564490-Reviews-British_Lawnmower_Museum-Southport_Merseyside_England.html

• Cidadania é aquele que participa da vida política de uma Pólis
• O cidadão é aquele que sabe tanto mandar quanto obedecer, e por isso é virtuoso, é um bom cidadão.
• os contratos que definem o que é ser cidadão ou não, dependem de cada caso.

A Família é a associação estabelecida por natureza, para atender as necessidades do dia-a-dia, em busca da ética. Como o crescimento dessas famílias e o surgimento de novas, se formam as aldeias, e posteriormente, as polis

¿Qué significa realmente "vivir en plenitud" y expresiones similares?

DOI: 10.1016/j.celrep.2024.114202

Resource: (Santa Cruz Biotechnology Cat# sc-836, RRID:AB_632445)

Curator: @scibot

SciCrunch record: RRID:AB_632445

What is this?

DOI: 10.1016/j.immuni.2024.04.018

Resource: (Thermo Fisher Scientific Cat# 47-0451-80, RRID:AB_1548790)

Curator: @scibot

SciCrunch record: RRID:AB_1548790

What is this?

Camponeses usam a terra, produzindo para o suserano em troca de proteção

• Denúncia feita por Sally Davis, ex-chefe do serviço médico da Inglaterra, aponta para o fado de que o abuso no uso de medicamentos como antibióticos pode levar a contra efeitos perigosos, como o desenvolvimento de microrganismos resistentes a eles
• Mais de 2/3 dos antibióticos são usados em animais de criação, para evitar contágio em ambientes insalubres e superlotadas
• Esses medicamentos contaminam o meio ambiente, especialmente por meio da excreção
• Fábricas que produzem antibióticos muitas vezes não cuidam adequadamente de seus efluentes, o que contamina de modo dramático o meio ambiente
• Infecções resistentes a medicamentos matam mais de 1,2 milhão de pessoas por ano
• Entre as ações para enfrentar este cenário, está a proibição de uso de medicamentos relevantes na medicina humana na agropecuária

Prisco combines the 'they're here but we can't see it' and dark forest answers to [[Fermi Paradox 20201123150738]].

Retos significativos para los humanistas digitales. No solo el uso y la compresión de herramientas tecnológicas, sino la clasificación y compresión de lo que se quiere mostrar o expresar.

Com o feedback o professor, através de uma avaliação qualitativa e descritiva do percurso realizado, ajuda o aluno a identificar as lacunas existentes, os aspectos a melhorar, de modo a que este desenvolva o seu potencial e efetue aprendizagens. Em ensino que assenta em ambientes virtuais o feedback, permite que o aluno se sinta “acompanhado” e motivado durante o processo de aprendizagem ( Fluminhan, Arara & Fluminhan; 2013). Um feedback construtivo deve dar ao aluno uma informação clara acerca dos progressos feitos e dos aspetos a melhorar.

I found this and I'm trying it out today \o/ I had exactly four ripe bananas.

Looking forward to seeing the results -- thanks @houshuang!

ASD u kobiet trudniej wykryć

Czynniki prawdopodobnie wpływające na różnice między grupami w bramkowaniu sensorycznym

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Reply to the reviewers

*Reviewer #1 (Evidence, reproducibility and clarity (Required)):

*The study examined the mechanisms behind the nuclear transport of capsid proteins of various flaviviruses. The study used mass spectrometry to identify the interaction partners of JEV capsid protein and found Importin 7 as the top hit. After validating this interaction with IP-western blotting, using IPO7 knock-out cells they showed that the nuclear accumulation of capsid is dependent on IPO7. Moreover, they also observed nearly 10-folds reduction in titre of virus produced from knock out cells without reduction in virus replication or particle assembly.

The study needs improvements to bring it to publication standards. Some overaarching problems include, all capsid localization studies being done with GFP-tagged capsid, and not wild type capsid produced during authentic infection, lack of quantitation of most of the localization data and not showing capsid localization from infection experiments in knock out cells, and no in-depth analysis of the potential mechanisms behind the observed reduction in titre in knock out cells etc.

Thank you for your constructive comments. We have sincerely answered all of them, as shown below. We hope you are satisfied with our additional data and the revised manuscript.

The major comments are

Fig 1B: Please add quantitation and statistical analyses of the ratio of nuclear and cytoplasmic capsid protein of all different capsids used. Also include western blot to prove that there is no cleavage between Capsid and GFP and the green signal indeed comes from the fusion protein. Ideally you should use capsid alone instead of a fusion protein for at least selected few constructs to prove that the Capsid-GFP behaves identical to Capsid alone.

Following the reviewer’s comments, we have added quantification and statistical data in Figure 1D. We have added CBB data and western blot data in Figures 1B and S1. Because recombinant proteins of low molecular weights were artificially translocated into the nucleus through diffusion, less than 20 kDa proteins are typically used as GFP or GST fusion proteins for the IJ and PM experiments. Instead of IJ and PM experiments, we have added data on the translocation of the non-tagged core using IFA and its statistical data in Figure 1A. Although in vitro data on the translocation of capsid protein differ somewhat from IFA data, the data on nuclear translocation of core proteins are consistent across different experiments.

Fig 1C: It is unclear from the figure legends the WT JEV capsid means GFP-Capsid or Capsid alone. You should clearly state the GFP part if the construct includes GFP. Quantitation and statistics are missing and the information on how many independent experiments were performed is also not included in the figure legend.

Following the reviewer’s suggestion, we have described that the JEV proteins fused GFP as follows: “AcGFP-JEVCoreWT or AcGFP-JEVCoreGP/AA” (Line. 771). We added quantification and statistical analysis as shown in Figure 1E. IJ and PM experiments were performed three times independently and described in the legend of Figure 1 in the revised manuscript (Lines 773–774).

Fig 2B: Quantitation and statistics are missing. Ideally, the data need to be reproduced with Capsid alone instead of Capsid-GFP. A positive control is needed for the activity of Bimax to prove that the drug was working in the assay.

We have added quantitative and statistical data in the revised Figure 2B. As mentioned above, capsid alone is potentially translocated into the nucleus artificially using the IJ and PM assay. Bimax binds to importin alpha but not importin beta, specifically inhibiting the importin alpha/beta pathway. The RanGTP mutant binds to the importin beta family, including importin beta 1, and widely inhibits importin beta-dependent nuclear import. These inhibitors are well-characterized and recognized in the field. We cited the following reference: Tsujii et al., JBC, 2015.

Fig 2C: How do you reconcile the IP mass spectrometry data that Importin b1 is the second strongest hit with the lack of IP interaction you observed in fig 2C?

As shown in Figure 2C, importin b1 does not interact with the JEV core. Importin b1 is the most abundant member of the importin beta family. Thus, it might be a non-specific interaction between importin b1 and the JEV core. Therefore, we excluded importin b1 from further analyses. We added a sentence to explain why importin b1 was excluded on Line 145.

Fig 3C: How many independent confirmations of this experiment was performed?

All IJ and PM experiments were performed thrice independently. We described this in the legend of Figure 3 in the revised manuscript (Line; 794).

Fig 4A and B: Add quantitation for the western blot. 4A-D Include data on the number of biological repetitions. 4C-D: Add quantitation and statistical analyses of the ratio of nuclear and cytoplasmic capsid protein.

We have added quantification data, as shown in Figures 4A and 4B. All experimental results shown in Figures 4A, 4B, 4C, and 4D were performed thrice independently, as described in the legend of Figure 4 of the revised manuscript (Lines; 810-812).

Fig 5B. This data should be shown in the context of infection with untagged Capsid at least for 1-2 viruses. This is a serious drawback of the present study as there is no clear evidence presented that the native capsid protein in an infection context depend on importin 7 for nuclear accumulation and behave similar to the GFP-Capsid constructs being used.

Following the reviewer’s concerns, we used an un-tagged JEV and DENV core to examine core translocation in WT or IPO7KO Huh7 cells. As shown in Figures 5C and 5D and their quantitative data, nuclear translocation of JEV and DENV core protein was inhibited in IPO7KO Huh7 cells. We tested the translocation of core protein upon infection with DENV as shown in Figure 5F. Although we could not examine ZIKV infection because we could not find appropriate antibodies against the ZIKV core, these data are consistent in that nuclear translocation of flavivirus core protein largely depends on IPO7.

Fig 5 A-D: Two repetitions are insufficient; a minimum of three biological repeats and statistical analysis need to be included. 5E-F: You cannot do statistics on two repeats, need minimum of three repeats to perform statistical analysis. 5G-H: I presume three repetitions based on the data points shown, this should be clearly stated in the figure legend.

We repeated three independent experiments, shown in Figures 5A and 5C-5F, and indicated them on Lines 823. We have added statistical data in Figures 5B-5F. We have corrected the statement of biological repeats in Figures 6A and 6B (Lines; 843-844).

Fig 5E-G: Taking the data of 5E and 5G together it seems Importin 7 functions as the level of particle release and not particle assembly or maturation. Have you checked for the specific infectivity of the particles released from knock out cells to determine the reason behind the reduction in virus titre? You could look at the prM maturation by furin cleavage to check it this is altered in the IPO7 knock out cells.

We determined the ratio of infectious titer per 103 copies of viral RNA in Figure 6F. The proportion of infectious viruses targeting extracellular JEV RNA was decreased in IPO7KO cells. Simultaneously, no difference was observed in the proportion of infectious viruses targeting intracellular JEV RNA between WT and IPO7KO cells. Although we could not find appropriate antibodies against the JEV core, we checked prM expression using the DENV virus. The expression of prM was slightly increased in JEV-infected IPO7-KO Huh7 cells (Figure S3D). This result suggests that the efficiency of prM cleavage by furin was partially involved in the impairment of infectious virus release in IPO7KO Huh7 cells.

Fig 5H: Have you checked if the observation regarding intracellular RNA levels in 5F is applicable to these viruses as well.

We checked the intracellular RNA levels of DENV and ZIKV-infected cells. In contrast to JEV, intracellular ZIKV or DENV RNA showed no difference in IPO7-KO Huh7 cells (Figure 6H). We discuss it in Discussion section (Lines; 269-271)

Fig 6: The figure legend "Data are representative of two (A, B) independent experiments and are presented as the mean {plus minus} SD of three independent experiments (C)" is confusing. The sentence should be reworded to state the repetitions separately for independent experiments. Fig 6C should show original titres and not percentages.

We have corrected Figure legends according to the reviewer’s comments. We have showed the original titers in Figures 6C and 6E.

Fig 7B: This experiment should be performed in IPO7 knock out cells to confirm that the observed reduction of core mutant is mainly contributed from its lack of interaction with IPO7 and not from any other confounding factors.

Following the reviewer’s suggestion, we performed SRIP experiments for GP/AA mutation using IPO7KO Huh7 cells. As shown in Figure 7C, the SRIPs harboring WT core were impaired in IPO7KO Huh7 cells; no difference was observed in the SRIPs harboring GP/AA mutations in WT and IPO7KO cells. These results suggest that IPO7-dependent nuclear translocation of core protein is important for the viral release.

Reviewer #1 (Significance (Required)): While the authors could convincingly demonstrate the interaction between capsid and IPO7, how that interaction results in the observed reduction in viral titre is largely unexplored. As all the localization data used a GFP-tagged capsid outside an infection context, this reviewer is not confident that all the reported observations will hold in an infection setting. This need to be urgently addressed to rise the confidence about the observation. The current data is insufficient to confidently attribute the change in titre to the interaction between capsid and IPO7 and the capsid localization to the nucleus. Knocking out IPO7 could have pleotropic effects independent of capsid nuclear accumulation that could lead to the observed titre reduction. This need to be addressed further before linking both these phenotypes. Certain key experiments needed to address these questions are currently missing. While the interaction of Capsid with IPO7 is certainly intriguing, the implications of this interaction on virus biology needed further investigation before clear conclusions can be drawn regarding this observation.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: In this study Itoh and colleagues investigate the mechanism, role and impact of the nuclear localization of the flavivirus core protein. The import of the core protein has long been observed and investigated and herein the authors use some novel approaches to identify potential cellular binding partners that facilitate nuclear import. Via proteomics and biochemical approaches they determine that importin-7 plays a crucial role in the import of the core protein that appears to be conserved across Flavivirus members. In general the findings and conclusions are sound but there are some significant omissions and caveats that warrant further investigation.

Major comments: - one of the major caveats of the study is that the flavivirus NS5 protein also translocates to the nucleus in an Importin-alpha/beta dependent manner. Therefore how can the authors discount any impact of preventing NS5 import, in addition to core, on virus and SRIP replication and production. Some discussion, if not additional experiments are required here ie. NS5 localization in the KO cells during virus infection

We examined the localization of NS5 using IPO7KO Huh7 cells. As shown in Figure S2D and S2E, we confirmed that IPO7 was not involved in the nuclear localization of NS5.

• the localization is predominantly nucleolus rather that nucleoplasm when compared to the SV40 NLS. What are the sequence differences between the flavivirus proteins that potentially could account for this? A protein known to localize solely to the cytoplasm should also be used eg. NS1 or NS3.

The JEV core does not contain a consensus nucleolar localization signal. Nuclear localization of NS5 depended on importin-α similar to the SV40 NLS, while flavivirus core proteins were independent of importin-α. Gly42 and Pro43 are critical amino acids for the nuclear localization of the core protein, as shown in Figures 1C and 1D. The Gly42 to Pro43 of core proteins were well-conserved in the core proteins of the Flaviviridae family.

• controls for Figure 2? Ie. a protein known to be inhibited by Bimax but not the RanGTP mutant and vice versa.

Bimax binds to importin alpha but not importin beta and specifically inhibits the importin alpha/beta pathway. The RanGTP mutant binds to the importin beta family, including importin beta 1, and widely inhibits importin beta-dependent nuclear import. These inhibitors are well-characterized and recognized in the field. Therefore, we have cited the following references: Tsujii et al., JBC, 2015.

• Fig 5. Difference with WNV and DENV in nucleoplasm localization but also WNV still appeared to have Core in the nucleus in the KO cells

We agree with the reviewer’s comment about differences in nuclear localization among the viruses using the IJ assay. We have added new data to examine the localization of the DENV core after DENV infection. Nucleolar localization of the DENV core following DENV infection was observed, as shown in Figure 5F. Therefore, differences in nucleoplasm or nucleolar localization among different viruses shown in Figure 1C and Figure 5B might be artifacts of recombinant proteins. One possibility is that the localization of core proteins using IJ assay was detected by anti-GFP antibodies. Although purified GFP-core proteins, as shown in Figure 1B and S1, were observed as a single band of fusion proteins, core proteins of WNV and DENV might be cleaved during IJ experiments, and GFP alone might be detected at nucleoplasm, as shown in Figure 5B. Because our study focused on the nuclear translocation of flavivirus core proteins, the detailed localization of each core protein in the nucleus will be studied in the future.

• Fig 5C still has substantial JEV and DENV core but not WNV and ZIKV. Why is the DENV and WNV localization pattern different to Fig 5B?

We appreciate the reviewer’s suggestion; we re-checked all our data presented in Figure 5B and other data shown in Figure 5B. We quantified the ratio of nuclear localization as shown in the right of Figure 5B. Our quantification data showed that the nuclear transport of all core proteins used in this study was dependent on IPO7. In contrast, Figure 5A shows that nuclear translocation of WNV core protein is partially dependent on IPO7. This discrepancy might be explained that nuclear translocation of WNV core protein might be regulated by several nuclear carriers. We described this in discussion section (Line; 250-254).

• Fig 5F, does the KO also restrict NS5 from entering the nucleus and could this then results in increase polymerase activity confined to the cytoplasm resulting in more viral RNA?

Following the reviewer’s suggestion, we examined NS5 localization during viral infection and plasmid transfection, as shown in Figure S2D and S2E. Previous data regarding the nuclear localization of NS5 depended on importin-α. Our data are consistent with previous reports that IPO7 was not involved in the nuclear localization of NS5. In contract to JEV, we also confirm that intracellular ZIKV or DENV RNA showed no difference in WT and IPO7-KO Huh7 cells (Figure 6H). As described in the discussion, other factors, such as antiviral factors, might be involved in IPO7-mediated nuclear transports in JEV infected cells (Line; 269-271).

• Why was WNV infection not performed in Fig 5H? What where the viral tires compared to for the relative % values?

Because our institution does not have a BSL3 facility, we could not use WNV. Following the reviewer’s comment, we showed viral titers in Figure 6G.

• Fig 6B, still a significant amount of core present in the nucleolus. Also WT cells have (almost?) no cytoplasmic staining for core where this could be clearly observed in the WT cells in Fig 5D. Why the difference?

Plasmid transfection of AcGFP-Core WT showed that almost all core proteins were located in the nucleus. We assumed that AcGFP might influence nuclear exports of core proteins or the efficiency of nuclear transports as shown in other data of in vitro experiments. However, our finding that IPO7 was involved in the nuclear transport of core proteins is consistent.

• In Fig 7B, D and E, when were the SRIPs collected and what was the time period after subsequent infection?

Following the reviewer’s comments, we have added more details on SRIP experiments in Materials & Methods (Line; 521-523).

• In Fig 7C was the luciferase measured from the initial transfection and how did it correlate with RNA production? A 15-fold increase in replicon RNA actually seems quite low over a 48h period

Because large amounts of in vitro-transcribed replicon RNA were injected into cells in this experiment, we observed that significant amounts of luciferase values were detected after 4 h. However, the 15-fold enhancement in luciferase value was consistent with previous reports (PMID: 30413742, PMID: 17024179). We have added references in the revised manuscript.

• quantitation is required throughout all of the experimental IFA data provided

Following reviewer comments, we have quantified all IFA data and showed their results.

Reviewer #2 (Significance (Required)):

The nuclear translocation of flavivirus protein has long been studied and it has been observed that the core, NS5 (RNA polymerase) and potentially the NS3 (helicase/protease) proteins all translocate the nucleus. Importin alpha and beta have been shown to facilitate this process. The authors aim to extend this to identify importin-7 as a major cellular factor enabling nuclear translocation. Overall the experiments have been performed well but there is a lack of quantitation for many of the results an suitable controls are required.

I am a researcher in the field of flavivirus replication

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In the presented study the authors identified and mechanistically investigated how Flaviviruses including Japanese encephalitis virus (JEV), Dengue virus (DENV), and Zika virus (ZIKV) commonly use importin-7 (IPO7), an importin-β family protein, as a cellular carrier protein to facilitate nuclear core protein translocation. The authors evaluated how the production of infectious viruses is regulated by IPO7 using cellular infection models including IPO7-deficient knockout cells. In the submitted manuscript, the authors provide evidence that IPO7 facilitates viral core protein import into the nucleus of infected cells, which is essential for effective Flavivirus replication. Taken together, the study is interesting to a broader readership with interest in molecular virology, and its findings are informative for potential future targeting of IPO7 to affect flavivirus replication using small molecule drugs. The manuscript is well-written and easy to follow, the methods are appropriate, the structure is logical, and statistical analysis is adequate.

Major comments:

• It is unclear why the authors specifically used Ala substitution at Gly42 anb Pro43 to obtain the abolishment of nuclear core protein localization. It would be helpful to put this into more context and explain the approach.

Mutations of Gly42 and Pro43 to Ala were previously reported and characterized by the same research group (PMID: 15731239). Following the reviewer’s comment, we have added more details of GP mutations in the text (Lines 66–70).

• In Figure 4, the authors claim that the binding between IPO7 and RPS7 is disrupted upon the addition of RanGTPQ69L. This is not clearly evident from the pulldown experiment and should be proven experimentally with additional experiments (e.g. by using an imaging approach) to underline the statement that the binding mode of IPO7 to the JEV core protein is similar to that of RPS7. Loading controls for pulldown blots should be added.

As described in response to the comment by reviewer#2 regarding Figure 2, the RanGTPQ69L mutant inhibits the interaction between the importin beta family, including IPO7 and its substrates, by directly binding to importin beta proteins. For the benefit of readers without knowledge of the typical Ran-dependent nuclear transport mechanism, we have described its effects with several cited references (Dickmanns et al., 1996; Tachibana et al., 2000). We referred to a study that showed that IPO7 transports RPL proteins, including RPS7 (Jäkel and Görlich, 1998). The data in Figures 4A and 4B demonstrate that adding RanGTPQ69L remarkably reduces the binding of IPO7 to the Core proteins and that the effect is more robust than that for RPS7. We believe that these results are experimentally valid, indicating that nuclear transport of Core proteins by IPO7 is achieved through a typical Ran-dependent pathway.

• Most methods used are presented logically but require some more details so that they can be reproduced. In particular, the difference between Figure 4 E and 4H is confusing. What is the difference? Is 4E showing intracellular viral titers and 4H infectious viral titers in the supernatant of cells? Clarification needed. Put relevance of these experiments in context of the hypothesis.

We apologize for the confusion regarding the data in Figures 5E and 5H (we assume). These data were derived from the same experiments, except for the time-course data presented in Figure 5E. We have removed Figure 5E to simplify our results.

• Identical phenotypes induced by IPO7 knockout in a number of HuH7 clones are shown in Figures 6A to 6C. This data does not add to the overall understanding and should be moved to supplementary figures. Why are 293T cells used in experiments shown in Figure 6D and 6E? What is the relevance of kidney cells to Flavirius infections?

Following the reviewer’s comments, we have moved Figure 6 to supplementary figures. We used 293T cells because of efficient JEV propagation and gene-deficient efficiency. We wanted to demonstrate that our data are not Huh7-dependent through experiments in 293T cells.

• Prior studies are referenced appropriately, however, in a recent study it was demonstrated that IPO7 is stabilized upon Epstein-Barr Virus infection and that IPO7 presence is required for the survival of host cells (Yang YC, Front Microbiol. 2021 Feb 16;12:643327. doi: 10.3389/fmicb.2021.643327).

We deeply appreciate the publications in these fields. Following the reviewer’s comment, we have cited these references.

This important study about the physiological relevance of IPO7 during viral infections has not been cited by Itoh and colleagues in the presented study. However, the results of the uncited study are very relevant to the provided manuscript, since Itoh and colleagues are using IPO7 knockout cells to investigate its function in Flavivirus core protein nuclear import. Hence, the authors should perform cell survival and cellular fitness experiments to demonstrate that observed phenomena of reduced viral replication and virus export in IPO7 knockout cells are independent of compromised cellular fitness due to IPO7 deficiency.

We evaluated cellular fitness between WT and IPO7KO Huh7 cells using PI (Propidium Iodide) staining through flow cytometry. As shown in Figure S2F, no differences were observed in cell viability between WT and IPO7KO Huh7 cells. It suggests that viral titers reduced in IPO7KO Huh7 cells are not involved in cellular fitness.

Minor comments:

• Describing Figure 3B, the authors state that they focused on IPO7 among the core binding proteins belonging to the importin-b family, because IPO7 "was identified the most peptides" in the mass spectrometry approach. This requires a more detailed explanation. Also, an explanation of why HEK293T cells were used for this approach and not HuH7 cells, as used predominately in most parts of the study, would provide more clarity to the reader.

We focused on IPO7 because it had the highest number of detected peptides, and we found that the second most detected peptide, IPOB1, did not bind to JEV core proteins as shown in Figure 2C. Therefore, we included the lack of interaction between IPO7 and IPOB1 as part of the rationale.

• In Figures 4E and 4F, colour coding is missing.

We have indicated color coding in this data. Thank you for your comments.

Reviewer #3 (Significance (Required)):

The provided manuscript 'Importin-7-dependent nuclear localization of the Flavivirus core protein is required for infectious virus production' by Itoh and colleagues investigates a topic with important scientific relevance. The presented study builds on previous findings by the authors where they have demonstrated that Flavivirus core protein nuclear localization is actually conserved among Flaviviridae and represents a potential target for broad-range antiviral small molecule drugs (Tokunaga et al., Virology, 2020 Feb;541:41-51). However, our understanding of Flavivirus core protein nuclear localization during viral replication and how the processes could potentially be targeted using novel therapeutic drugs remains elusive. Here, the provided manuscript addresses a mechanistic investigation of how the Flavivirus core protein is actually translocated from the cytoplasm to the nucleus of infected cells. The study is informative particularly for virologists with expertise in Flavivirus replication.

However, from my point of view as a virologist investigating host-pathogen interactions with a strong interest in clinical translational, the manuscript requires a more careful evaluation and interpretation of some results of key experiments. In addition, some of the results need to be more precisely described for clearer understanding by a broader readership.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Summary: In the manuscript entitled "Importin-7-dependent nuclear localization of the Flavivirus core protein is required for infectious virus production", by combining proteomics, CRISPR/Cas9 gene KO, CLSM and standard virology techniques, Yumi Itoh report novel data concerning the involvement of IPO7 in the nuclear and nucleolar localization of Flaviviridae core nuclear and nucleolar localization and viral particle release. Surprisingly, IMPa/b1 inhibition via Bimax2 does not affect core nuclear transport, whereas both RanQ69L and WGA did so. The authors try to identify the cellular transporters involved in core nuclear import, and to this end performed a MS spec analysis of JEV core interactors, which yielded IPO7 as the most likely candidate. After confirming the result by Co-IP, the authors go on showing most core proteins require IPO7 for nuclear delivery using Huh7 and HEK7 IPO7-KO cells, with the exception of WNV core which was able to partially enter the nucleus. In such cells, upon infection, extracellular (but not intracellular) viral titers were strongly reduced, a phenotype which was observed with a JEV core mutant bearing the Gly42 and Pro43 to Ala substitutions in a previous study.

Major comments: - The major conclusions of the study are:

1.IPO7 is the main driver of core nuclear transport 2.Core nuclear localization is somehow important for viral particle release Both conclusions are well-supported by experimental evidence.

Methods are clear and precise, the study appears to have been produced with high quality standards, and so is the presentation of the results. A few controls however should be added to increase the reliability of the results presented here (see below)

Since the authors attempt to link the phenotype observed on virus release upon IPO7 KO to defects on core nuclear import by making a parallelism with core GP/AA mutant, it would be important to know the behavior of such virus in Huh7 wt and Huh IPO7 KO cells. In other words, is GP/AA JEV released efficiently in Huh7 IPO7 KO cells?

We have added new data examining the propagation of the GP/AA JEV mutant in IPO7KO Huh7 cells (Figure 6F). Our new data showed that there were no differences in the propagation of the GP/AA mutant in WT and IPO7-KO Huh7 cells.

A similar approach can be applied to data shown in Figure 7 (effect on release on a capsid nuclear deficient mutant). This would help understand if IPO7 KO, viral release defects and core nuclear import are somehow linked.

We produced SRIPs harboring GP/AA core using WT and IPO7KO Huh7 cells and demonstrated that the number of infectious viruses produced by WT and IPO7KO Huh7 cells was the same (Figure 7C).

Minor comments:

INTRODUCTION • “Flaviviruses...are mosquito-borne human pathogens" What about tick borne encephalitis virus?

We have corrected it (Line; 43-44).

• " replication.... occur in the endoplasmic reticulum (ER)" This sentence is a bit inaccurate. Flaviviridae RNA replication occurs in so-called viral replication factories, double membrane vesicles which are partly derived from the ER. see "PMID: 26958917".

We have corrected this sentence according to the reviewer’s comment (Line; 60-62).

• "it is known that some flavivirus core proteins are translocated from the cytoplasm into the nucleus" o I think the first evidence of core in the nucleus dates back to 1989, and here it might be appropriate to cite the original reference: "PMID: 2471810". o It might be worth mentioning that NS5 has also been reported in the nucleus (See "PMID: 28106839")

We have corrected the sentence according to the reviewer’s comment (Line; 63-65).

• "In the cytoplasm, NLS-containing proteins are recognized by importin-α " o This is true only for classical NLSs, not every NLS binds IMPa, as the authors confirm in this study! Indeed, we have also PY-NLS, IPO7 specific NLSs, IPOb1 NLSs, etc. I therefore suggest rephrasing.

Thank you for pointing out the exact description of NLS. We agree with the reviewer’s comment that “NLS” includes all types of signal sequences, such as PY-NLS. To clearly distinguish between the CLASSICAL nuclear transport pathway by importin α/β1 and the various nuclear transport pathways by the importin β family, such as transportin, we refer to NLS as classical NLS (cNLS) in the document. We have modified the following sentence by adding “such as transportin” and “without importin-α.”

RESULTS

• Fig. 1. o it is not clear what is new here, with respect to what has been already published. The authors should clearly differentiate novel findings from confirmatory results

Thank you for your suggestion. We would like to introduce our new assay using recombinant virus core proteins, as shown in Figures 1C and 1D. The data shown in Figure 1 are crucial for understanding our data in Figure 2, and we believe this figure is required for broad-ranging readers.

Fig. 2 and 4 o Proteins whose nuclear transport is dependent on IMPa/IMPb1 (such as SV40 NLS) are lacking here

Bimax binds to importin alpha but not to importin beta and specifically inhibits the importin alpha/beta pathway. The RanGTP mutant binds to the importin beta family, including importin beta 1, and widely inhibits importin beta-dependent nuclear import. These inhibitors are well-characterized and recognized in the field. Therefore, we have cited the following references: Tsujii et al., JBC, 2015.

• Fig.5 o It would be important to know the effect on total virus infectivity (intracellular + extracellular) and total viral RNA. It would also be important the effect on RNA replication by using a subgenomic viral replicon (with deletion of the env gene for example). The question here is if IPO7 depletion affects to any extent viral genome replication, and this is impossible to assess in a fully assembling system. We determined the ratio of infectious titer per 103 copies of viral RNA in Figure 5D. The proportion of infectious viruses targeting extracellular JEV RNA was decreased in IPO7KO cells, and there was no difference in the proportion of infectious viruses targeting intracellular JEV RNA between WT and IPO7KO cells. We examined the effects of IPO7 on viral RNA replication of subgenomic replicon. We showed that the deficiency of IPO7 enhanced viral RNA replication as shown in Figure 7E. As described in the Discussion section, IPO7 may transport other factors possessing antiviral activity against flaviviruses. These data will be investigated in the future.

o Panels A-F legend is missing, consider adding it?

We have added more details to Figure 5A-5F following the reviewer’s suggestion.

• Fig.7 o I did not completely understand how NLuc is the readout here To quantify RNA replication, we quantified Nluc values using a plate reader. We have added more details on the reporter assay in Materials and Methods (Line; 521-523).

o Also, I do not understand if the effect of GP/AA substitution of panel B has already been reported or if it is a novel finding

Previous reports regarding the effect of GP/AA substitution of JEV showed the impairment of infectious virus release. However, the SRIP assay was performed to examine the viral release step. Our detailed data showed that the lack of IPO7-mediated nuclear transport of core proteins impaired infectious viral release, and our new results using SRIPs harboring GP/AA core showed that the lack of nuclear transport of core proteins also impaired the release of infectious viruses. Our data strongly suggest that the lack of nuclear transport of core proteins influences the viral release.

• All CLSM figures lack quantification (Fn/c; Fno/n)

We have added quantitative data for IFA experiments in our revised manuscript.

DISCUSSION

• "The nuclear entry of viral genomic DNA has been demonstrated to involve IPO7" o It would be nice to know which viruses the authors are freeing to here

We have added the virus name and corresponding references.

• "While RNA viruses, including flaviviruses, are considered to replicate in the cytoplasm of mammalian cells, increasing evidence suggests nucleolar localization of the viruses " o I suspect Rawlinson did not propose the viruses localize to the nucleolus, as this sentence seems to imply. Rather, a trafficking of viral proteins to nucleoli, to manipulate cell function, is more realistic. I suggest considering rephrasing. We have corrected this sentence.

Reviewer #4 (Significance (Required)):

SECTION B - Significance ========================

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. As alluded to above, this work presents several advances of current knowledge in the field of viral proteins nuclear trafficking, and in Flavivirus biology. The finding of most core proteins depending on IPO-7 is novel and intriguing, and opens the question of what makes WNV core special. Indeed, this protein nuclear targeting is only partially inhibited in IPO7 deficient cells. The fact that the authors extend their findings to several Flaviviruses adds significance. The role of nuclear core for virus release is also intriguing, but appears poorly characterized. In this respect a mechanistic explanation of the phenomenon would be highly desirable to increase the significance of the work presented here.

In this context I would have a few suggestions:

A) The authors performed MS spec on JEV core, this most likely resulted in a long list of "hits". However, they only report IMPb superfamily members. This is perfectly fine, since they focus at identifying partners responsible for nuclear import. However, it might be helpful for understanding the role of nuclear core. By comparing MS of wt core and GP/AA core, and or wt core in wt and IPO7KO cells, authors could identify core biding partners in the nucleus (in the nucleolus?) which are important for virus release. This could be subsequently addressed by knocking down these factors and study the effect on virus life cycle.

We appreciate the reviewer’s valuable comments. We did not perform MS analysis on GP/AA core protein and core protein using WT or IPO7KO Hun7 cells. To report IPO7-mediated core translocation simply, we would like to cite our manuscript focusing on IPO7. To clarify the importance of nuclear transport of core protein on the viral life cycle, we will perform wide-ranging proteomics.

1. B) Further, the authors should try to address the role of core in the nucleus (and nucleolus). Does it interact with cellular/nucleolar proteins? Does it deliver viral RNA to sites of assembly? Does it interfere with rRNA synthesis? All these findings would be easily obtainable using the GP/AA virus and/or Huh7 KO cells, and tremendously increase the impact of the study, which at the moment is limited at points 1 and 2 in the first section of the current report.

Thank you for your valuable comments. We agree that we should clarify the roles of the nucleus or nucleolar localization of the core protein. We tested the effects of rRNA synthesis on JEV core expression. Our data showed that core protein expression slightly impaired the maturation of rRNA synthesis, as shown here. However, the core expression did not influence protein translation. We focused on the phase separation capacity of core protein localized in the nucleolar or nucleus. From our accumulating data, we hypothesized that the acquisition of phase separation capacity of core protein might be involved in an efficient virus release step. We hope that these data will be reported in the near future.

Overall, this work should be interesting for both cell biologists interested in trafficking of viral proteins, and virologists interested in virus-host interactions. The antiviral approach at the moment is a bit less convincing, but the manuscript might be interesting for scientists trying to develop new antiviral strategies. (In this context it might be worth reading and possible discussing the very recent paper from the Bartenschlager group "PMID: 37702492." Also, I think that it would be worth discussing the recent discovery that a closely related virus belonging to the Hepacivirus genus within the Flaviviridae family, mediated re-localization of Nups to viral replication factories, where they are believed to control access to DMVs interior, thereby regulating virus replication and assembly. Could the core IPO7-interaction have any role in core delivery to DMVs? See "PMID: 26150811".

Thank you for your valuable comments. We have added several sentences in the Discussion section (Line; 297-305). We will investigate the role of nuclear transports in viral life cycles in the future.

Since I am a molecular virologist studying viral nucleocytoplasmic trafficking, virus-host interactions, and antiviral drug-discovery I think I have sufficient expertise for an informative and helpful revision of this work.

Przetwarzanie sensoryczne jest związane z uwagą, równoważeniem motoryki dużej i tonusem mięśni. Są stymulowane przez aktywność fizyczną

Związek przetwarzania sensrycznego i lęku oraz, nieuwagi (głównie, deficytów w percepcji). - Deficyty w percepcji (oraz dysregulacja emocjonalna, jako coś dodadanego, albo skutek), deficyty w pamięci i koncentracji tworzą podstawy do rozwoju i nasilania się lęku, oraz lęku panicznego

Przedsionkowe przetwarzanie i integracja sensoryczna/wrażenia sensoryczne