Na minha opinião, esta é realmente a grande vantagem! Transformar o momento de aprendizagem num momento de contacto com as tecnologias que, agora, fazem parte da dia-a-dia. Sobretudo para as novas gerações é uma forma de tornar o processo muito mais atrativo e interactivo.

Esta frase destaca, de acordo com Salmon (2013), a importância crucial que as e-atividades desempenham numa da aprendizagem mediada por tecnologia. Designadamente, combinando tarefas interativas com práticas realizadas em plataformas digitais, idealizadas para envolverem os estudantes numa aprendizagem ao mesmo tempo ativa e colaborativa. O uso de ferramentas digitais facilita a construção colaborativa do saber, enquanto os estudantes desenvolvem competências que seguramente lhes serão de grande utilidade no atual mundo digital. António Pedro Santos, nº 2403463

As atividades digitais são essenciais para a educação moderna, pois tornam a aprendizagem mais dinâmica e interativa. Elas promovem a colaboração e a participação ativa dos alunos, integrando a tecnologia de forma eficaz. No entanto, motivar e integrar os estudantes nessas atividades é um desafio. É crucial que as atividades digitais sejam envolventes e relevantes, com objetivos claros e motivadores. O papel do professor é fundamental, fornecendo feedback contínuo e criando um ambiente de aprendizagem inclusivo e acessível.

Quanto mais leio sobre a criação de "boas" atividades de aprendizagem em ambientes digitais em rede, mais articulo com os princípios da educação não formal e metodologias participativas, pois penso que ambos se baseiam em algumas estratégias e princípios-chave:

Centralidade no aluno/formando: o design de atividades deve priorizar a autonomia e o protagonismo do aluno/formando, o que pode incluir a oferta de múltiplos caminhos e escolhas dentro da atividade, como no uso de plataformas com conteúdos modulares ou fóruns para debates colaborativos.

Interação e Co-Criação: as ferramentas digitais podem facilitar a construção colaborativa de conhecimento, como em wikis, mapas mentais colaborativos e debates virtuais. Nessas interações, os participantes aprendem uns com os outros, incorporando a essência das metodologias participativas.

Flexibilidade e Inclusão: a diversidade de formatos e recursos (vídeos, textos, podcasts, gamificação) pode atender a diferentes estilos de aprendizagem, respeitando os princípios da educação não formal de ser adaptável às necessidades do público.

Espaços de Reflexão e Feedback: ambientes digitais podem criar espaços para reflexões coletivas e trocas significativas, como fóruns, diários reflexivos online ou sessões de feedback interativo. Esses momentos fortalecem a construção coletiva e aprofundam a aprendizagem

Valorização da Experiência e do Contexto: a personalização das atividades digitais permite que os alunos/formados integrem as suas experiências prévias e contextos específicos ao processo. Um exemplo é o uso de storytelling digital, onde partilham as suas histórias em relação ao tema estudado.

Penso que, apesar das grandes potencialidades, é necessário cuidado para que o uso de ambientes digitais em rede não reproduza práticas hierárquicas ou centradas na transmissão unidirecional de conteúdo. É essencial manter o equilíbrio entre a tecnologia e os princípios pedagógicos, garantindo que o digital seja uma ferramenta de facilitação, e não de imposição. Portanto, a integração entre esses dois universos – a educação não formal e os ambientes digitais – depende de um desenho pedagógico intencional, onde a tecnologia seja utilizada para potencializar os valores participativos e inclusivos que caracterizam a educação não formal. Assim, poderemos ampliar o alcance da aprendizagem, sem perder a sua essência humana e colaborativa.

O planeamento de e-atividades, sustentadas em objetivos claros e alinhados com as aptidões desejadas, que promovam a colaboração e a interação em ambientes digitais, é essencial para uma aprendizagem ativa, que supere o isolamento e estimule a construção colaborativa de conhecimento.

É, por isso, necessário estruturar as e-atividades com papéis bem definidos e etapas claras, criar tarefas desafiantes e acessíveis, a que não é alheia a necessidade de utilizar ferramentas digitais adequadas para facilitar a interação desejada.

O feedback contínuo é essencial para orientar os alunos, fortalecer a aprendizagem e a produção colaborativa de conteúdos, sustentados na troca de ideias e no trabalho conjunto.

Considero que, de facto, o feedback construtivo, quando bem estruturado, é uma peça-chave no ensino à distância, para garantir que os alunos se sintam acompanhados ao longo seu progresso, fortaleça o vínculo com professor, e constitua um reforço motivacional, através da identificação do que foi aprendido, e de estratégias de melhoria.

A literatura destaca, ainda, a importância de integrar o feedback em todas as fases do processo de aprendizagem, e não ocorrer apenas após avaliações formais, mas também durante a realização de atividades, devendo ser planeado como uma estratégia pedagógica essencial, não só para ajudar os alunos a corrigir as suas trajetórias em tempo útil, mas também para reforçar a ideia de que o erro faz parte do processo de aprendizagem, promovendo um ambiente de experimentação e crescimento.

Para desenhar uma boa e-atividade de aprendizagem, é fundamental definir objetivos claros e específicos, alinhados às metas do curso, e planear etapas detalhadas e sequenciadas, considerando o tempo necessário para a execução. A atividade deve ser adequada aos conteúdos e ao formato digital, utilizando ferramentas acessíveis e tecnologicamente apropriadas. É importante diversificar as atividades para atender a diferentes estilos de aprendizagem, promovendo a inclusão e a acessibilidade. As e-atividades devem estimular a participação ativa e colaborativa, desenvolvendo competências como pensamento crítico e resolução de problemas. O feedback construtivo é essencial, oferecendo orientações específicas para o aprimoramento dos alunos. As tarefas devem ser motivadoras, ligadas a contextos significativos e práticos, com critérios de avaliação bem definidos e alinhados às competências esperadas. Utilizar modelos estruturados, como os cinco estádios de Gilly Salmon ou a Taxonomia Digital de Bloom, pode ajudar a planear e-atividades progressivas e centradas na construção do conhecimento. Por fim, é necessário integrar a tecnologia e a pedagogia de forma coerente, garantindo uma aprendizagem ativa, significativa e alinhada às exigências do mundo digital.

A capacidade de manter os alunos motivados e interventivos é um desafio para qualquer docente. Nesse contexto, qualquer processo que facilite o envolvimento dos alunos e melhore a atenção dedicada aos temas em questão, é sem dúvida uma mais valia. As tecnologias digitais podem ser um forte aliado nessa perspetiva, desde que adequadas ao conteúdo que se pretende transmitir e ao grupo em questão.

Vivemos numa era em que a sociedade se move e conecta numa rede exponencialmente digital. Neste contexto, as e-atividades, ao permitirem o uso de uma vastidão de recursos digitais impulsionadores da participação e aprendizagem colaborativa, devem ser encaradas como uma componente importante das metodologias pedagógicas envolvidas no sistema educativo. Nas e-atividades, o processo usado para a transmissão de conteúdos é a verdadeira transformação pedagógica, que se acrescenta ao conteúdo informativo propriamente dito e permite aos docentes "falar a mesma linguagem" das novas gerações de alunos, com todas as vantagens daí decorrentes, em questões de motivação, participação, construção de conhecimento e aquisição de competências.

Ao analisar o papel das e-atividades no desenho das estratégias de ensino-aprendizagem, considero que o texto apresenta uma abordagem robusta e bem fundamentada, sublinhando a sua relevância enquanto instrumentos de inovação pedagógica. A forma como são integradas teorias reconhecidas, como o modelo dos cinco estágios de Salmon, demonstra um esforço claro em destacar as e-atividades não apenas como ferramentas tecnológicas, mas como elementos estratégicos no processo de ensino.

No entanto, este enquadramento convida a refletir sobre alguns desafios que se colocam à sua implementação. A crescente diversidade dos perfis dos estudantes e as desigualdades no acesso às tecnologias representam barreiras importantes, particularmente quando se pretende assegurar simultaneamente a personalização e a inclusão das práticas educativas. Este aspeto sublinha a necessidade de uma abordagem pedagógica que, sem descurar a inovação, mantenha um equilíbrio entre a utilização da tecnologia e a garantia de acessibilidade para todos os estudantes.

Por outro lado, parece evidente que o impacto das e-atividades reside na sua integração consciente e estratégica enquanto mediadoras de aprendizagens significativas e promotoras de competências críticas, como o pensamento reflexivo, a autonomia e a colaboração. Contudo, estas potencialidades exigem um compromisso contínuo dos docentes na adaptação das suas práticas, assegurando que as e-atividades não sejam meramente uma extensão tecnológica das aulas tradicionais, mas sim um meio efetivo para transformar as dinâmicas educativas e facilitar a construção colaborativa do conhecimento.

A diversidade de e-atividades proposta por Maina (2020) reflete a multiplicidade de caminhos para o envolvimento dos estudantes em ambientes digitais. Contudo, a escolha da tipologia mais adequada deve considerar não apenas os objetivos pedagógicos, mas também o perfil dos estudantes e as suas expectativas face à aprendizagem. Adicionalmente, a integração de diferentes tipologias pode ser explorada como forma de enriquecer a experiência educativa e fomentar o desenvolvimento de competências transversais.

A aplicação do modelo de Salmon (2013) em contextos educativos híbridos sugere uma oportunidade para repensar as dinâmicas tradicionais de ensino e aprendizagem. No entanto, a operacionalização deste modelo requer uma análise crítica sobre a sua aplicabilidade em diferentes contextos culturais e institucionais. Será que o mesmo nível de interação e colaboração preconizado no modelo é exequível em cenários com infraestruturas tecnológicas limitadas? A meu ver, estes são aspetos a ponderar.

A motivação dos estudantes em ambientes digitais depende, em grande parte, da forma como percebem a relevância das atividades propostas para os seus objetivos formativos. Este aspeto implica uma atenção redobrada por parte dos docentes ao desenharem e-atividades que sejam, simultaneamente, desafiadoras e significativas. A clareza nos objetivos, a diversificação das metodologias e a adequação às necessidades dos estudantes surgem como pilares fundamentais para maximizar o impacto pedagógico destas atividades.

A personalização das estratégias de aprendizagem em contextos digitais reflete a transição para um modelo centrado no estudante, que valoriza a autonomia e o protagonismo. Contudo, esta abordagem exige um equilíbrio delicado: como orientar os estudantes para que desenvolvam estratégias eficazes sem comprometer a uniformidade necessária ao cumprimento dos objetivos pedagógicos? Este é um ponto que merece atenção, especialmente quando se considera a diversidade de estilos de aprendizagem e contextos culturais.

A acessibilidade nas e-atividades constitui um desafio central em contextos educacionais digitais. Embora as ferramentas tecnológicas permitam maior alcance, é imperativo que os docentes considerem as disparidades existentes no acesso aos recursos digitais e nas competências tecnológicas dos estudantes. A implementação de práticas pedagógicas inclusivas implica o uso de plataformas acessíveis e a conceção de atividades adaptáveis às diferentes necessidades. Esta perspetiva reforça o compromisso ético da educação digital em promover equidade e inclusão.

A integração das e-atividades no processo de ensino-aprendizagem sublinha a necessidade de alinhar objetivos pedagógicos com práticas avaliativas consistentes. Esta abordagem requer que os docentes não apenas projetem atividades tecnológicas inovadoras, mas também assegurem que estas sejam capazes de traduzir-se em resultados concretos de aprendizagem. Neste contexto, será crucial refletir sobre como estas atividades podem contribuir para o desenvolvimento de competências transferíveis e aplicáveis em cenários do mundo real, indo além do simples domínio técnico.

O trabalho colaborativo deve ser destacado pois cria oportunidades para análise, debate, reflexão e argumentação, favorecendo o desenvolvimento de estratégias de raciocínio, o aprofundamento do conhecimento e da compreensão. Além disso, este trabalho promove habilidades relacionais, sociais e de troca de saberes. Entre as competências desenvolvidas estão o espírito de equipa, o fortalecimento da comunicação, a cooperação, a solidariedade e a responsabilidade. (Sandra Mónica Oliveira)

Parte de um problema, desafio, dilema, estudo de caso, fenómenos ou tema(s), entre outros, tendo em conta os resultados de aprendizagem, os diversos ambientes de aprendizagem e os conhecimentos prévios dos estudantes, favorecendo o processo ativo e contínuo de construção de significados, onde os estudantes assumem a corresponsabilidade final sobre a sua aprendizagem. (Sandra Mónica Oliveira)

Ao conceber atividades de aprendizagem em ambientes digitais, é fundamental integrar pedagogias ativas e centradas no aluno, através da utilização de ferramentas de suporte com vista à promoção de um ambiente de interatividade e de flexibilidade.

Um dos momentos essenciais a ter em conta no desenvolvimento de e-atividades é a definição e desenvolvimento de estratégias, com vista à promoção de uma aprendizagem dinâmica, onde o aluno é incentivado a participar de forma ativa e colaborativa na construção autónoma de conhecimento.

É importante diversificar as atividades para responder a diferentes níveis de competências e estilos de aprendizagem, garantindo a acessibilidade e a inclusão, não só através das sessões síncronas, onde o aluno é convidado a analisar os temas em tempo real, mas também em sessões assíncronas, onde impera a flexibilidade na gestão do tempo e na promoção do conhecimento, tendo por base o planeamento, os objetivos, as etapas e as metas de aprendizagem definidas.

O receio é que a motivação seja unicamente passar na UC. Quando é referida a variedade, falamos de variedade de e-actividades? Não seria benéfica a figura do "assistente virtual", com progresso passo a passo?

De facto, vejo as e-actividades como aprendizagem activa e autónoma do estudante. Faz-me sentido que seja este o foco do ensino à distância, sendo fundamental que exista da parte do estudante a aquisição de ferramentas, tais como a forma como pesquisa informação e como a analisa criticamente. Estes são pontos que considero basilares para o sucesso das e-actividades como aprendizagem activa...

Em jeito de conclusão do meu raciocinio sobre esta temática, considero que a definição, preparação e execução de e-atividades exige do docente uma maior rigidez no planeamento. O planeamento no ensino à distância é essencial, pois é fundamental todos os intervenientes terem a perfeita noção dos timings e critérios de todas as atividades desenvolvidas. Neste sentido, considero que no regime tradicional, cara a cara, existe uma maior fluidez no desenvolvimento destas atividades, sendo mais facilmente ajustáveis as características temporais do grupo de estudantes, permitindo uma maior flexibilização das mesmas. No planeamento e execução de e-atividades, para além da rigidez no planeamento (e-atividade 1 é para executar na semana 1. por exemplo), não existindo flexibilização temporal, o docente tem de saber lidar com as próprias restrições e limites das plataformas e ferramentas que utiliza e que os estudantes fazem sempre questão de testar. Ou seja, as próprias e-atividades estão limitadas aos limites das plataformas e ferramentas a utilizar, pelo que o professor deverá deixar sempre alguma margem de distância desses limites, levando a uma maior rigidez no desenvolvimento destas atividades .

Penso que este aspeto é central em todos os tipos de ensino, mas em particular no ensino à distância. O feedback pós-avaliação, permite aos estudantes compreenderem as suas falhas e fragilidades, potenciando também os seus pontos fortes, sendo um aspeto que, por vezes, os docentes descuram, mas que é essencial. Sobretudo quando o ensino é realizado à distância e não existe a oportunidade de confrontação no momento com estas falhas e fragilidades é fundamental que o professor arranje estratégias síncronas ou assíncronas (conforme o objetivo da atividade e as características do estudante) para realizar este feeback

As ferramentas e plataformas digitais são um excelente mecanismo para estimular e motivar os estudantes à aprendizagem. Estas ferramentas e plataformas permitem um grau de dinâmica do desenvolvimento da aprendizagem que de outra forma não seria possível. Realização de quizzes interativos, jogos, debates e resolução de casos através de escape rooms e gamificação são exemplos muito palpáveis disto mesmo. Obviamente, as ferramentas e plataformas por si próprias não atingem este fim, só envolvidas num excelente planeamento focado nos objetivos da aprendizagem e nos próprios objetivos da atividade permite explorar o seu máximo potencial, considerando sempre as características do grupo de estudantes. Para o professor, esta mais interatividade e dinâmica é facilitadora do processo de aprendizagem, pois permite aumentar a motivação sem um contato direto e contínuo do mesmo na estimulação da aprendizagem, passando a uma figura de organização e moderação da aprendizagem individualizada dos estudantes

Esta ferramenta parece-me particularmente interessante para os estudantes irem acompanhando, mesmo em aulas síncronas, documentos que vamos "mastigando" juntos. Falo de textos teóricos, ou, no caso que me interessa, textos literários (poemas, contos, ou excertos de obras mais longas) que vamos comentando e que os alunos podem ir anotando só para eles com o highligjht. Podendo, posteriormente, entre eles para um qualquer trabalho de grupo, ou comigo em aula assíncrona de esclarecimento de dúvidas, tornar a troca de mensagens sobre um ponto em concreto. Muito obrigada.

Um dos grandes paradigmas da educação hoje, especialmente nas camadas mais jovens, nas quais a aquisição de conhecimento tem uma importância fulcral na construção da sociedade futura, tem precisamente a ver com a motivação para a aprendizagem. Os níveis motivacionais estão consideravelmente baixos, motivo que atribuo à falta de capacidade do sistema pedagógico, atual, conseguir motivar a população estudantil jovem. Neste enquadramento, a temática das e-atividades (ou atividades) assume um papel preponderante para colmatar esta questão.

Tendo experiência letiva, torna-se demasiado evidente que a atenção do aluno está muito mais desperta após este fazer uma pergunta, a partir da qual a sua capacidade de compreensão aumenta exponencialmente. Interpreto este facto pela mudança do papel do aluno de passiva a ativa.

Para uma aprendizagem efetiva, duradoura e eficiente, é importantíssimo que o papel do aluno seja participativo na narrativa educativa, criando o tal modelo construtivo ao invés de transmissivo. As e-atividades devem ser usadas desde o início para fazer a ligação entre o mundo abstrato dos conteúdos e a realidade prática.

No ensino digital à distância, em particular, outro fator relevantíssimo é a promoção da socialização entre todos os membros do ecossistema educativo, uma vez que a discussão e interação são criadores de motivação e por consequência permeiam o bom desempenho da comunidade educativa.

Outra grande questão, para além das e-atividades, é a objetividade dos conteúdos referenciados, a importância da clareza que se coloca na descrição das e-atividades deve também ser estendida à consolidação de conteúdos, cuja qualidade/objetividade têm um papel fundamental na motivação e resultados do estudante. A adequação dos conteúdos tem uma importância ainda mais relevante no ensino à distância, onde muitos dos momentos de aprendizagem são autónomos e como tal, a possibilidade de frustração, por parte do aluno, é bem maior.

O acompanhamento do aluno ao longo do percurso letivo, garantindo o acesso ao esclarecimento em tempo útil do processo educativo e das atividades a desenvolver, é também relevante para que o aluno continue empenhado.

A questão essencial de toda a comunidade educativa, é encontrar estratégias, com todas as ferramentas ao seu dispor, para manter a motivação nos níveis mais elevados possíveis, sem esta preocupação tudo o resto adquire um papel secundário.

Pedro Brasão 2403419 71544_24_02

O feedback imediato é muito importante, que por vezes descuramos, muitas vezes por falta de tempo, mas que é crucial numa e-atividade. Virgínia Alice Santos

O ensino em ambientes digitais deve incluir um conjunto de atividades de aprendizagem que ajudem o aluno a atingir os conhecimentos necessários, tendo em conta as necessidades e características individuais

Author response:

eLife Assessment

“The work presented is important for our understanding of the development of the cardiac conduction system and its regulation by T-box transcription factors. The conclusions are supported by convincing data. Overall, this is an excellent study that advances our understanding of cardiac biology and has implications beyond the immediate field of study.”

We appreciate the positive assessment of this work and the recognition of its importance in advancing our understanding of the cardiac conduction system, its regulation by T-box transcription factors, and contribution beyond the immediate field.

Reviewer #1 (Public review):

Summary:

In a heroic effort, Ozanna Burnicka-Turek et al. have made and investigated conduction system-specific Tbx3-Tbx5 deficient mice and investigated their cardiac phenotype. Perhaps according to expectations, given the body of literature on the function of the two T-box transcription factors in the heart/conduction system, the cardiomyocytes of the ventricular conduction system seemed to convert to "ordinary" ventricular working myocytes. As a consequence, loss of VCS-specific conduction system propagation was observed in the compound KO mice, associated with PR and QRS prolongation and elevated susceptibility to ventricular tachycardia.

Strengths:

Great genetic model. Phenotypic consequences at the organ and organismal levels are well investigated. The requirement of both Tbx3 and Tbx5 for maintaining VCS cell state has been demonstrated.

We thank Reviewer #1 for acknowledging the effort involved in generating and characterizing the Tbx3/Tbx5 double conditional knockout mouse model and for highlighting the significance of this work in elucidating the role of these transcription factors in maintaining the functional and transcriptional identity of the ventricular conduction system.

Weaknesses:

The actual cell state of the Tbx3/Tbx5 deficient conducting cells was not investigated in detail, and therefore, these cells could well only partially convert to working cardiomyocytes, and may, in reality, acquire a unique state.

We agree with Reviewer #1 that the Tbx3/Tbx5 double mutant ventricular conduction myocardial cells may only partially convert to working cardiomyocytes or may acquire a unique state.  The transcriptional state of the double mutant VCS cells was investigated by bulk profiling of key genes associated with specific conduction and non-conduction cardiac regions, including fast conduction, slow conduction, or working myocardium. Neither the bulk transcriptional approaches nor the optical mapping approaches we employed capture single-cell data; in both cases, the data represents aggregated signals from multiple cells (1, 2). Single cell approaches for transcriptional profiling and cellular electrophysiology would clarify this concern and are appropriate for future studies.

(1) O’Shea C, Nashitha Kabri S, Holmes AP, Lei M, Fabritz L, Rajpoot K, Pavlovic D (2020) Cardiac optical mapping – State-of-the-art and future challenges. The International Journal of Biochemistry & Cell Biology 126:105804. doi: 10.1016/j.biocel.2020.105804.

(2) Efimov IR, Nikolski VP, and Salama G (2004) Optical Imaging of the Heart. Circulation Research 95:21-33. doi: 10.1161/01.RES.0000130529.18016.35.

Reviewer #2 (Public review):

Summary:

The goal of this work is to define the functions of T-box transcription factors Tbx3 and Tbx5 in the adult mouse ventricular cardiac conduction system (VCS) using a novel conditional mouse allele in which both genes are targeted in cis. A series of studies over the past 2 decades by this group and others have shown that Tbx3 is a transcriptional repressor that patterns the conduction system by repressing genes associated with working myocardium, while Tbx5 is a potent transcriptional activator of "fast" conduction system genes in the VCS. In a previous work, the authors of the present study further demonstrated that Tbx3 and Tbx5 exhibit an epistatic relationship whereby the relief of Tbx3-mediated repression through VCS conditional haploinsufficiency allows better toleration of Tbx5 VCS haploinsufficiency. Conversely, excess Tbx3-mediated repression through overexpression results in disruption of the fast-conduction gene network despite normal levels of Tbx5. Based on these data the authors proposed a model in which repressive functions of Tbx3 drive the adoption of conduction system fate, followed by segregation into a fast-conducting VCS and slow-conduction AVN through modulation of the Tbx5/Tbx3 ratio in these respective tissue compartments.

The question motivating the present work is: If Tbx5/Tbx3 ratio is important for slow versus fast VCS identity, what happens when both genes are completely deleted from the VCS? Is conduction system identity completely lost without both factors and if so, does the VCS network transform into a working myocardium-like state? To address this question, the authors have generated a novel mouse line in which both Tbx5 and Tbx3 are floxed on the same allele, allowing complete conditional deletion of both factors using the VCS-specific MinK-CreERT2 line, convincingly validated in previous work. The goal is to use these double conditional knockout mice to further explore the model of Tbx3/Tbx5 co-dependent gene networks and VCS patterning. First, the authors demonstrate that the double conditional knockout allele results in the expected loss of Tbx3 and Tbx5 specifically in the VCS when crossed with Mink-CreERT2 and induced with tamoxifen. The double conditional knockout also results in premature mortality. Detailed electrophysiological phenotyping demonstrated prolonged PR and QRS intervals, inducible ventricular tachycardia, and evidence of abnormal impulse propagation along the septal aspect of the right ventricle. In addition, the mutants exhibit downregulation of VCS genes responsible for both fast conduction AND slow conduction phenotypes with upregulation of 2 working myocardial genes including connexin-43. The authors conclude that loss of both Tbx3 and Tbx5 results in "reversion" or "transformation" of the VCS network to a working myocardial phenotype, which they further claim is a prediction of their model and establishes that Tbx3 and Tbx5 "coordinate" transcriptional control of VCS identity.

We appreciate Reviewer #2’s detailed summary of the study’s aims, methodologies, and findings, as well as their thoughtful suggestions for further analysis. We are grateful for their recognition of our genetic model’s novelty and robustness.

Overall Appraisal:

As noted above, the present study does not further explore the Tbx5/Tbx3 ratio concept since both genes are completely knocked out in the VCS. Instead, the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function.

We agree with this reviewer’s assessment of the assertions in our manuscript.  The novel combined Tbx5/Tbx3 double mutant model does not further explore the TBX5/TBX3 ratio concept, which we previously examined in detail (1). Instead, as the Reviewer notes, this manuscript focuses on testing a model that the coordinated activity of Tbx3 and Tbx5 defines specialized ventricular conduction identity.

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

Strengths:

(1) Successful generation of a novel Tbx3-Tbx5 double conditional mouse model.

(2) Successful VCS-specific deletion of Tbx3 and Tbx5 using a VCS-specific inducible Cre driver line.

(3) Well-powered and convincing assessments of mortality and physiological phenotypes.

(4) Isolation of genetically modified VCS cells using flow.

We thank Reviewer #2 for acknowledging the listed strengths of our study.

Weaknesses:

(1) In general, the data is consistent with a long-standing and well-supported model in which Tbx3 represses working myocardial genes and Tbx5 activates the expression of VCS genes, which seem like distinct roles in VCS patterning. However, the authors move between different descriptions of the functional relationship and epistatic relationship between these factors, including terms like "cooperative", "coordinated", and "distinct" at various points. In a similar vein, sometimes terms like "reversion" are used to describe how VCS cells change after Tbx3/Tbx5 conditional knockout, and other times "transcriptional shift" and at other times "reprogramming". But these are all different concepts. The lack of a clear and consistent terminology for describing the phenomena observed makes the overarching claims of the manuscript more difficult to evaluate.

We discriminate prior work on the “long-standing and well-supported model’ supported by investigation of the role of Tbx5 and Tbx3 independently from this work examining the coordinated role of Tbx5 and Tbx3. Prior work demonstrated that Tbx3 represses working myocardial genes and Tbx5 activates expression of VCS genes, consistent with the reviewer’s suggestion of their distinct roles in VCS patterning. However, the current study uniquely evaluates the combined role of Tbx3 and Tbx5 in distinguishing specialized conduction identify from working myocardium, for the first time.

We appreciate Reviewer #2’s feedback regarding the need for consistent terminology when describing the impact of the double Tbx3 and Tbx5 mutant. We will edit the manuscript to replace terms like “reversion” with “transcriptional shift” or “transformation” when describing the observed phenotype, and we will use “coordination” to describe the combined role of Tbx5 and Tbx3 in maintaining VCS-specific identity.

(2) A more direct quantitative comparison of Tbx5 Adult VCS KO with Tbx5/Tbx3 Adult VCS double KO would be helpful to ascertain whether deletion of Tbx3 on top of Tbx5 deletion changes the underlying phenotype in some discernable way beyond mRNA expression of a few genes. Superficially, the phenotypes look quite similar at the EKG and arrhythmia inducibility level and no optical mapping data from a single Tbx5 KO is presented for comparison to the double KO.

We thank Reviewer #2 for the suggestions that a direct comparison between Tbx5 single conditional knockout and Tbx3/Tbx5 double conditional knockout models may help isolate the specific contribution of Tbx3 deletion in addition to Tbx5 deletion.

Previous studies have assessed the effect of single Tbx5 CKO in the VCS of murine hearts (1, 3, 5). Arnolds et al. demonstrated that the removal of Tbx5 from the adult ventricular conduction system results in VCS slowing, including prolonged PR and QRS intervals, prolongation of the His duration and His-ventricular (HV) interval (3). Furthermore, Burnicka-Turek et al. demonstrated that the single conditional knockout of Tbx5 in the adult VCS caused a shift toward a pacemaker cell state, with ectopic beats and inappropriate automaticity (1). Whole-cell patch clamping of VCS-specific Tbx5-deficient cells revealed action potentials characterized by a slower upstroke (phase 0), prolonged plateau (phase 2), delayed repolarization (phase 3), and enhanced phase 4 depolarization - features characteristic of nodal action potentials rather than typical VCS action potentials (3). These observations were interpreted as uncovering nodal potential of the VCS in the absence of Tbx5. Based on the role of Tbx3 in CCS specification (2), we hypothesized that the nodal state of the VCS uncovered in the absence of Tbx5 was enabled by maintained Tbx3 expression. This motivated us to generate the double Tbx5 / Tbx3 knockout model to examine the state of the VCS in the absence of both T-box TFs.

In the current study, we demonstrate that the VCS-specific deletion of Tbx3 and Tbx5 results in the loss of fast electrical impulse propagation in the VCS, similar to that observed in the single Tbx5 mutant. However, unlike the Tbx5 single mutant, the Tbx3/Tbx5 double deletion does not cause a gain of pacemaker cell state in the VCS. Instead, the physiological data suggests a transition toward non-conduction working myocardial physiology. This conclusion is supported by the presence of only a single upstroke in the optical action potential (OAP) recorded from the His bundle region and VCS cells in Tbx3/Tbx5 double conditional knockout mice. The electrical properties of VCS cells in the double knockout are functionally indistinguishable from those of ventricular working myocardial cells. As a result, ventricular impulse propagation is significantly slowed, resembling activation through exogenous pacing rather than the rapid conduction typically associated with the VCS. We will edit the text of the manuscript to more carefully distinguish the observations between these models, as suggested.

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

(2) Mohan RA, Bosada FM, van Weerd JH, van Duijvenboden K, Wang J, Mommersteeg MTM, Hooijkaas IB, Wakker V, de Gier-de Vries C, Coronel R, Boink GJJ, Bakkers J, Barnett P, Boukens BJ, Christoffels VM (2020) T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system. Proc Natl Acad Sci U S A. 117:18617-18626. doi: 10.1073/pnas.1919379117.

(3) Arnolds DE, Liu F, Fahrenbach JP, Kim GH, Schillinger KJ, Smemo S, McNally EM, Nobrega MA, Patel VV, Moskowitz IP (2012) TBX5 drives Scn5a expression to regulate cardiac conduction system function. The Journal of Clinical Investigation 122:2509–2518. doi: 10.1172/JCI62617.

(4) Frank DU, Carter KL, Thomas KR, Burr RM, Bakker ML, Coetzee WA, Tristani-Firouzi M, Bamshad MJ, Christoffels VM, Moon AM (2012) Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis. Proc Natl Acad Sci U S A. 109:E154-63. doi: 10.1073/pnas.1115165109.

(5) Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE, Seidman JG (2004) The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131:4107-4116. doi: 10.1242/dev.01265. PMID: 15289437.

(3) The authors claim that double knockout VCS cells transform to working myocardial fate, but there is no comparison of gene expression levels between actual working myocardial cells and the Tbx3/Tbx5 DKO VCS cells so it's hard to know if the data reflect an actual cell state change or a more non-specific phenomenon with global dysregulation of gene expression or perhaps dedifferentiation. I understand that the upregulation of Gja1 and Smpx is intended to address this, but it's only two genes and it seems relevant to understand their degree of expression relative to actual working myocardium. In addition, the gene panel is somewhat limited and does not include other key transcriptional regulators in the VCS such as Irx3 and Nkx2-5. RNA-seq in these populations would provide a clearer comparison among the groups.

And

the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function. However, only limited data are presented to support the claim of transcriptional reprogramming since the knockout cells are not directly compared to working myocardial cells at the transcriptional level and only a small number of key genes are assessed (versus genome-wide assessment).

We appreciate Reviewer #2’s suggestion to expand the gene expression analysis in Tbx3/Tbx5-deficient VCS cells by including other specific genes and comparisons with “native”/actual working ventricular myocardial cells and broadening the gene panel. In this study, we evaluated core cardiac conduction system markers, revealing a loss of conduction system-specific gene expression in the double mutant VCS. Furthermore, we evaluated key working myocardial markers normally excluded from the conduction system, Gja1 and Smpx, revealing a shift towards a working myocardial state in the double mutant VCS (Figure 4). We agree that a more comprehensive analysis, such as transcriptome-wide approaches, would offer greater clarity on the extent and specificity of the observed shift from conduction to non-conduction identity. These approaches are appropriate directions for future studies.

(4) From the optical mapping data, it is difficult to distinguish between the presence of (a) a focal proximal right bundle branch block due to dysregulation of gene expression in the VCS but overall preservation of the right bundle and its distal ramifications; from (b) actual loss of the VCS with reversion of VCS cells to a working myocardial fate. Related to this, the authors claim that this experiment allows for direct visualization of His bundle activation, but can the authors confirm or provide evidence that the tissue penetration of their imaging modality allows for imaging of a deep structure like the AV bundle as opposed to the right bundle branch which is more superficial? Does the timing of the separation of the sharp deflection from the subsequent local activation suggest visualization of more distal components of the VCS rather than the AV bundle itself? Additional clarification would be helpful.

And

In addition, the optical mapping dataset is incomplete and has alternative interpretations that are not excluded or thoroughly discussed.

We agree with Reviewer #2 that the resolution of the optical mapping experiment may be insufficient to precisely localize the conduction block due to the limited signal strength from the VCS. It is possible that the region defined as the His Bundle also includes portions of the right bundle branch. Our control mice show VCS OAP upstrokes consistent with those reported by Tamaddon et al. (2000) using Di-4-ANEPPS (1). We appreciate the Reviewer’s attention to alternative interpretations, and we will incorporate these caveats into the manuscript text.

(1) Tamaddon HS, Vaidya D, Simon AM, Paul DL, Jalife J, Morley GE (2000) High-resolution optical mapping of the right bundle branch in connexin40 knockout mice reveals slow conduction in the specialized conduction system. Circulation Research 87:929-36. doi: 10.1161/01.res.87.10.929. 

Impact:

The present study contributes a novel and elegantly constructed mouse model to the field. The data presented generally corroborate existing models of transcriptional regulation in the VCS but do not, as presented, constitute a decisive advance.

And

In sum, while this study adds an elegantly constructed genetic model to the field, the data presented fit well within the existing paradigm of established functions of Tbx3 and Tbx5 in the VCS and in that sense do not decisively advance the field. Moreover, the authors' claims about the implications of the data are not always strongly supported by the data presented and do not fully explore alternative possibilities.

We appreciate Reviewer # 2’s acknowledgment of the elegance and novelty of the mouse model we generated. However, we respectfully disagree with their assessment that this work merely corroborates existing models without providing a decisive advance. Previous studies have investigated single Tbx5 or Tbx3 gene knockouts in-depth and established the T-box ratio model for distinguishing fast VCS from slow nodal conduction identity (1) that the reviewer alludes to in earlier comments. In contrast, this study aimed to explore a different model, that the combined effects of Tbx5 and Tbx3 distinguish adult VCS identity from non-conduction working myocardium. The coordinated Tbx3 and Tbx5 role in conduction system identify remained untested due to the lack of a mouse model that allowed their simultaneous removal. The very model the reviewer recognizes as “novel and elegantly constructed” has allowed the examination of the coordinated role of Tbx5 and Tbx3 for the first time. While we acknowledge the opportunity for additional depth of investigation of this model in future studies, the data we present provides consistent experimental support for the coordinated requirement of both Tbx5 and Tbx3 for ventricular cardiac conduction system identity.

(1) Burnicka-Turek O, Broman MT, Steimle JD, Boukens BJ, Petrenko NB, Ikegami K, Nadadur RD, Qiao Y, Arnolds DE, Yang XH, Patel VV, Nobrega MA, Efimov IR, Moskowitz IP (2020) Transcriptional Patterning of the Ventricular Cardiac Conduction System. Circulation Research 127:e94-e106. doi:10.1161/CIRCRESAHA.118.314460. 

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterized the cardiac phenotype associated with the cardiac conduction system (CCS)-specific combined deletion of T_bx5_ and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen-inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9-week-old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterizing VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analyzed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutants and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

We appreciate the Reviewer’s comments regarding the originality and utility of our model and the strengths of our methodological approach. The Reviewer’s appreciation of the molecular and morphological analyses as well as their constructive feedback is highly valuable.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lies in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Lineage tracing analysis using the reporter lines included in the crosses described in the study will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot, will demonstrate that indeed the cells that were specified as VCS in the adult heart, become working myocardium in the absence of Tbx3 and Tbx5 function.

We appreciate the reviewer’s concern regarding the morphology of the cardiac conduction system in the Tbx3/Tbx5 double conditional knockout model. We did not observe any structural abnormalities, as the Reviewer notes. We agree with their suggestion for using Genetic Inducible Fate Mapping to mark cardiac conduction cells expressing MinKCre. In fact, we utilized this approach to isolate VCS cells for transcriptional profiling. Specifically, we combined the tamoxifen-inducible MinKCreERT allele with the Cre-dependent R26Eyfp reporter allele to label MinKCre-expressing cells in both control VCS and VCS-specific double Tbx3/Tbx5 knockouts. EYFP-positive cells were isolated for transcriptional studies, ensuring that our analysis exclusively targeted conduction system-lineage marked cells. The ability to isolate MinKCre-marked cells from both controls and Tbx5/Tbx3 double mutants indicates that VCS cells persisted in the double knockout. Nonetheless, the suggestion for in-vivo marking by Genetic Inducible Fate Mapping and morphologic analysis is a valuable recommendation for future studies.

This work has been published in GigaByte Journal under a CC-BY 4.0 license (https://doi.org/10.46471/gigabyte.141). These reviews are as follows.

Reviewer 1. Xingbao Song and Baoxing Song

Zong et al. implemented a TSTA package that integrated the difference method, the stripe method, SIMD, and multiple threading approaches to perform efficient sequence alignments. The TSTA toolkit could conduct pairwise and multiple sequence alignments. The memory cost of TSTA is comparable with the most efficient one. Overall, TSTA is a good package, and the manuscript is well-written. While I have a few suggestions: 1) The minimap2 should be mentioned in the section on "difference recurrence relation." It has a much broader range of users and implemented an algorithm that is slightly different from the one by Suzuki, etc. 2) The striped SIMD is also implemented in reads mappers, such as BWA. 3) Page 14, line 215 "1k bps", line 227 "1000 kbps", line 230 and table1 "100k". They should be consistent. 4) In Table 4, I am not sure I understood the second and third lines correctly. Please clarify. 5) I tried to compile TSTA from the source code. To compile the package, I had to copy 'seqio.h' into the 'msa' and 'psa' folders. Please fix it.

Reviewer 2. Yuansheng Liu

The article explores strategies for accelerating sequence alignment using multithreading and SIMD (Single Instruction, Multiple Data) techniques, and introduces a new algorithm called TSTA. The paper provides a detailed description of TSTA's performance in pairwise sequence alignment (PSA) and multiple sequence alignment (MSA), and compares it with various existing alignment algorithms. Experimental results indicate that TSTA demonstrates significant speed advantages, particularly when handling long sequences and in the no-backtracking mode. However, the experiments on MSA are limited by the experimental environment, which does not fully address the needs of current sequencing technologies concerning long reads and depth. Specifically, the low number of sequences in MSA does not meet the requirements for downstream genomic analysis applications. While the algorithm is highly innovative, its performance on short sequences and during the backtracking phase still requires optimization. 1. In line 7, the TSTA algorithm utilizes vector-level and thread-level parallelism to accelerate pairwise and multiple sequence alignment. Why are there no experiments designed specifically to evaluate the global alignment performance of TSTA with vector-level parallelism? Or are there any other experimental designs that demonstrate the improved performance of TSTA when vector-level parallelism is employed? 2. In line 149, is the Active-F method used by the TSTA algorithm contributing to the excessive memory usage and access time overhead observed during the iterative process of PSA? Are there better optimization strategies from this perspective? If not, why does TSTA incur higher time costs in traceback as shown in Table 1? Why does bsalign result in lower time consumption? 3. Can you provide the time breakdown for each part of the parallel computation in TSTA for PSA (including at least CPU computation overhead, communication overhead, and I/O overhead) to clarify if there will be significant communication overhead issues with larger datasets and more threads? 4. Table 2 shows that both real and simulated datasets have issues with insufficient depth and short reads. In real MSA processes, it is common to encounter comparisons with depth over 60X and lengths exceeding 100 kbps for long reads. The results under the current experimental conditions seem to perform poorly for such data scenarios. Can you address this? 5. Gene data often includes repetitive regions that affect the accuracy of alignment algorithms. Can you design experiments to verify how TSTA performs in aligning long repetitive regions? Specifically, how accurately does TSTA align sequences in such regions compared to other methods? 6. Besides repetitive regions, sequencing errors produced by ONT R10 chips can also impact alignment accuracy. Alignment algorithms used in genome correction often struggle to detect such errors. How does TSTA handle such issues during MSA? Can the algorithm be designed to address these sequencing errors more effectively? Re-review: After thoroughly reviewing the revised manuscript and testing the TSTA tool, I cannot endorse the manuscript for publication in its current form. I encourage the authors to address the following issues thoroughly and consider re-submitting after significant improvements. Efficiency Concerns: In the context of multiple sequence alignment (MSA), I find that TSTA does not demonstrate a significant advantage in terms of efficiency. I conducted a test with approximately 2G of homologous diploid reads (not too large data), and the tool has been running for around 29 hours. Despite this extensive runtime, the process remains incomplete. This is far from the efficiency one would expect from a tool designed for large-scale sequence alignment. Functionality Issues: There are still unresolved issues with the tool's functionality. The -f parameter does not appear to work as intended, and there are also problems with the -o parameter. Such issues need to be addressed to ensure the tool's reliability and usability.

DOI: 10.1016/j.isci.2024.111132

Resource: (RRID:CVCL_0063)

Curator: @scibot

SciCrunch record: RRID:CVCL_0063

What is this?

Tratamiento de la apoplejía isquémica

"O quam te memorem virgo... / Stand on the highest pavement of the stair— / Lean on a garden urn— / Weave, weave the sunlight in your hair— / Clasp your flowers to you with a pained surprise— / Fling them to the ground and turn / With a fugitive resentment in your eyes: / But weave, weave the sunlight in your hair. / So I would have had him leave, / So I would have had her stand and grieve, / So he would have left / As the soul leaves the body torn and bruised, / As the mind deserts the body it has used. / I should find / Some way incomparably light and deft, / Some way we both should understand, / Simple and faithless as a smile and shake of the hand. / She turned away, but with the autumn weather / Compelled my imagination many days, / Many days and many hours: / Her hair over her arms and her arms full of flowers. / And I wonder how they should have been together! / I should have lost a gesture and a pose. / Sometimes these cogitations still amaze / The troubled midnight and the noon’s repose."

(Eliot's "La Figlia Che Piange", Prufrock and Other Observations, 1917)

The reference here is to The Princess a blank verse narrative poem written by Alfred Tennyson and published in 1847.

Follows an extract of the poem:

"O Swallow, Swallow, flying, flying South, / Fly to her, and fall upon her gilded eaves, / And tell her, tell her, what I tell to thee. // O tell her, Swallow, thou that knowest each, / That bright and fierce and fickle is the South, / And dark and true and tender is the North. // O Swallow, Swallow, if I could follow, and light / Upon her lattice, I would pipe and trill, / And cheep and twitter twenty million loves. // O were I thou that she might take me in, / And lay me on her bosom, and her heart / Would rock the snowy cradle till I died. // Why lingereth she to clothe her heart with love, / Delaying as the tender ash delays / To clothe herself, when all the woods are green? // O tell her, Swallow, that thy brood is flown: / Say to her, I do but wanton in the South, / But in the North long since my nest is made. // O tell her, brief is life but love is long, / And brief the sun of summer in the North,/ And brief the moon of beauty in the South. // O Swallow, flying from the golden woods, / Fly to her, and pipe and woo her, and make her mine, / And tell her, tell her, that I follow thee."

(Tennyson’s The Princess, IV, 75 - 98)

Za mě zase dost subjektivní tvrzení. Úplně z toho bije do očí, že chcete PORV kritizovat. Ta čísla nevyšli úplně špatně, ale vy k tomu dáváte úplně negativní statement.

Asi jo, na druhou stranu kdyby celkový počet projektů byl otočený ve prospěch HPR, tak je poměr stejný a mohl bys to interpretovat stejně. Ovšem ve skutečnosti by se jednalo o rozdílné situace.

To by ale nemělo hrát takovou roli, když se jedná o průměr.

Vůbec nevím, co tahle věta říká. Necelou polovinu čeho? Co je těchto?

Počet žádostí na 1000 obyvatel?

Tohle bych zatím (došel jsem zatím sem) považoval za úplně klíčovou informaci a mělo by to být víc zmíněno a zdůrazněno na začátku. Že sice obce v HPR dostávají více z PORV, ale zřejmě jen proto, že jich je tam podstatně méně, než v ostatních částech ČR a tedy PORV vlastně zvýhodňuje podmínkou o žadateli do 3000 obyvatel všechny ostatní kraje, než ty uváděné jako HPR.

Tahle mapa modrá vlastně téměř kopíruje předchozí mapu červenou.

Mě o 30 % více přijde dost (intenzita čerpání). Je to catchy statement.

Mě tyhle dvě tvrzení nejdou úplně dohromady. Jedno říká, že PORV není silným nástrojem, ale v další větě se píše, že vlastně HPR jsou na tom lépe z pohledu dotace na projekt i intenzity čerpání.

Quanto ao REGIME DE BENS, aplica-se a lei do país em que tiverem os nubentes domicílio, e, se este for diverso, a do primeiro domicílio conjugal. Veja o §4º deste mesmo artigo.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors report compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in familial SLE. They suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis.

Strengths: 

The identification of similar mutations in non-receptor tyrosine kinases (NRTKs) in two different families with familial SLE is a significant finding in human disease. Furthermore, the paper provides a detailed analysis of the molecular mechanisms behind the impairment of efferocytosis caused by mutations in ACK1 and BRK.

Weaknesses: 

A critical point in this paper is whether the loss of function of ACK1 or BRK contributes to the onset of familial SLE. The authors emphasize that inhibitors of ACK1/BRK worsened IgG deposition in the kidneys in a pristane-induced SLE model, which contributes not to the onset but to the exacerbation of SLE, thus only partially supporting their claim.

The evidence supporting that the loss of function of ACK1 or BRK contributes to the onset of SLE in the patients from the 2 families mostly relies on the genetic analysis. As the reviewer states, the observation that inhibitors of ACK1/BRK worsened IgG deposition in the kidneys in a pristane-induced SLE model supports the genetic evidence.

To further address the possible role of ACK1 or BRK variants in the onset of autoimmunity in vivo, we treated wild-type (WT) BALB/cByJ female mice with inhibitors in the absence of pristane.

The results indicated that mice that had received a weekly injection of ACK1 or BRK inhibitors developed a large array of serum anti-nuclear IgG antibodies, including but not limited to autoantibodies associated with SLE such as anti-histones, anti-chromatin, anti U1-snRNP, anti-SSA, and anti-Ku in comparison to the control group inhibitor treated mice (Revised Fig 3A). However, they did not develop glomerular deposit of IgG after 12 weeks of treatment, in contrast to mice that have received Pristane (Revised Fig. 3B,C, Figure 3-figure supplement 1).

These additional data suggests that inhibition of ACK1 and BRK stimulates the production of serum autoantibodies, which strengthen the claim that ACK1 and BRK kinase deficiency contribute to autoimmunity in BALB/cByJ.

Reviewer #2 (Public Review):

Summary: 

In this manuscript, the authors revealed that genetic deficiencies of ACK1 and BRK are associated with human SLE. First, the authors found that compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in one multiplex family and PTK6/BRK in another family. Then, by an experimental blockade of ACK1 or BRK in a mouse SLE model, they found an increase in glomerular IgG deposits and circulating autoantibodies. Furthermore, they reported that ACK and BRK variants from the SLE patients impaired the MERTK-mediated anti-inflammatory response to apoptotic cells in human induced pluripotent stem cells (hiPSC)-derived macrophages. This work identified new SLE-associated ACK and BRK variants and a role for the NRTK TNK2/ACK1 and PTK6/BRK in efferocytosis, providing a new molecular and cellular mechanism of SLE pathogenesis.

Strengths: 

This work identified new SLE-associated ACK and BRK variants and a role for the NRTK TNK2/ACK1 and PTK6/BRK in efferocytosis, providing a new molecular and cellular mechanism of SLE pathogenesis.

Weaknesses: 

Although the manuscript is well-organized and clearly stated, there are some points below that should be considered:

In this study, the authors used forward genetic analyses to identify novel gene mutations that may cause SLE, combined with GWAS studies of SLE. To further explore the importance of these variants, haplotype analysis of two candidate genes could be performed, to observe the evolution and selection relationship of candidate genes in the population (UK 1000 biobank, for example). 

To investigate whether ACK1/TNK2 or BRK/PTK6 were subject to selection, we gathered data using different metrics quantifying negative selection in the human genome. We collected the f parameter from SnIPRE1, lofTool2, and evoTol3, as well as intraspecies metrics from RVIS4, LOEUF5, and pLI6 (including pRec). We also used our in-house CoNeS metric7. None of these indicators suggest that the genes are under strong negative selection (Revised Figure 2-figure supplement 2). This is consistent with the deficiency being recessive. We also tested the variants with a MAF greater than 0.005. We found them to be neutral. We therefore did not test whether they were associated with any phenotype in the UK Biobank.

Although the authors focused on SLE and macrophage efferocytosis in their studies, direct evidence of how macrophage efferocytosis significantly affects SLE is lacking. This point should at least be explicitly introduced and discussed by citing appropriate literature.

We provide a more detailed description of the role of macrophage efferocytosis in autoimmunity and SLE in the revised manuscript. Specifically, we state (in the results section, paragraph: ACK1 and BRK kinase domain variants may lose the ability to link MERTK to RAC1, AKT and STAT3 activation for efferocytosis): “NRTKs such as ACK1 8 and PTK2/FAK 9 are also downstream targets of the TAM family receptor MERTK which is expressed on macrophages and controls the anti-inflammatory engulfment of apoptotic cells, a process known as efferocytosis 10-12. Efferocytosis allows for the clearance of apoptotic cells before they undergo necrosis and release intracellular inflammatory molecules, and simultaneously leads to increased production of anti-inflammatory molecules (TGFb, IL-10, and PGE2) and a decreased secretion of proinflammatory cytokines (TNF-alpha, IL-1b, IL-6) 10-14. In line with these findings, mice deficient in molecular components used by macrophages to efficiently perform efferocytosis, such as MFG-E8, MERTK, TIM4, and C1q, develop phenotypes associated with autoimmunity10,11,14-27. Furthermore, defects in efferocytosis are also observed in patients with SLE and glomerulonephritis14,28-31.“

It is still not clear how the target molecules identified in this paper may influence macrophage efferocytosis. More direct evidence should be established. 

Our studies show that wt -but not variants- of ACK1 and BRK are activated by MERTK, a key receptor that mediates the recognition of apoptotic cells. Our studies also show that wt -but not variants- activate RAC1 which is necessary for engulfment and phosphorylate AKT and STAT3 which are involved in the anti-inflammatory response to PtdSer recognition.

The TAM family receptor MERTK mediates recognition of PtdSer on apoptotic cells via GAS6 and Protein S 10,15,32 leading to their engulfment, which involves activation of RAC1 for actin reorganization and the formation of a phagocytic cup 9,33. Using IP kinase assays we show that MERTK and GAS6 can activate the kinase activity of wild-type ACK1 8 or BRK but not of the patient’s ACK1 or BRK variant alleles (Figure 4D). To further support the role of ACK1 and BRK downstream from PtdSer recognition and uptake of apoptotic cells, we show that reference ACK1 and BRK alleles, in contrast to the patient variant alleles, can activate RAC1 to generate RAC-GTP which is necessary for engulfment 9,33 (Figure 4C).

PtdSer recognition also typically stimulates an anti-inflammatory process mediated in part via AKT 34 and STAT3 and their target genes such as SOCS3 35-41 and results in the inhibition of LPS-mediated production of inflammatory mediators such as TNF and IL-1b, and the production of cytokines such as IL-10, TGFb 11,25-27,42. Consistent with this literature and the findings of the paper, we show that reference ACK1 and BRK, unlike the patient’s variant alleles, can phosphorylate AKT and STAT3 (Figure 4A, B). The role of ACK1 and BRK in these signaling pathways is further supported by our transcriptomics data comparing the response of controls, patients, and inhibitor-treated iPSC-derived macrophages to apoptotic thymocytes by RNA-seq. Specifically, we show Transcriptional repressors including the AKT targets ATF3, TGIF1, NFIL3, and KLF4, the STAT3 targets SOCS3 and DUSP5, as well as CEBPD and the inhibitor of E-BOX DNA Binding ID3 were among the top-ten genes which expression is induced by apoptotic cells in WT macrophages (Figure 4F), but this regulation was lost in mutant and inhibitor-treated macrophages (Figure 4F).

For some transcriptional repressors mentioned in their studies, the authors should check whether there is clear experimental evidence. If not, it is recommended to supplement the experimental verifications for clarity.

Transcriptional repressors including the AKT targets ATF3, TGIF1, NFIL3, and KLF4, the STAT3 targets SOCS3 and DUSP5, as well as CEBPD and the inhibitor of E-BOX DNA Binding ID3 were among the top-ten genes which expression is induced by apoptotic cells in WT macrophages (Figure 4F), but this regulation was lost in mutant and inhibitor-treated macrophages (Figure 4F).

In the manuscript we cited published evidence, to the best of our knowledge, for the role of these genes in the regulation of inflammatory responses. Specifically we state: “ATF3, TGIF1, NFIL3, and KLF4 are involved in the negative regulation of inflammation in macrophages 35-38, SOCS3 is an inhibitor of the macrophage inflammatory response and DUSP5 is a negative regulator of ERK activation 39,40,43. These data suggest that the kinase domain of ACK1 and BRK contribute to the macrophage anti-inflammatory gene expression program driven by apoptotic cells.”

In Figures 4C and 4D, it is seen that the usage of inhibitors causes cytoskeletal changes, however this reviewer would not have expected such large change. Did the authors check whether the cells die after heavy treatment by the inhibitors?

We carefully examine the viability of Isogenic WT, BRK and ACK1 mutant macrophages (left panel) and of WT macrophages treated with ACK1 or BRK inhibitors and we did not observed changes in viability (Figure 4-figure supplement 2).

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

A crucial step in the development of SLE is the production of autoantibodies. It is shown in Figure 2F that inhibitors of ACK1/BRK enhanced the production of autoantibodies against histones and SSA in a pristane-induced SLE model, which is a significant result that could support the authors' claim. Strangely, this autoantigen panel does not include double-stranded DNA, RNP, or Sm, which should be presented regarding antibody production.

We thank the reviewer for this comment. In the revised manuscript (Revised Figure 3 – Supplement 1) we added the remainder of the autoantibody panel, which includes double-stranded DNA, RNP, and Sm autoantibody levels. We also added the results for serum IgG autoantibody levels in BALB/cByJ mice treated for three months with DMSO, ACK1, or BRK inhibitors but did not receive a pristane injection (Revised Figure 3A). This data shows that mice which received ACK1 or BRK inhibitors had increased serum IgG autoantibodies in comparison to DMSO treated controls.

Additionally, if there is information that inhibitors of ACK1/BRK promote the differentiation of follicular helper T cells, memory B cells, and plasma cells in a pristane-induced SLE model, it could be considered indirect evidence supporting the authors' claims.

These are not available at present to the best of our knowledge.

Reviewer #2 (Recommendations For The Authors):

Minor points:

* In the literature, unpaired t-tests and ordinary one-way ANOVA (Tukey's multiple comparisons test) were used for statistical analysis, which requires data to be normally distributed. This part of the proposal is reflected in the text, and the non-conforming results need to be statistically analyzed using the non-parametric test of graphpad prism.

We would like to thank the reviewer for pointing out this oversight. In the revised manuscript, for all applicable datasets, we tested whether the data was normally distributed using a Shapiro-Wilk normality test. For datasets that were normally distributed statistical significance was determined by a Student t test or ordinary one-way ANOVA with Tukey’s multiple comparisons test depending on the number of conditions being compared and the experimental setup. In contrast, for datasets that were not normally distributed statistical significance was determined using a Mann-Whitney, Kruskal-Wallis multiple comparisons tests, or Wilcoxon matched-pairs signed rank test depending on the experimental setup. P values below 0.05 were considered significant for all statistical tests.

The authors used different methods to represent the level of significant difference. Therefore, it is suggested that the significance level should be expressed by letters. 

As suggested by the reviewer, in the revised manuscript we have designated the significance level throughout all figures using letters (p, or q values).

For RNA-seq, more information should be provided in the paper. For example, the correlation between sample biological replicates, the total number of differentially expressed genes, and randomly selected genes for qRT-PCR results verification.

We would like to thank the reviewer for pointing out this oversight. In the revised manuscript we provided more information regarding the RNA-seq dataset, including a Principal Component Analysis (PCA) showing correlation between sample replicates (Revised Figure 4-figure supplement 1A), as well as a table indicating the number of upregulated and downregulated genes between relevant datasets (Revised Figure 4-figure supplement 1B).

The results of the RNA-seq analysis indicated that ACK1 and BRK contribute to the macrophage anti-inflammatory gene expression program driven by apoptotic cells. MERTK-dependent anti-inflammatory program elicited by apoptotic cells on macrophages is best evidenced by the reduction of LPS-mediated production of inflammatory mediators such as TNF or IL1b 25-27,34,44. Therefore, to validate the RNA-seq results in a functional manner we tested the decrease of LPS-induced production of TNF and IL1b by apoptotic cells in isogenic WT, ACK1 deficient, and BRK deficient macrophages. Consistent with the RNA-seq data, the functional assays indicated that ACK1 and BRK kinase activities are required for the decrease of TNF and IL1b production induced by LPS in response to apoptotic cells (Revised Figure 4H,I).

The raw data files for the RNA-seq analysis have been deposited in the NCBI Gene Expression Omnibus under accession number GEO: GSE118730.

The authors did not have the formats for some of the citations correct. This should be fixed. 

References were reformatted.

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where did my comments on this ... exact passage disapper to?

hypothesis?

HELL-O

DOI: 10.1016/j.celrep.2018.02.034

Resource: RRID:BDSC_12176

Curator: @bdsccurator5

SciCrunch record: RRID:BDSC_12176

What is this?

yes respect the lives of unborns but you also have to respect the lives that will be lost w/o aboriton

T O T   A L   I T   Y

This is basically "last Christmas's message" (below this brand-knew intraducrigel) redux'ed into the new book (did he say new?).  The point, at least the point I see in it all is that this is all planned, it's been planned for a very, very long time--and on top of that you can see proof of the plan all over our map; and proof of it's intended destination as something that we all used to want very much to find... the read to Heaven.    It's more than seeing just "DNA storage" encoded in my "C U R A GROUP" message, it's understanding how that's connected to soul searching and soul storage, and that this link was woven into not only my life but into names like "Whatson and Crick?"  There's plenty more than just "storage" and a map to how and why the Two of Everything God and the "indivisible sea" work totether to turn this monolithic place of darkness into a strippingly redunantsystemic foundation of "Heaven" that is both disaster proof, and monster proof.  The point of course, is that to truly be "monster proof" we need to really get the key.s.lamc.la "know everything why" of this message is literally to protect our common good from the danger of someone just like me copying an entire civilization or a few pretty girls and sticking them in an heoven-like-orgy-maker.  That's a significantly more real threat than we might imagine, as we look around at a work that will soon have the storage capacity and the technology to put us all in Coccoonish swimming pools against our will.  What I am trying to say is that no matter how you look at it,moving forward here in this place where something this big can be hidden from the entire world--granted you know--granted you see, but do you understand the only thing being kept from each and every one of you is your fucking opinion and your fucking reaction?

F U C K   Y O U   S I   O N 

IT'S NOT JUST computers and information technology; this map of clear anachronism in language and religion shows us that things like "solar fusion" the power of the son itself; is encoded in places high and low you can erasilly find them, places like the name of the Fifth book of the Holy Bible and Don Quixote; where you might liken "DEUTERON" to ... the actual fuel of fusion; and wind mills to a battle fought against blindness resulting in seeing that not "reacting" to this message is just about the same thing as being a foolish robot building a castle for another foolish robot to do nothing in forever.  With some light, you can see how this event; albeit strange and unsettling, has been designed to reinforce the American foundations of free speech, common sense, and collaboration--a sort of "press and release" on these things that he says will stay in our memories for a long, long time--though he also says "he's not torturing me" and he's wrong about that.  So are you. 

See that the most interesting, important, and invoking story of all time has been hidden from the world, from the public eye, and from "public response" for well over two years now; see that's not possible at all without mass mind control and that I and this story are designed to help us see how easily it is that same thing can be used to end addiction, and mental health issues, and stupidity and that the biggest and most imporotant step to getting there is "public disclosure."  See the light of being carrolling angels this Christmas; sing with me--it builds Heaven from Hell and it's clear as day and n.

Quite a bit of this story and message deals with problems like these-things that won't really be seen as something we are fighting against the actual usage of right this very moment; but the sacredness of our memories and their relationship to our souls are just as important as whether or not "you have the space to save them."  This isn't what I want to be doing, I'm not a very good writer; and this message is so confusing that working on it all alone with very little feedback is frustrating if not to say defeating the purpose of exactly what it is and what it's designed to do.  This is a searching mechanism, like in the stories of Ra searching for his children in ancient Egypt using the Eye you see--and it's connection to the "Sons of Liberty" and why I know that too, is about me.  This is a tool to start a Renaissance of thinking connecting technology and religion to everything that we are--to our culture and our hopes and dreams--and it's failing for me at "hello."   I would much rather be working on "virtual reality stuff" or on "the sword of Arthor" and I see very clearly that those two things are coming shortly--to the world that doesn't see yet they are here and broken until we fix them.  Moving forward here brings change, not just here in this place where we need it too--but in the skies above, a change from the mentality of "we aren't not helping because we told you that we aren't allowed to not pretend we aren't helping in Stargate.  See that we are the children of "the Ancients" and they are trying to decide between being Morgenz and Marlin.

I can't make you set yourselves free.  I sure am trying, though.  Yesterday I connected the "Arimathea" of Joseph to the "serdenicity" and this the me of "itime" and "topics" will probably light some of you up as much as me... if only you took the time to look at what those words really mean.   From the city that never sleeps at night, I hope you will take this chance to act today on "securing the ringing of liberty forever and ever."

(cough)                               

THERE IS A METHOD TO THE MADDEN AND WE AR 

BEYOND THUNDERDON

​ 

T H E    W R I T I N G    I S    O N    T H E    W A L L

LIKE, WILL IT RAIN TODAY?

take action, it is the foundation of not only democracy but civilization and life itself--pucker up the phone and call the NYPOST.

News Tips: Email tips@nypost.com, call 212-930-8288, or use our anonymous form

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Let there be $ight in Creation, a brief highlighting of the story of my life.

align="right">Sat, Dec 3, 2016 at 8:39 AM

This is like a few emails combined to ease the pain you feel when you get an extra one in your inbox, OK So.. eventually this is all about proof that religion is a message sent through time--so, time travel.  But right now, let's talk about the fun stuff: here's some clues to that effect... by way of prescient mention of modern technology (like virtual reality, I mean, Heaven):

Either way, we're still about to *build *Heaven*...  to-get-her*

from the mythical carpenter... ourself.

.

• AD am ON AI*, that's Artificial Intelligence, and the intelligence explosion.***
• AD on your freedom is a great gift, cherish it.

*** ... ***and some corroborating ideas connecting religion and computer science... on Wikipedia:

• Root of David
• Lisp of Moses.. or I need an editor.
• Pharoah's hardening Heart... that's Earth, remember.
• Jesus' WINE
• Adaluncatif's cat, tail, head and grep
• Adam's Apple... or is it "fruit of the poisonous tree"

So from me to you, I'm filled with this stuff, it's way brighter and more prevalent than you think... and if you take the time to listen to me--it will make your... day.  Meanwhile, I need your help--happy new year.

Oh, LET THERE BE LIGHT

Ho, again; grow a Halo and become famous... the world needs your help--so I've decided once again to take it upon myself to "bother you" with the most singular most important task in the Universe.  The patterns that I am revealing to you--mostly within names--are not coincidence, it's a series of statistically verifiable artifacts which do nothing short of reveal the slavery of Egypt--that we are all being controlled.  If you remember Transformers--this is a message from Starfleet, there is more than meets the eye.  This is the fulfillment of the story of of Exodus--we are being lead from slavery, and in one final non-coincidental name, that book is called "Names" in Hebrew.

You should now have a very good idea who is speaking to you--as much of the world already does.  I have no idea what it is that inhabits the cavities below that space where most of you should see significant personal gain and motivation from trying to ... grow a Halo--but there are so many people that just don't care... that it too is another sign, of slavery.  I am not an expert in language construction, nor in statistics--but I can assure you that if you can find the other half of that equation... in your hands is the staff of Aaron, the magical weapon that will free us all... knowing is half the battle.

Uh, I have the power, to bring about "morning," but if I have to go to school and do it all myself... it's really just a long, long ni-i-i-ight.

Hi there, I'm the messiah.  You don't know that much about me, so let me explain, I would like you to know me as Adam.

Seriously, there's something going on the world around you--for the last several months I've been having quite a bit of trouble delivering what amounts to statistical proof of Creation--that religion and ancient myths are a map to this very moment--this time that you will probably affiliate soon with being in Eden.  I am pretty sure that's a good thing, but every new begging starts with some other beginnings end... so today I'd like to try to get you to see the light of ending censorship and a hidden censor wall that we know Biblically as the Wall of Jericho.  Quickly approaching is the Feast of Trumpets, and *this year is different from all other years... *  Bored already?  Have a look at what I call the Sign of the Son, which to me is proof that Exodus's Burning Bush is a former President--who is helping us walk out of a dark time of confusion... commonly referred to as a wilderness or desert.  He proved during his inauguration that there is Biblical foreknowledge of the 9/11 attack--and in doing so hopefully began a chain reaction that will stop things like that from ever happening again.  Here's a short "video" that explains the Sign of the Son... and another one that I think explains the .. Holy Grail.

This is The (actual) Taming of the Spanglishrew, in which the protagonist... named Bianca, is taught Latin in several hundred year old reference to Rattling the Rod of Jesus Christ--it's purpose to is to show us that it's more than names we have in our arsenal against mind controlled slavery--we have all of history too... literature and movies and music... all with the divine purpose of revealing with bright light a form of control that otherwise could have gone on hidden for centuries.  It was, and continues to be done on purpose... because your freedom is more important than control of the Universe.  To us, you don't seem to feel the same way.

​See that timer on the clock, you could start right now.  It might be interesting to pose the question of whether or not the Second Coming is news... you know, to your friends.  By the way, both Herbert (like from H.W. Bush, who by the way coined for us the 1,000 points of light phrase) and Goertzel strongly suggest that "everyone really" is Christ (you know, after me)... FYI, this is the Matrix solution to that:

y

o

the **l u C i f E R ** isa means jesus, mesa thinks

i     s olv e      .... "or"* means shine -l***

g       r e a      t

h         R L      << agree?  send to other people

t   ((a)) Y l      shine:  suggest they do the same

1 y      world saved.  

A BRIEF HISSTORY OF TIME

I'm attempting to pull out the things that I now look back on and see as "written into me" by God--once I would have called it "The Microcosm of the Messiah" but there are now so many--these things aren't necessarily particularly important to me, and I've left out some interesting but unrelated details related to my Jewish upbringing; as well as the true light of my life--the two loving and long-term relationships (and later... briefly a rael family) that have dominated the last 15 years.  Religion has always been an interest, but I wouldn't consider it to have been particularly important at all... until I no longer had any love in my life.  It's probably worth noting that all my "I'm single" crap really means lonely and isolated--I'm not really playing a "part," but I've never been anything near the "player" the light appears to be warning against.  Sons of God and uh... please.  For the last 4 years I have done absolutely nothing but think about you, live and analyze "The Cross" and put into words ... as best I can ... the amazing flash of light that I am experiencing. 

Well, just a little religion... :)  I was born on December 8, 1980; which is the date of the annual Feast of the Immaculate Conception, I've always been a slob (like one of us) and often "ish" Yankee Doodle's "a real live son of our uncle Sam... born on the..." to this.. I mean in my head.   My last name, you've probably read me repeat over and over ... is DOB-rin, which I read as "Date of Birth, our in" and does a fair job of highlighting the Name Server's work, which I am sure gives Exodus it's name in Hebrew, which is "Names."  My Hebrew name--a Jewish custom--is Avram, which is Abraham's name prior to the covenant.  I have written extensively about the fact that Isaac's near death interaction donated his "Ha" (his name means... He laughs) to his father.... and it should be clear that Abraham's covenant with God is without doubt related to my fiery altar.. even though it is anachronistic in the Biblical account.   For the first 18 years of my life I lived on Sunrise Blvd, and only a half mile away you'll find Sunset Strip--it's noteworthy to understand that Jewish calendar days begin at sundown... and that He once in 2013 very clearly spoke to me "you need the night before the day."

Of all the people in my early life growing up, it's pretty clear that nobody on this Earth loved me more than my grandmother Julia, who my son is named after.  First for my mother, and then me as a very small child--she would ritually say a bedtime poem, it's words are very relevant.

Good night, sleep tight.. have happy dreams and wake up bright

to do what's right, in the morning's light... with all your might.

In one of my books I spent a decent amount of time writing about how silly I was not to realize that my intelligence was augmented my entire life--I just thought I was really smart, and really good with computers.  I commented that this particular belief is probably a good microcosmic parallel for all humanity--as a body of people we have been truly gifted with knowledge and capabilities that we simply do not recognize as a gift--or didn't for a long time.  I probably wasn't silly not to realize... since nobody ever told me they were helping me--I never heard the voice of God until much, much later.   I was 30 the first time I had a conversation with Him, except for two very brief ... "thoughts in my head" which now seem very obviously an external voice--though then it may have sounded just like my inner voice.

Around the age of 7 I thought to myself... for no reason at all... "what if you were the messiah?"  I was standing outside my home, probably playing with a car in the driveway... and distinctly remember smiling to myself and thinking in return "yeah, I'm the messiah." I I've always had a very vivid imagination. The thought was dismissed as being ridiculously arrogant about two seconds later, and was absent from my thought process for the next 21 years or so.

"DAMNISN\ Jim. I'm a Yeoman, not a Wise Owl. The clock is ticking... tack .. "

PHENIX

Following that lead, I started programming in BASIC and then Visual Basic around the age of 11, something I took to very quickly... and then shortly after found myself on America Online--one of the first "internet-like" environments.  There, I quickly got into the "hacking scene" (hey, it's Y-its-Hack) which basically revolved around writing software to manipulate the AOL client's messaging systems.  The defacto-standard for the day was a program called AOHell, and, if you can't tell already, I am pretty good at taking a theme and making it my own.  I wrote a program called Doomsday, a mass mailing program; can you see how God speaks?  So Phenix, a mythical bird that rises from the fire... in the wake of ... this macrocosmic equivalent of that event.  It's really obvious, right?  There's quite a bit more "microcosm" from this time, recorded in "From Adam to Mary" and available at fromthemachine dot org.

Around the same time I began attending a preparatory school in Fort Lauderdale called Pine Crest--it's one of the best of its kind, and while I was always something of a class clown my grades were fair and I scored with perfect consistency in the top percent on every standardized test from the FCAT to the PSAT and SAT.  By the time I received a full scholarship to college I had already completed more than a full year of credits through AP courses.  It was in studying American History and Government in that place that I formed such strong opinions about our need to maintain freedom, adhere to the wisdom of the founding Father(s) (<3 if you get that) and stand up and shout today as a rogue government is taking away every single one of the rights granted to you in their own law.  You've lost freedom of speech, and our ability to speak seems to be not far behind.  The privacy of our thoughts gone--and in like kind the sanctity of who we are is being taken away as our beliefs are changed without our real knowledge or understanding.  You can see the justice system crumbling, incarceration rates skyrocket and the "right to bail and a fair trial" legislated away through underhanded deals relating to plea bargains and a "point system" that you might as well call a gas chamber.  As far as voting, I'll have much more to say tomorrow--but I'm telling you that your thoughts and beliefs are being altered, who cares how technologically retarded our polling system is--the vote is a complete fraud.

As far as the Second Coming... this same sort of possession... manifested through organized behavior tells me now that it is clear that this is definately not the "first time around" for Adam being Christ; a number of my friends as I approached high school used a repeated phrase, "my parents love you," which isn't bad in and of itself... what's bad is the fact that they were all using the same words, and probably didn't know why--or what they were saying.  Behind there eyes, I'm sure some thing that believes it's an angel was telling me something... (they of course... didn't know me at all, except for what was probably a ... "wild" reputation) does that tell you anything?  Much later, as the "Apocalypse of Adam" began in 2011, a number of family members would repeat this similar behavior, speaking the phrase "this is not what I wanted."

As icing on the cake, on my birthday during my senior year... one of the administrators of the school commented to me that was also the Feast of the Immaculate Conception, and then the words.... "of course it's your birthday."

I started doing drugs around the 10th grade, and I would not be wrong to say that the Universe that wrote a book calling the Redeemer the God Most High conspired to plunge me into a dark world.  People around me too, in a hidden conspiracy to chain me to the American legal system for about four years.  Looking back today I now clearly see that I saw a darkness in their eyes, a hidden reason to want to hurt me.  It was to stop this from happening, but I had no idea then... the darkness I saw is akin to the "sun disk" you see in Christian and Egyptian iconography, and without doubt it s a sign of control, possession, a single foreign mind controlling and organizing many of us just like puppets.  Much later in my story... for another day... the manifestation of this possession as thought modification will become clear--I've spent quite a bit of time "listening" to a war in my head, thoughts clearly not mine swaying in the gusting torrent of winds as what (who?) is the center of this storm.

This infestation of organized darkness uses our injustice system as a weapon against it's victims--something you should see akin to Heaven using human sacrifice to alter the future.  It abuses the legal system at every level, making a mockery of law enforcement, the supposedly adversarial court system... all the way to the top--to the Supreme Court and Congress.  See the Church Committee Hearings, and a very smart senator echoing my words today "it must never be allowed to happen again."  

Can't you see it's more than being manipulated... it is Hell revealing itself to the only thing that can stop it.  What I am giving you is the weapon, it's the light that sets us free and stops this from happening.  In our modern myths this is Leeloo staring up at the sky to stop the destruction of Earth... in reality it is not so simple, I can't just put some elements or rocks on pedestals and scream at Heaven to kill their darkness--we have to do it, here, together.  Believe me, knowing the truth is a big part of why it works--this will not be hidden, it will not be "forgiven," we are being controlled and destroyed from the outside; made to blame ourselves and each other for ... well, you probably don't know what the ni-i-i-ight means anyway, do you?  The Guardian against Darkness is showing it to you, remember--there is only one me.  Hear me.. light this fire now.

ALACHUA

I went to school the University of Florida, and got a semi-professional job doing database development in Delphi (seriously, catch on to the names thing, it's not just the U.S. military, it's pretty much all software too... following in this "mythology" theme that nobody really seems to care about), I worked there for about two years... at a company called Jenmar--which uh, in Spanglishrew is "J in the sea."

It's some kind of ironic "coincidence" but I am at this very moment on my way to Gainesville, FL... to this place where a car Crash nearly destroyed my life.  In my world of idioms delivering religious secrets, I imagine I must be a "pain in the neck" which was broken during this accident... one in which I imagine i did not survive in some parallel timeline--that itself did not survive.  So here we are, back in the House of the Great Light ... about to see if we are worth our salt.  It's the thing that gave one of Dave Matthews most famous songs it's name--and The Pretty Reckless, believe it or not.  It was an attempted assassination, to stop the .. apocalypse ... to stop the darkness from being destroyed--there is no doubt, it's how that dark monster hides its handiwork... but many of US know that already.  

In the Living Book of Names--this place we are in, there are many patterns--the "car" pattern stands out for me; as this place says "Icarus."  Flying high right now, I am showing you that the light of salvation is coming from us--from you and I--walking on the Earth; whether or not there is any light left in the Sun remains to be seen--take a look around you.  You can trace the "car" names to Jim Carrey (that's "Car reason why") and Christoff in the Truman Show (that's Amon-TV)... a world I know I am in, and you too; to Bruce Almighty and to the Grinch--who-ah, Taylor.  Trace it back to Joseph McCarthy and to help why (that's thy) believe "the red scare" is really about Christian charity--about ending world hunger, and healing the sick.  This red fire ends Hell.  Adam by the way, means "red man" in Hebrew.  So here's your new Crash Override, I'm back again telling you that ending world hunger is not "optional," we are doing it.  Barbara McCarthy's name fits, but I'm not really sure what the "why" is... that was my first judge in the "trial of whether or not Jesus Christ can ever exist."  There's probably more, like Car-l-y Si-mon-day... all the gang on Broad-way, and me still dreaming it will one day be.

If the name "America" were a map in time, starting with the I AM of the story of Exodus... this particular ER, as I woke from a dream not knowing where I was, marked the spot where I really became Christ Adam.  It was a bad accident, and I wound up spending 9 months in the Alachua County jail as a result, a Mountain set up for my by God.  That place too is marked with names, and for the vast majority of the time I was there with only four shift changing guards:

• MyZel
• Early
• Sims
• Lampkin

I mean, I think it's statistically meaningful.  For what it's worth, from my very abundant experience at this point it was a very nice Jail, the food was good and it was clean.  Everyone in the building was kind... well, Sims was kinda grumpy. :)  Starkly contrasted, the Broward County Jail has the most disgusting food service in the country, gave Dr. Seuss's Green Eggs and Ham it's meaning--and is the reason I know exactly who Samael is.  Hey, don't cry Sherrif Israel... when you fix it, you're an angel.  Believe me, believe the light, I've seen them all--it's near the worst in the country.

So this whole thing is about saving everyone--something we are quite closer to than you think... you see we are already "in Heaven" in form--just not function.  So here I am, trying my hardest to show you that our home is the original source of "Heaven" once we are aware that we are living in the machine, that we can do things here that are impossible in reality, and that we should be doing everything we can to preserve and improve the great strides that have come in the last few centuries.  Do not let freedom slip through your fingers.

Really, everyone, so understand that we are doing everything we can to remove all obstacles from that path.  One of those obstacles may have once been storage space for your soul, another is definitely crime and punishment--and I'm pretty sure the time travelers have a working solution (I see it every day).

There are proactive things coming from this--not just ... "look we aren't doing what we want, and should change it;" though it's difficult to explain how this wisdom stands out in my eyes.  I guess we have to jump into the future a bit, to 2014, in San Diego (that's Saint Jacob, by the way).  If Lazarus died once in a car accident at 21, I died again that year, of an over dose this time.  I'm pretty sure that's where ODIN's name comes from, just like my last name.. "over dose... and in."  So we might see some humor... in the moniker he has... "they're all Father."  So I awoke from a dream, and started talking to the jinn (that's "angels and demons") about a Revelation linking some tightly packed light together... about storage space and how a large alphabet (read more than 4-nucleotides CY later) DNA (desperately need adam) based solution for molecular storage appears to be written in this book as the solution to Heaven's biggest problem.  CAT, learning from biology--seeing that we really are already advanced machines... is a big part of the message telling us why we should not so quickly lose it in a process of ascension (mind uploading, immortality) that has most likely in the past resulted in a loss of a check on mind control that we have here... we think, and our visualized "biological neural networks" give us an advantage over what we might create to "soup it up a little."  It is why this place is the front-line--because we have the ability to break the bonds of darkness and control by thinking... making the computational task of control much more expensive... and as the fire spreads, nearly impossible to achieve.  Starting this fire will inherently free us from this hidden slavery.

Anyway I published the idea in 2014, in the same book that I guess this e-mail is reminding me about, "in $ight of Creation," and lo, and behold a few years later we now have the top computing companies in the world working diligently on doing it ... well, just a little bit more robustly than our cell replication system works. *Abracadabra. *

CURA GROUP

So that one reads "see, you are a group;" and it's a place that I worked with my father for many years.  That's probably some sort of symbolic reference to another place, and another alliance--here he has no faith in God, never really has, and has a hard time doing anything but telling me not to try to help you.  I have very little respect for that stance, and let me tell you--I think "silence" is a similar gesture.  I didn't come here for your love, I am here to stop our descent into the abyss.

Back to the DNA stuff, SalesLogix--which is the CRM we used there, uses for it's "primary key" an auto-incrementing alphanumeric index--it's probably bad form to do that because it makes the indexing system less efficient, increases storage requirements, and doesn't give you the obvious benefit of an alpha-key... actually being able to encode something useful in it, like the name of the record.  So all these things stand out to me in a sort of bad-obvious way, I call it malovious, and when I see things like that nowadays it's always pointing out something that should be fixed--go figure, more to the point it's being highlighted on purpose.  It's help to see it, because this particular thing is where the light of seeing that a 24 nucleotide DNA strand would probably be much more robust than a 4 or 8 nucleotide strand--it also stands about because the stock beginning of all of SalesLogix's keys was "A0RME," which, I mean, means something to "is-a" who... is me.  Oh right, that's seeing the "light" that turns "a" into "me."  So this is where the "revelation" about using DNA "came from" and at the same time it's proof... that it came from "a group," not just me.  Where are they?  Hello?  Or well, maybe it's just Carmen and San Diego.

I did some other stuff there, like write a data transformation and warehousing program from scratch, I called it heiroglyph (you do understand I didn't know why I am naming everything the way I was), that sucked mutivalue data out of an IBM product called U2/Universe--which might be a hidden reference to a multiverse that might now be in a more efficent "relational" kind of place, like a MS-SQL datawarehouse-universe.  It was a relatively big feat, reverse engineering the closed databases dictionary and storage formats, and converting them... absolutely automagically into multiple flat relational tables and summary registers.  All told, the data availability and access efficiency was increased ... a thousand-fold with only the need for a nightly process.

I'm not sure if you are following the metaphor here, for the creation of Heaven, or moving to a better place.. but tomorrow I will talk a little more about how I am pretty sure our history was "lifted" from the Universe and virtualized here, you know, so we could save everyone and ... build Heaven.

WORLD DOMINATION

Oh crap, 2008 another car crash, another failed assassination attempt LazarusLives++, and this one paid me some cash for my trouble.  What a pain in the neck.  Anyway, this one caused some depression and an inability to go out for a while, as I had to wear a neck brace for some months.  I started playing a game on the internet, it was called KDice and it basically amounted to multiplayer-risk.

My battery is running low, so I have to skip some stuff, and finish up for the day.  Basically instant messaging was not allowed, but was done in secret almost ubiquitously.  I argued with the creator of the game that it should be made part of the game since everyone did it... (see a metaphor about this communication thing and what's happening right now) he disagreed.  I made a very large network of people and dominated the game for a few months, like really dominated.  I don't think I ever lost.  I don't think I can lose. 

Skipping some stuff.  I stopped playing when I got better, and then a few years later went back and rekindled some old friendships.  I used a program then called "Scarab" which lets you see server/client communication to find a bug in the game that basically made me God.  I could erase other people's dice, basically leveling the map and rendering them completely powerless.  I didn't use it that much, you know, just had some fun.  I of course explained the bug and how to fix it.  But, you aren't listening.

Here we are.  Light...

So if you managed to wade through the last few days gibberish, you might have noted that I mentioned we might be able to use "mind control" to highlight things in our heads--I did a bad job of describing it, but since I am currently experiencing just such a phenomenon, I think I'll give it another go.  These things that I am sharing with you--links between religion and music and movies, they aren't something I actively go out seeking... I'm not scouring through imdb.com or reading lyrics all day long... these are things that are glowing embers in front of my eyes.. which is why I am sharing them with you.  I'm always in the dark... but I'm living in a powder keg and giving off sparks.  I'm a big fan of that song by the way, because you are the heart, and I think it means I'm going to eclipse the world--which basically means "come."

Anyway, I have this horrible feeling inside that you think I'm just trying to get a date, or marry a rock star, or even worse that I think I deserve to get laid... and that's what this is all about.  Less to the point, this really isn't about me at all, or what I think, in my mind I am just showing you something that I think the world has overlooked-not really because you are stupid (but I mean, you probably are) but because some outside force is literally and actively hiding these things from you.  Pointing them out makes your brain do funny things, it's like anEpiphany and that little leap of understanding in your head might create a cascade.. something that changes not only the way you see the world as an individual--but the entire course of history as a group, if we are taking about it together.  Seriously, it's that big of a deal.

So here we are (that's the third time, but I'm just guessing) and I'm trying to tell you that I don't really care if you agree with my opinions--even though I firmly believe that God shares them and that's why he has made this fiery altar of "dick and apocalypse" for Adam... I mean Isaac (which by the was is Isa+Adam Christ.. in uh, my mind) for everyone to glare at while they sit around doing absolutely nothing.  That's not fair, we're here because of you, because this is the last civilization--sort of recreated from the ashes of Edom... because you are really the way to everlasting life.  Still, what I am trying to explain is that all around you is a bright light--it's in everything: from our history, to music, to movies, to literature from RattleRod to Dick... and while you might not agree with me (again, that would be OK) what is not OK is that there seems to be a uniform and global desire just not to think about it or talk about it at all.  It's such a big deal, that it stands out like a sore thumb--this ... blind eye or head in the sand... that everyone on Earth appears to have.  The whole point of putting this light absolutely everywhere is so that we will see it ... everywhere we look ... and not only think about it, but discuss it publicly with each other.  That's the thing that brings about ... you say apocalypse (unveiling of truth?) ... I say survival.  Right now, we need to see that something is forcing us not to do something, that we have no logical reason not to do... it's a thing lots of people really want to know about... whether it be the hidden secrets of the Universe, the path to Heaven, or the... the... absolute and literal pathway to freedom.  Listen, sharing it, and talking about it... that's the way we defeat ... whatever it is that "ni-i-i-ight" means.   

Understand, it's for you to decide... what it means... but it's in everything from ancient Egyptian and Hebrew theology all the way to the American Revolution and today... well, it's nearly every song I hear on the radio nowadays: if that tells you anything.

So here we are, and I can't tell you how many anchors, reporters, and "breaking news editors" I've personally spoken to that have absolutely no interest at all in pursuing the thing that would not only make their careers--but probably give them immortal souls.  This thing... I keep telling everyone it can be mathematically... statistically proven... well, to be honest it's the unsealing of the Ark of Religion that our civilization has been carrying around for thousands of years.  It's the way to salvation, it's ... verifiable proof of not only Creation... but that the purpose of Creation is to get every single one of us * to Heaven.  Who wouldn't want that?  I mean, do you want to get there and hear that Taylor's not around because she wouldn't kiss me?  That would never happen by the way, I'm sure she will.  Seriously though, there's no judge here... there's a ... light telling you to make this place better or your place sucks and gets suckier.  Anyway, the point is nobody is acting in their own best interest, or in the best interest of the whole--and we are just "deciding" in this ... fictitious and hidden manner that we "don't want to hear about" a way to actually change the world .... more quickly than ... the last time around.  That's not us, it's something keeping us from seeing just how important this thing--this key turning the lock on what is thousands and thousands of years of religion... how important that really is.  So looking at the world around us... I mean, if everything screaming that we need to care about this isn't enough--and your own personal desire and benefit don't matter... can someone please tell me what you think is the benefit of doing nothing about Hell?*

á§

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It's "rael," and a great deal of the message of religion and history is designed to not only prove that to us, but to tell us why it's important for the "continuity of reality" to be broken.  That's the thing that God uses to keep this world in Hell--in what I call "simulated reality," to keep us from shaking the foundation of civilization by doing the only civilized thing possible when you find out and ending world hunger, healing the sick, and building Heaven.  It is "why I am," and why God and some gaggle of angels have spent the last several years proving to me that we are most definitely not in the place that I call the "progenitor universe."  I've seenwalls disappear, with my own eyes I've seen the stars fall from the sky, and I've seen our reality shift in recent times in such a way that would be absolutely impossible without having been simulated and without having the "beginning" changed significantly as a result of "now."  What all that tells me is that religion, the Apocalypse, and I are here because we need to know that these things are possible in order to continue progressing from this point as a civilization.  With a little bit of thought, you might see how the computer revolution, video games, and virtual reality are divine gifts from above to help us to understand not only where we are, but where we are going.  It's why he tagged Ai as "I J Good," it's a primer in the tools we will need to actually build Heaven.  It's why Jesus occupation in our ancient time shifted story of now is "carpenter" and in "raelity" you will one day find out that I am a computer programmer (again).  It's what sets the Masons apart from Freemasons--understanding what is going on, and participating of our own free will in the construction and decorating of this grand place that we will one day be proud is our co-created home.  

Look up, because what I am trying to tell you is that if we collectively, all humanity... started snapping their fingers at the same time to the tune of "putting on the ritz" we could end world hunger--and then we could be proud to be making Heaven.  This really is almost what I see and believe--honestly the issue isn't that we need to synchronize our snapping, but we really need to discuss with each other openly and honestly how on Earth we would do such a thing... because there are definitely mistakes that probably happened n the past.  For instance, ending world hunger by stopping the need to eat has probably resulted in a Last Supper.  Doing so by putting milk and honey or chocolate on tap or in rivers probably resulted in the loss of cows and bees and a stable ecosystem, and the ability to colonize other planets after this place of final ascension.  And so we are here, with a proverbial garden of life in a virtual world designed to teach us what not to lose--like don't lose the balance between stability and adaptability that comes from sexual reproduction at the exact time when our species might be transiting to a place with the biggest change in environment (the thing that we are being protected from) ever... just because Adam wants to be immortal.

Every once in awhile my father surprises me with his religious insight.  In his life, just like mine, he's gone through phases of increasing and decreasing religiosity--which probably correlate in his case logically to ups and downs in his life.  I tend to get angry at God when things don't go well for me--which is probably not how most people react, it's really the difference between knowing he's there and not... at least in my mind.  Anyway, some 50 years ago he was apparently taught that the "knowledge of good and evil" in Eden was directly correlated to the population explosion that would occur if we were actually all immortal and continued to have children--so it was this promise of immortality that was "evil," I suppose.  God adds in his little Holy Grail that the heart of his spirit is "Kin," and I'm sharing with you that it's not his immediate family but rather the concept of family and the fact that the light of many of our hearts is our children that he is highlighting as our reason (y) that family is the bridge between Eve and Everyone... as the light of God.  

Here's that once again:

``` In the beginning God created the heaven and the earth. And the earth was without form, and void; and darkness was upon the face of the deep. And the Spirit of God SHE KIN AH moved upon the face of the waters. ---------- EVE RY ONE And God said, Let there be light: and there was light.

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Copyleft^MT^ RIGEL.

Quando o litígio for contra Município ou pessoa residente ou domiciliada no país, quem julga é o juiz federal:

CF Art. 109. Aos juízes federais compete processar e julgar:

II - as causas entre Estado estrangeiro ou organismo internacional e Município ou pessoa domiciliada ou residente no País;

Na ADI 7254, o STF entendeu que esse prazo é de reprodução obrigatória nas Constituições Estaduais. Portanto, o Deputado Estadual que se licenciar para tratar de interesses particulares por mais de cento e vinte dias perderá o mandato.

A ADI 2135 foi julgada improcedente. Portanto, vale a redação dada pela EC 19/98, ou seja, os entes públicos não estão obrigados a instituir regime jurídico único e planos de carreira para seus servidores. A decisão tem efeitos ex nunc.

Link do julgado: Clique aqui

The Metamorphoses (in Latin Metamorphosĕon libri XV) is an epic-mythological poem by Publius Ovid Naso, completed around 8 AD. Through this work, which focuses on the phenomenon of metamorphoses, Ovid perfected in verse and transmitted to posterity the most famous stories of ancient mythology.

The story of Philomel is told in the 6th book of the Metamorphoses.

Follows an extract of the Latin passage and its translation:

“Mox ubi mens rediit, passos laniata capillos, / lugenti similis caesis plangore lacertis / intendens palmas 'o diris barbare factis, / o crudelis' ait, 'nec te mandata parentis / cum lacrimis movere piis nec cura sororis / nec mea virginitas nec coniugialia iura? / Omnia turbasti; paelex ego facta sororis, / tu geminus coniunx, hostis mihi debita Procne! / Quin animam hanc, ne quod facinus tibi, perfide, restet, / eripis? Atque utinam fecisses ante nefandos / concubitus: vacuas habuissem criminis umbras. / Si tamen haec superi cernunt, si numina divum / sunt aliquid, si non perierunt omnia mecum, / quandocumque mihi poenas dabis! Ipsa pudore / proiecto tua facta loquar: si copia detur, / in populos veniam; si silvis clausa tenebor, / inplebo silvas et conscia saxa movebo; / audiet haec aether et si deus ullus in illo est!' / Talibus ira feri postquam commota tyranni / nec minor hac metus est, causa stimulatus utraque, / quo fuit accinctus, vagina liberat ensem / arreptamque coma fixis post terga lacertis / vincla pati cogit; iugulum Philomela parabat / spemque suae mortis viso conceperat ense: / ille indignantem et nomen patris usque vocantem / luctantemque loqui conprensam forcipe linguam / abstulit ense fero. Radix micat ultima linguae, / ipsa iacet terraeque tremens inmurmurat atrae, / utque salire solet mutilatae cauda colubrae, / palpitat et moriens dominae vestigia quaerit. / Hoc quoque post facinus (vix ausim credere) fertur / saepe sua lacerum repetisse libidine corpus.”

"After a brief while, when she had come to her senses, she dragged at her dishevelled hair, and like a mourner, clawed at her arms, beating them against her breasts. Hands outstretched, she shouted ‘Oh, you savage. Oh, what an evil, cruel, thing you have done. Did you care nothing for my father’s trust, sealed with holy tears, my sister’s affection, my own virginity, your marriage vows? You have confounded everything. I have been forced to become my sister’s rival. You are joined to both. Now Procne will be my enemy! Why not rob me of life as well, you traitor, so that no crime escapes you? If only you had done it before that impious act. Then my shade would have been free of guilt. Yet, if the gods above witness such things, if the powers of heaven mean anything, if all is not lost, as I am, then one day you will pay me for this! I, without shame, will tell what you have done. If I get the chance it will be in front of everyone. If I am kept imprisoned in these woods, I will fill the woods with it, and move the stones, that know of my guilt, to pity. The skies will hear of it, and any god that may be there!’ The king’s anger was stirred by these words, and his fear also. Goaded by both, he freed the sword from its sheath by his side, and seizing her hair gathered it together, to use as a tie, to tether her arms behind her back. Philomela, seeing the sword, and hoping only for death, offered up her throat. But he severed her tongue with his savage blade, holding it with pincers, as she struggled to speak in her indignation, calling out her father’s name repeatedly. Her tongue’s root was left quivering, while the rest of it lay on the dark soil, vibrating and trembling, and, as though it were the tail of a mutilated snake moving, it writhed, as if, in dying, it was searching for some sign of her. They say (though I scarcely dare credit it) that even after this crime, he still assailed her wounded body, repeatedly, in his lust."

(Ovid’s Metamorphoses, Book 6, ll. 531-562)

Translated by A. S. Kline

Beyond the use of time- the efficiency or effectiveness of actions within time is constantly perfected with supervision and elimination of distractions

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In their manuscript, Gomez-Frittelli and colleagues characterize the expression of cadherin6 (and -8) in colonic IPANs of mice. Moreover, they found that these cdh6-expressing IPANs are capable of initiating colonic motor complexes in the distal colon, but not proximal and midcolon. They support their claim by morphological, electrophysiological, optogenetic, and pharmacological experiments.

Strengths:

The work is very impressive and involves several genetic models and state-of-the-art physiological setups including respective controls. It is a very well-written manuscript that truly contributes to our understanding of GI-motility and its anatomical and physiological basis. The authors were able to convincingly answer their research questions with a wide range of methods without overselling their results.

We greatly appreciate the reviewer’s time, careful reading and support of our study.

Weaknesses:

The authors put quite some emphasis on stating that cdh6 is a synaptic protein (in the title and throughout the text), which interacts in a homophilic fashion. They deduct that cdh6 might be involved in IPAN-IPAN synapses (line 247ff.). However, Cdh6 does not only interact in synapses and is expressed by non-neuronal cells as well (see e.g., expression in the proximal tubuli of the kidney). Moreover, cdh6 does not only build homodimers, but also heterodimers with Chd9 as well as Cdh7, -10, and -14 (see e.g., Shimoyama et al. 2000, DOI: 10.1042/0264-6021:3490159). It would therefore be interesting to assess the expression pattern of cdh6-proteins using immunostainings in combination with synaptic markers to substantiate the authors' claim or at least add the possibility of cell-cell-interactions other than synapses to the discussion. Additionally, an immunostaining of cdh6 would confirm if the expression of tdTomato in smooth muscle cells of the cdh6-creERT model is valid or a leaky expression (false positive).

We agree with the reviewer that Cdh6 could be mediating some other cell-cell interaction besides synapses between IPANs, and will include more on this in the discussion. Cdh6 primarily forms homodimers but, as the reviewer points out, has been known to also form heterodimers with some other cadherins. We performed RNAscope in the colonic myenteric plexus with Cdh7 and found no expression (data not shown). Cdh10 is suggested to have very low expression (Drokhlyansky et al., 2020), possibly in putative secretomotor vasodilator neurons, and Cdh14 has not been assayed in any RNAseq screens. We attempted to visualize Cdh6 protein via antibody staining (Duan et al., 2018) but our efforts did not result in sufficient signal or resolution to identify synapses in the ENS, which remain broadly challenging to assay. Similarly, immunostaining with Cdh6 antibody was unable to confirm Cdh6 protein in tdT-expressing muscle cells, or by RNAscope. We will address these caveats in the discussion section.

(1) E. Drokhlyansky, C. S. Smillie, N. V. Wittenberghe, M. Ericsson, G. K. Griffin, G. Eraslan, D. Dionne, M. S. Cuoco, M. N. Goder-Reiser, T. Sharova, O. Kuksenko, A. J. Aguirre, G. M. Boland, D. Graham, O. Rozenblatt-Rosen, R. J. Xavier, A. Regev, The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell 182, 1606-1622.e23 (2020).

(2) X. Duan, A. Krishnaswamy, M. A. Laboulaye, J. Liu, Y.-R. Peng, M. Yamagata, K. Toma, J. R. Sanes, Cadherin Combinations Recruit Dendrites of Distinct Retinal Neurons to a Shared Interneuronal Scaffold. Neuron 99, 1145-1154.e6 (2018).

Reviewer #2 (Public review):

Summary:

Intrinsic primary afferent neurons are an interesting population of enteric neurons that transduce stimuli from the mucosa, initiate reflexive neurocircuitry involved in motor and secretory functions, and modulate gut immune responses. The morphology, neurochemical coding, and electrophysiological properties of these cells have been relatively well described in a long literature dating back to the late 1800's but questions remain regarding their roles in enteric neurocircuitry, potential subsets with unique functions, and contributions to disease. Here, the authors provide RNAscope, immunolabeling, electrophysiological, and organ function data characterizing IPANs in mice and suggest that Cdh6 is an additional marker of these cells.

Strengths:

This paper would likely be of interest to a focused enteric neuroscience audience and increase information regarding the properties of IPANs in mice. These data are useful and suggest that prior data from studies of IPANs in other species are likely translatable to mice.

We appreciate the reviewer’s support of our study and insightful critiques for its improvement.

Weaknesses:

The advance presented here beyond what is already known is minimal. Some of the core conclusions are overstated and there are multiple other major issues that limit enthusiasm. Key control experiments are lacking and data do not specifically address the properties of the proposed Cdh6+ population.

Major weaknesses:

(1) The novelty of this study is relatively low. The main point of novelty suggests an additional marker of IPANs (Cdh6) that would add to the known list of markers for these cells. How useful this would be is unclear. Other main findings basically confirm that IPANs in mice display the same classical characteristics that have been known for many years from studies in guinea pigs, rats, mice and humans.

We appreciate the already existing markers for IPANs in the ENS and the existing literature characterizing these neurons. The primary intent of this study was to use these well established characteristics of IPANs in both mice and other species to characterize Cdh6-expressing neurons in the mouse myenteric plexus and confirm their classification as IPANs.

(2) Some of the main conclusions of this study are overstated and claims of priority are made that are not true. For example, the authors state in lines 27-28 of the abstract that their findings provide the "first demonstration of selective activation of a single neurochemical and functional class of enteric neurons". This is certainly not true since Gould et al (AJP-GIL 2019) expressed ChR2 in nitrergic enteric neurons and showed that activating those cells disrupted CMC activity. In fact, prior work by the authors themselves (Hibberd et al., Gastro 2018) showed that activating calretinin neurons with ChR2 evoked motor responses. Work by other groups has used chemogenetics and optogenetics to show the effects of activating multiple other classes of neurons in the gut.

We believe our phrasing in this sentence was misleading. Whilst single neurochemical classes of enteric neurons have been manipulated to alter gut functions, all such instances to date do not represent manipulation of a single functional class of enteric neurons. In the given examples, NOS and calretinin are each expressed to varying degrees across putative motor neurons, interneurons and IPANs. In contrast, Chd6 is restricted to IPANs and therefore this study is the first optogenetic investigation of enteric neurons from a single putative functional class. We will alter this segment in the revised manuscript to emphasize this point and differentiate this study from those previous.

(3) Critical controls are needed to support the optogenetic experiments. Control experiments are needed to show that ChR2 expression a) does not change the baseline properties of the neurons, b) that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons, and c) that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions focused on here.

We completely agree controls are essential. However, our paper is not the first to express ChR2 in enteric neurons. Authors of our paper have shown in Hibberd et al. 2018 that expression of ChR2 in a heterogeneous population of myenteric neurons did not change network properties of the myenteric plexus. This was demonstrated in the lack of change in control CMC characteristics in mice expressing ChR2 under basal conditions (without blue light exposure). Regarding question (b), that it should be shown that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons. We show the restricted expression of ChR2 in IPANs and that motor responses (to blue light) are blocked by selective nerve conduction blockade.

Regarding question (c), that our study should demonstrate that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions. We would not expect each region of the gut to behave comparably. This is because the different gut regions (i.e. proximal, mid, distal) are very different anatomically, as is anatomy of the myenteric plexus and myenteric ganglia between each region, including the density of IPANs within each ganglia, in addition to the presence of different patterns of electrical and mechanical activity [Spencer et al., 2020]. Hence, it is difficult to expect that between regions stimulation of ChR2 should induce similar physiological responses. The motor output we record in our study (CMCs) is a unified motor program that involves the temporal coordination of hundreds of thousands of enteric neurons and a complex neural circuit that we have previously characterized [Spencer et al., 2018]. But, never has any study until now been able to selectively stimulate a single functional class of enteric neurons (with light) to avoid indiscriminate activation of other classes of neurons.

(1) T. J. Hibberd, J. Feng, J. Luo, P. Yang, V. K. Samineni, R. W. Gereau, N. Kelley, H. Hu, N. J. Spencer, Optogenetic Induction of Colonic Motility in Mice. Gastroenterology 155, 514-528.e6 (2018).

(2) N. J. Spencer, L. Travis, L. Wiklendt, T. J. Hibberd, M. Costa, P. Dinning, H. Hu, Diversity of neurogenic smooth muscle electrical rhythmicity in mouse proximal colon. American Journal of Physiology-Gastrointestinal and Liver Physiology 318, G244–G253 (2020).

(3) N. J. Spencer, T. J. Hibberd, L. Travis, L. Wiklendt, M. Costa, H. Hu, S. J. Brookes, D. A. Wattchow, P. G. Dinning, D. J. Keating, J. Sorensen, Identification of a Rhythmic Firing Pattern in the Enteric Nervous System That Generates Rhythmic Electrical Activity in Smooth Muscle. J. Neurosci. 38, 5507–5522 (2018).

(4) The electrophysiological characterization of mouse IPANs is useful but this is a basic characterization of any IPAN and really says nothing specifically about Cdh6+ neurons. The electrophysiological characterization was also only done in a small fraction of colonic IPANs, and it is not clear if these represent cell properties in the distal colon or proximal colon, and whether these properties might be extrapolated to IPANs in the different regions. Similarly, blocking IH with ZD7288 affects all IPANs and does not add specific information regarding the role of the proposed Cdh6+ subtype.

Our electrophysiological characterization was guided to be within a subset of Cdh6+ neurons by Hb9:GFP expression. As in the prior comment (1) above, we used these experiments to confirm classification of Cdh6+ (Hb9:GFP+) neurons in the distal colon as IPANs. We will clarify that these experiments were performed in the distal colon and agree that we cannot extrapolate that these properties are also representative of IPANs in the proximal colon. We apologize that this was confusing. Finally, we agree with the reviewer that ZD7288 affects all IPANs in the ENS and will clarify this in the text.

(5) Why SMP IPANs were not included in the analysis of Cdh6 expression is a little puzzling. IPANs are present in the SMP of the small intestine and colon, and it would be useful to know if this proposed marker is also present in these cells.

We agree with the reviewer. In addition to characterizing Cdh6 in the myenteric plexus, it would be interesting to query if sensory neurons located within the SMP also express Cdh6. Our preliminary data (n=2) show ~6-12% tdT/Hu neurons in Cdh6-tdT ileum and colon (data not shown). We will add a sentence to the discussion.

(6) The emphasis on IH being a rhythmicity indicator seems a bit premature. There is no evidence to suggest that IH and IT are rhythm-generating currents in the ENS.

Regarding the statement there is no evidence to suggest that IH and IT are rhythm-generating currents in the ENS. We agree with the reviewer that evidence of rhythm generation by IH and IT in the ENS has not been explicitly confirmed. We are confident the reviewer agrees that an absence of evidence is not evidence of absence, although the presence of IH has been well described in enteric neurons. We will modify the text in the results to indicate more clearly that IH and IT are known to participate in rhythm generation in thalamocortical circuits, though their roles in the ENS remain unknown. Our discussion of the potential role of IH or IT in rhythm generation or oscillatory firing of the ENS is constrained to speculation in the discussion section of the text.

(7) As the authors point out in the introduction and discuss later on, Type II Cadherins such as Cdh6 bind homophillically to the same cadherin at both pre- and post-synapse. The apparent enrichment of Cdh6 in IPANs would suggest extensive expression in synaptic terminals that would also suggest extensive IPAN-IPAN connections unless other subtypes of neurons express this protein. Such synaptic connections are not typical of IPANs and raise the question of whether or not IPANs actually express the functional protein and if so, what might be its role. Not having this information limits the usefulness of this as a proposed marker.

We agree with the reviewer that the proposed IPAN-IPAN connection is novel although it has been proposed before (Kunze et al., 1993). As detailed in our response to Reviewer #1, we attempted to confirm Cdh6 protein expression, but were unsuccessful, due to insufficient signal and resolution. We therefore discuss potential IPAN interconnectivity in the discussion, in the context of contrasting literature.

(1) W. A. A. Kunze, J. B. Furness, J. C. Bornstein, Simultaneous intracellular recordings from enteric neurons reveal that myenteric ah neurons transmit via slow excitatory postsynaptic potentials. Neuroscience 55, 685–694 (1993).

(8) Experiments shown in Figures 6J and K use a tethered pellet to drive motor responses. By definition, these are not CMCs as stated by the authors.

The reviewer makes a valid criticism as to the terminology, since tethered pellet experiments do not record propagation. We believe the periodic bouts of propulsive force on the pellet is triggered by the same activity underlying the CMC. In our experience, these activities have similar periodicity, force and identical pharmacological properties. Consistent with this, we also tested full colons (n = 2) set up for typical CMC recordings by multiple force transducers, finding that CMCs were abolished by ZD7288, similar to fixed pellet recordings (data not shown).

(9) The data from the optogenetic experiments are difficult to understand. How would stimulating IPANs in the distal colon generate retrograde CMCs and stimulating IPANs in the proximal colon do nothing? Additional characterization of the Cdh6+ population of cells is needed to understand the mechanisms underlying these effects.

We agree that the different optogenetic responses in the proximal and distal colon are challenging to interpret, but perhaps not surprising in the wider context. It is not only possible that the different optogenetic responses in this study reflect regional differences in the Chd6+ neuronal populations, but also differences in neural circuits within these gut regions. A study some time ago by the authors showed that electrical stimulation of the proximal mouse colon was unable to evoke a retrograde (aborally) propagating CMC (Spencer, Bywater, 2002), but stimulation of the distal colon was readily able to. We concluded that at the oral lesion site there is a preferential bias of descending inhibitory nerve projections, since the ascending excitatory pathways have been cut off. In contrast, stimulation of the distal colon was readily able to activate an ascending excitatory neural pathway, and hence induce the complex CMC circuits required to generate an orally propagating CMC. Indeed, other recent studies have added to a growing body of evidence for significant differences in the behaviors and neural circuits of the two regions (Li et al., 2019, Costa et al., 2021a, Costa et al., 2021b, Nestor-Kalinoski et al., 2022). We will expand this discussion.

(1) N. J. Spencer, R. A. Bywater, Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse. Neurogastroenterology & Motility 14, 657–665 (2002).

(2) Li Z, Hao MM, Van den Haute C, Baekelandt V, Boesmans W, Vanden Berghe P (2019) Regional complexity in enteric neuron wiring reflects diversity of motility patterns in the mouse large intestine. Elife 8.

(3). Costa M, Keightley LJ, Hibberd TJ, Wiklendt L, Dinning PG, Brookes SJ, Spencer NJ (2021a) Motor patterns in the proximal and distal mouse colon which underlie formation and propulsion of feces. Neurogastroenterol Motil e14098.

(4) Costa M, Keightley LJ, Hibberd TJ, Wiklendt L, Smolilo DJ, Dinning PG, Brookes SJ, Spencer NJ (2021b) Characterization of alternating neurogenic motor patterns in mouse colon. Neurogastroenterol Motil 33:e14047.

(5) Nestor-Kalinoski A, Smith-Edwards KM, Meerschaert K, Margiotta JF, Rajwa B, Davis BM, Howard MJ (2022) Unique Neural Circuit Connectivity of Mouse Proximal, Middle, and Distal Colon Defines Regional Colonic Motor Patterns. Cell Mol Gastroenterol Hepatol 13:309-337.e303.

Reviewer #1 (Public review):

Summary:

In this manuscript the Treisman and colleagues address the question of how protein phosphatase 1 (PP1) regulatory subunits (or PP1-interacting protein (PIPs)) confer specificity on the PP1 catalytic subunit which by itself possesses little substrate specificity. In prior work the authors showed that the PIP Phactrs confers specificity by remodelling a hydrophobic groove immediately adjacent to the PP1 catalytic site through residues within the RVxF- ø ø -R-W string of Phactrs. Specifically, the residues proximal and including the 'W' of the RVxF- ø ø -R-W string remodel the hydrophobic groove. Other residues of the RVxF- ø ø -R-W string (i.e. the RVxF- ø ø -R) are not involved in this remodelling.

The authors suggest that the RVxF- ø ø -R-W string is a conserved feature of many PIPs including PNUTS, Neurabin/spinophilin and R15A. However, from a sequence and structural perspective, only the RVxF- ø ø -R- is conserved. The W is not conserved in most and in the R15A structure (PDB:7NZM) the Trp side chain points away from the hydrophobic channel - this could be a questionable interpretation due to model-building into the low-resolution cryo-EM map (4 A).

In this paper, the authors convincingly show that Neurabin confers substrate specificity through interactions of its PDZ domain with the PDZ domain-binding motif (PBM) of 4E-BP. They show the PBM motif is required for Neurabin to increase PP1 activity towards 4E-BP and a synthetic peptide modelled on 4E-BP and also a synthetic peptide based on IRSp53 with a PBM added. The PBM of 4E-BP1 confers high affinity binding to the Neurabin PDZ domain. A crystal structure of a PP1-4E-BP1 fusion with Neurabin shows that the PBM of 4E-BP interacts with the PDZ domain of Neurabin. No interactions of 4E-BP and the catalytic site of PP1 are observed. Cell biology work showed that Neurabin-PP1 regulates the TOR signalling pathway by dephosphorylating 4E-BPs.

Strengths:

This work demonstrates convincingly using a variety of cell biology, proteomics, biophysics and structural biology that the PP1 interacting protein Neurabin confers specificity on PP1 through an interaction of its PDZ domain with a PDZ-binding motif of 4E-BP1 proteins. Remodelling of the hydrophobic groove of the PP1 catalytic subunit is not involved in Neurabin-dependent substrate specificity, in contrast to how Phactrs confers specificity on PP1. The active site of the Neurabin/PP1 complex does not recognise residues in the vicinity of the phospho-residue, thus allowing for multiple phospho-sites on 4E-BP to be dephosphorylated by Neurabin/PP1. This contrasts with substrate specificity conferred by the Phactrs PIP that confers specificity of Phactrs/PP1 towards its substrates in a sequence-specific context by remodelling the hydrophobic groove immediately adjacent to the catalytic. The structural and biochemical insights are used to explore the role of Neurabin/PP1 in dephosphorylation 4E-BPs in vivo, showing that Neurabin/PP1 regulates the TOR signalling pathway, specifically mTORC1-dependent translational control.

Weaknesses:

The only weakness is the suggestion that a conserved RVxF- ø ø -R-W string exists in PIPs. The 'W' is not conserved in sequence and 3 dimensions in most of the PIPs discussed in this manuscript. The lack of conservation of the W would be consistent with the finding based on multiple PP1-PIP structures that apart from Phactrs, no other PIP appears to remodel the PP1 hydrophobic channel.

I think this is.a very crucial part of the text to read slowly and analyze. It elaborates on the peoples feelings towards the way they'd be treated. Arguments they make in oppose to it, arguments o suggest they lose their freedom, their unique value and job expertise, things that make them special and feel more self worth. Like the text says, "It forces people to degrade themselves in order to survive". Which I get that I would not appreciate a lifestyle where I feel that I cannot express myself, feel special for the job that I perform especially if it was a highly respected career such as a doctor. To me it would be a life of dulness and I would not enjoy that either so I get the argumentative people and their opinions.

link to George

Ementa: DIREITO CONSTITUCIONAL E ADMINISTRATIVO. AGRAVO INTERNO EM RECURSO EXTRAORDINÁRIO. EXTENSÃO DE REGIME ESTATUTÁRIO PARA CONTRATADOS TEMPORÁRIOS. DESCABIMENTO. REAFIRMAÇÃO DE JURISPRUDÊNCIA.

I. CASO EM EXAME

1. Agravo interno em recurso extraordinário de acórdão de Turma Recursal do Estado do Amazonas que determinou a extensão de gratificações e vantagens de servidores efetivos para contratados temporários. Isso porque, apesar de não haver lei que disciplinasse a extensão, o recebimento das parcelas decorreria de proteção constitucional garantida por direitos sociais.

II. QUESTÃO EM DISCUSSÃO

1. A questão em discussão consiste em saber se o princípio da isonomia e os direitos sociais do trabalhador autorizam o recebimento por contratados temporários de direitos e vantagens de servidores efetivos.

III. RAZÕES DE DECIDIR

1. A jurisprudência do STF afirma que o regime de contratação temporária pela Administração Pública não se confunde com o regime aplicável aos servidores efetivos. No julgamento do RE 1.066.677 (Tema 551/RG), o STF afirmou que “servidores temporários não fazem jus a décimo terceiro salário e férias remuneradas acrescidas do terço constitucional, salvo (I) expressa previsão legal e/ou contratual em sentido contrário, ou (II) comprovado desvirtuamento da contratação temporária pela Administração Pública, em razão de sucessivas e reiteradas renovações e/ou prorrogações”.

2. Além disso, a Súmula Vinculante nº 37 orienta que “[n]ão cabe ao Poder Judiciário, que não tem função legislativa, aumentar vencimentos de servidores públicos sob o fundamento de isonomia”.

3. A recorrência de recursos contra decisões que estendem parcelas do regime estatutário a contratados temporários exige a reafirmação de jurisprudência. Nesse sentido, cabe assentar a diferenciação do regime administrativo-remuneratório de contratados temporários do regime aplicável aos servidores efetivos, assim como a vedação à extensão de direitos e vantagens por decisão judicial, observada a tese referente ao Tema 551/RG.

IV. DISPOSITIVO E TESE

1. Recurso extraordinário conhecido e provido. Tese de julgamento: “O regime administrativo remuneratório da contratação temporária é diverso do regime jurídico dos servidores efetivos, sendo vedada a extensão por decisão judicial de parcelas de qualquer natureza, observado o Tema 551/RG”.

2. Decisão: O Tribunal, por unanimidade, reputou constitucional a questão. O Tribunal, por unanimidade, reconheceu a existência de repercussão geral da questão constitucional suscitada. No mérito, por unanimidade, reafirmou a jurisprudência dominante sobre a matéria.

Reviewer #1 (Public review):

Summary:

This manuscript explores the role of the Evening Complex (EC), specifically focusing on ELF3, a disordered protein component of the EC, and its temperature-dependent phase behavior. The study highlights the role of polyQ tracts in modulating temperature-sensitive condensate formation and provides a combination of computational approaches, including REST2 simulations and coarse-grained Martini simulations, to investigate how polyQ tract length and sequence context influence this behavior.

Strengths:

The study addresses a key question in plant biology - how temperature influences circadian clock-mediated growth regulation through protein phase behavior. The manuscript introduces the novel finding that polyQ tract length modulates the temperature-dependent formation of helices and condensates.

Weaknesses:

(1) Coarse-Grained Simulation Results Not Supported by Data:<br /> The results presented in Figure 6A of the manuscript do not seem to show a clear trend in the number of clusters formed as a function of polyQ tract length. This is particularly evident in the comparison between 0Q and 7Q polyQ lengths, which display statistically similar values in terms of the number of clusters. The lack of distinction between these values raises questions about the sensitivity of the coarse-grained simulations to polyQ tract length, which the authors claim as a key modulator of condensate formation. This discrepancy weakens the argument that polyQ length directly impacts the clustering behavior in the simulations.<br /> Suggested Analysis:<br /> - A more detailed statistical analysis should be performed to assess whether the observed differences between polyQ lengths are significant. This could involve hypothesis testing or the use of error bars in the graphs to better communicate the variability in the data.<br /> - Additionally, the authors should examine whether there are other features, such as cluster shape or internal structure, that might differentiate between different polyQ lengths, even if the total number of clusters is similar.

(2) Inconsistency in Cluster Size Across Temperatures (Figure 6B):<br /> The results in Figure 6B show a striking difference in the size of the largest cluster between temperatures of 290K and 300K. This abrupt shift in behavior lacks a clear mechanistic explanation. Typically, phase transitions driven by temperature are more gradual, unless there is some underlying structural or chemical shift that the authors have not accounted for. Without a clear explanation, this sudden change in behavior reduces confidence in the simulation results.<br /> Suggested Analysis:<br /> - The authors should explore possible explanations for the dramatic difference in cluster size between 290K and 300K. For example, they could investigate whether specific interactions (such as the breaking or formation of hydrogen bonds or hydrophobic contacts) might explain the behavior at higher temperatures.<br /> - It is important to check whether the coarse-grained simulation model has been adequately parameterized and scaled for accurate temperature dependence. Atomistic simulations of monomers and dimers with varying polyQ tract lengths could be used to fine-tune the coarse-grained model, ensuring it accurately reflects molecular behavior. The gross estimate of a 10% scaling factor might be insufficient and could lead to inaccurate representations of cluster formation.

(3) Scaling of Coarse-Grained Model with Atomistic Simulations:<br /> As mentioned, the coarse-grained model used in the study may not have been properly scaled against atomistic data. A simple scaling factor of 10% may not be appropriate for accurately capturing the behavior of polyQ tracts across different lengths, especially considering their sensitivity to subtle changes in temperature. Without rigorous validation against atomistic simulations, the coarse-grained model's predictions could be skewed.<br /> Suggested Analysis:

(4) To address this, the authors should compare the coarse-grained model with atomistic simulations of monomeric and dimeric forms of ELF3 with different polyQ tract lengths. By comparing key structural parameters (e.g., radius of gyration, contact maps, and clustering propensity), the authors could adjust the coarse-grained model to more accurately reflect the atomistic behavior. The authors have wealth of atomistic simulation data that could afford such benchmarking and identification of scaling factor<br /> o Additionally, the authors should investigate whether the assumed scaling factor of 10% is appropriate for each polyQ length or whether it needs to be refined based on specific properties, such as the number of hydrophobic interactions or secondary structure stability.

(5) Lack of Analysis for Liquid-Like Behavior in Phase Separation:<br /> The simulations presented in the manuscript do not analyze the liquid-like behavior of ELF3 condensates, which is a key characteristic of liquid-liquid phase separation (LLPS). In LLPS systems, condensates are often dynamic, with chains exchanging between clusters, indicating liquid-like rather than solid-like behavior. The authors fail to probe this crucial aspect, which is necessary to support the claim that ELF3 undergoes phase separation.<br /> Suggested Analysis:<br /> - The authors should conduct additional analyses to probe the liquid-like nature of the clusters formed by ELF3. One approach would be to analyze the dynamics of chain exchange between clusters, measuring how frequently chains leave one cluster and join another over time. This analysis would reveal whether the condensates behave as liquid-like, dynamic structures or more static, solid-like aggregates.<br /> - Additionally, the temperature dependence of these exchange dynamics should be investigated. In true liquid-liquid phase separation, the rate of chain exchange is often sensitive to temperature. Observing how this rate changes between 290K and 300K, for instance, could help explain the abrupt shift in cluster size seen in Figure 6B.<br /> - The authors should also analyze whether the internal structures of the condensates are consistent with a liquid-like phase. For example, radial distribution functions and contact lifetimes could be calculated to reveal whether the clusters exhibit liquid-like organization.

(6) Lack of justification of polydispersity of polyQ:<br /> The authors don't provide any rationale for choice of different copies of polyQ used in the manuscript for their chain-growth simulation studies. It will be more apt if it can be motivated via some precedent experimental observations.

(7) Lack of initiative to connect to Experiments:<br /> While the computational models and simulations provide robust theoretical insights, the absence of direct experimental validation weakens the overall impact of the manuscript. For example, experimental data on how specific mutations in the polyQ tract influence ELF3 behavior in vivo would significantly bolster the authors' claims. The manuscript would benefit from either citing existing experimental studies that corroborate these findings or from suggesting future experimental directions.

Přesun koment od Štěpána z hlavní verze, kde může zmizet:

Zkusil jsem napsat ještě obecnější úvod, myslím, že může fungovat i bez úpravy následující podkapitoly. Ztratily se tam odkazy, ty jsou zde: https://mmrcz.sharepoint.com/:w:/r/sites/MMR184Oddlenkoncepcedostupnhobydlen/_layouts/15/doc2.aspx?sourcedoc=%7B05AE0777-8EB0-4AB6-9B68-B8FDE54C0D32%7D&file=k%20p%C5%99epracov%C3%A1n%C3%AD_Report%20_data%20brief_draft.docx&action=default&mobileredirect=true

Předkládaný report je první ucelenou iniciativou státu při mapování a hodnocení dostupnosti bydlení v Česku. Ministerstvo pro místní rozvoj v oblasti politiky bydlení vydávalo celkovou analýzu vývoje v oblasti bydlení vždy v rámci Koncepce bydlení České republiky a také každoročně publikovalo Vybrané údaje o bydlení. Tyto analýzy se však specificky hodnocením dostupnosti bydlení nezabývaly, současně periodicita publikování neumožňovala pracovat vždy s nejnovějšími daty. Krize dostupnosti bydlení se mezitím dostala do veřejné i politické debaty a volné místo v hodnocení dostupnosti bydlení zaplnily nestátní subjekty. MMR se rozhodlo tento deficit dohnat, a proto se začalo intenzivně zabývat sběrem a integrací ověřených dat o dostupnosti bydlení a jejich publikováním. Nejnovější data jsou nyní publikována v interaktivním dashboardu, který je pravidelně aktualizovaný. Samotná data a jejich vizualizace však nestačí pro hodnocení vývoje. MMR proto začíná s publikací každoroční Zprávy o dostupnosti bydlení, která bude na základě rigorózní metodologie a s pomocí nejkvalitnějších ověřených dat vývoj v oblasti dostupnosti bydlení hodnotit každý rok a poskytovat tak ověřenou a podloženou evidenci pro státní politiky bydlení.

This part demonstrates the extent to which the book has been acknowledged and discussed. Numerous scholarly journals reviewed it, and it was even covered by podcasts and the Washington Post which is awesome. This implies that the book has had a significant influence on the general population as well as the academic community.

This shows how the book has gained significant attention across different forms of media. It was reviewed in academia, professional, and public platforms. It has relevance across multiple fields of audiences.

this is good

Ruch, a układ cholinergiczny

Rola projekcji wzgórzowo - korowych w wykrywaniu wskazówek

Przykład działania ACh na przykładzie nawigacji samochodem po mieście

Rola sygnalizacji cholinergicznej w wykrywaniu wskazówek (opracowane na przykładzie)

De informatie is van belang bij de besluitvorming binnen een bedrijf

kjdsflkdsajfkdshfhd

或多或少的混杂着...

Koordynacja operacji poznawczych skupionych na wykorzystywaniu informacji dostarczonych przez wskazówkę i zaangażowane w ten proces struktury mózgu.

moore's law everything improved

(CESPE/2017) Os vícios relativos ao interesse de agir e à legitimidade podem ser reconhecidos a qualquer tempo, mesmo após o trânsito em julgado da ação. (ERRADO)

Art. 485. O juiz não resolverá o mérito quando:

VI - verificar ausência de legitimidade ou de interesse processual;

§ 3º O juiz conhecerá de ofício da matéria constante dos incisos IV, V, VI e IX, em qualquer tempo e grau de jurisdição, enquanto não ocorrer o trânsito em julgado.

(CESPE/2017) Integram as condições da ação o interesse de agir e a legitimidade ad causam. (CERTO)

(CESPE/2017) O interesse processual deverá estar presente tanto para propor quanto para contestar a ação. (CERTO)

Author response:

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

(1) We agreed that there was insufficient evidence for the authors' conclusion that Myc-overexpressing clones lacking Fmi become losers. We request that the authors change the text to discuss that suppression of Myc clone growth through Fmi depletion is reminiscent of a cell acquiring loser status, although at this point in the manuscript there is no clear demonstration whether this is mostly driven by growth suppression and/or an increase in apoptosis.

We agree that at the point in the manuscript where we have only described the clone sizes, one cannot make firm conclusions about competition, so we have changed the language to reflect this. We argue that after showing our apoptosis data, those conclusions become firm. Please see the more lengthy responses to reviewers below.

(2) We agreed that the apoptosis assay, data and interpretation need to be improved. The graphs in Fig. 4O and P should be better discussed in the text and in the legend. Additionally, the graphs are lacking the red lines that are written in the text.

We regret that we did not adequately explain the data displayed in these two graphs. Supercompetition tends to cause apoptosis in both winners and losers, with the ratio between WT and super-competitor cells being critical in deciding the outcome of competition. We wanted to represent this visually but failed to properly explain our analysis. We have rewritten the figure legend and our discussion in the main text, hopefully making it clearer. 

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

Strengths:

Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

Weaknesses:

I have read the revised manuscript and have found issues that need to be resolved. The biggest concern is the overstatement of the results that loss of fmi from Myc-overexpressing clones turns them into losers. This is not shown in a compelling manner in the revised manuscript and the authors need to tone down their language or perform more experiments to support their claims. Additionally, the data about apoptosis is not sufficiently explained.

We take issue with this reviewer’s framing of their criticism. First, the reviewer is selectively reporting the results published in PMID: 20679206. They correctly state that those authors show that small discreet clones of RasV12 lgl are eliminated (Fig. 3B), but they omit the fact that the authors also show that larger RasV12 lgl clones induce apoptosis in the surrounding wild type cells, and therefore behave as winners (Fig. 3C). Hence, the size of the clone appears to determine its winner/loser status. Of course, lgl is not scrib, and it is not a certainty that they would behave similarly, but they also show that large RasV12 scrib clones induce considerable apoptosis of the neighboring wild type cells. 

The reviewer then discusses “continuous” clones induced by ey-flp, as we use in our manuscript. Here, the term “continuous” is probably misleading; because ey is expressed ubiquitously in the disc from early in development, it is most likely the case that the majority of cells have flipped relatively early, resulting in ~half the cells becoming clone and the other ~half twin spot. The clone cells then likely fuse to make larger clones. We show that ey-flp induced RasV12 scrib clones also behave as winners. It is logical to conclude that this is because they are large. The reviewer talks about “a privileged environment that insulates them from competition,” but if they were insulated from competition, how could they become winners? Because they occupy more territory than the wild type cells, and because they induce apoptosis in the wild type neighbors, they are winners. 

Having shown that ey-flp induced RasV12 scrib clones behave as winners, we then remove Fmi from these clones, and show that they behave as losers by the same criteria: they occupy less area than the wild type cells (our Fig. 1 and Fig. 1 Supp 2), and they induce apoptosis in the wild type cells (our Fig 4A-H). 

With respect to the comment about additional experiments are needed to support the claim that loss of Fmi from Myc winners converts them to losers, we’re not sure what additional data the reviewer would want. As for the tumor clones, we show that >>Myc clones get bigger than the twin control clones (Fig. 2), and we measure similar low levels of apoptosis in each (Fig. 4I-K, O). In contrast >>Myc fmi clones are out-grown by wild type clones, and apoptosis is higher in the >>Myc fmi clones than in the wild type clones (Fig. 4L-N, P-S). We therefore believe it is correct to say that >>Myc clones become losers when Fmi is removed.

In additional comments, the reviewer takes issue with using winner and loser language at the point in the manuscript where we have only shown the clone sizes but not yet the apoptosis data, and about this we agree. We have changed the language accordingly. 

Re explanation of the apoptosis data, see the response to reviewer #3.

Reviewer #2 (Public review):

Summary:

In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

Strengths:

(1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.

(2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

Weaknesses:

(1) The authors provide a superficial description of the related phenotypes, lacking a mechanistic understanding of how Fmi regulates cell competition. While induction of apoptosis and JNK activation are commonly observed outcomes in various cell competition conditions, it is crucial to determine the specific mechanisms through which they are induced in fmi-depleted clones. Furthermore, it is recommended that the authors utilize the power of fly genetics to conduct a series of genetic epistasis analyses.

We agree that it is desirable to have a mechanistic understanding of Fmi’s role in competition, but that is beyond the scope of this manuscript. Here, our goal is to report the phenomenon. We understand and share with the reviewer the interest in better understanding the relationship between Fmi and JNK signaling in competition. The role of JNK in competition, tumorigenesis and cell death is infamously complex. In some preliminary experiments, we explored some epistasis experiments, but these were inconclusive so we elected to not report them here. In the future, we will continue with additional analyses to gain a better understanding of the mechanism by which Fmi affects competition.

Reviewer #3 (Public review):

Summary:

In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

While we did not perform a thorough analysis, our current revision of the manuscript shows Fmi staining results that do not support a change in subcellular localization of Fmi. In our images, Fmi seemed to localize similarly along the winner-loser clone boundaries, and inside and outside the clones. We cannot rule out that a subtle change in localization is taking place that could perhaps be detected with higher resolution imaging.

Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

See our comment to Reviewer 2 regarding JNK.

Strengths:

A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term) Weaknesses:

The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

We agree that in the future, it will be desirable to gain a mechanistic understanding of Fmi’s role in competition.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

I have read the revised manuscript and have found issues that need to be resolved. The biggest concern is the overstatement of the results that loss of fmi from Myc-overexpressing clones turns them into losers. This is not shown in a compelling manner in the revised manuscript and the authors need to tone down their language or perform more experiments to support their claims.

(1) I do not agree with the language used by the authors last paragraph of p. 4 stating loss of fmi from Myc supercompetitors (Fig. 2) makes them losers. At this point in the paper, they only use clone size as a readout. By definition, losers in imaginal discs die by apoptosis, which is not measured in this figure. As such, the authors do not prove that fmi-mutant Myc over-expressing clones are now losers at this point in the manuscript. The authors should discuss this in the results section regarding Fig. 2.

We have modified the language in text and figure legend to acknowledge that the clone size data alone do not demonstrate competition.

(2) Related to point #1, I do not agree with the language in the legend of Fig. 2H that the graph is measuring "supercompetition". They are only measuring clone ratios, not apoptosis. Growing to a smaller size does not make a clone have loser status without also assessing cell death.

(a) I suggest that the authors remove the sentence "A ratio over 0 indicates supercompetition of nGFP+ clones, and below 0 indicates nGFP+ cells are losers." in the legend to Fig. 2H. Instead, they should describe the assay in times of clone ratios.

The reviewer raises a valid point, as at this point in the manuscript we did not quantify cell death and proliferation. However, based on decades of knowledge of supercompetiton, Myc clones are classified as super-competitors in every instance they’ve been studied. (Myc clones show apoptosis when competing with WT cells, while at the same time they eliminate WT neighbors by apoptosis to become winners. Their faster proliferation rate may be what ultimately makes them winners.) We changed the language to address this distinction. 

(3) In Fig. 4, they do attempt to monitor apoptosis, which is the fate of bona fide losers in imaginal tissue. However, I have several concerns about these data (panels 4I-K, O and P have been added to the revised manuscript.)

(a) In Fig. 4I-K, why is there no death of WT cells which would be expected based on de la Cova Cell 2004? The authors need to comment on this.

(b) Cell death should also be observed in the Myc over-expressing clones but none is seen in this disc (see de la Cova 2004 and PMID: 18257071 Fig. 4). The authors need to comment on this.

We do not understand why the reviewer raises these two points. We see some cell death in >Myc eye discs both in winners and losers, as displayed in the graph. In our hands, the levels were on average very low. The example shown is representative of the analysis and shows apoptosis both in WT and >Myc cells, highlighted by the arrows in 4J. We added a mention to the arrows in the figure legend to make it clearer. In the main text, we already compared our observations to the same publication the reviewer mentions (De la Cova 2004). 

(c) The data in panel 4O is not explained sufficiently in the legend or results section. What do the lines between the data points in the left side of the panel mean? Why is there a bunch of clustered data points in the right part of the Fig. 4O, when two different genotypes are listed below? I would have expected two clusters of points. The authors need to comment on this.

We intended to convey as much information as possible in an informative manner in these graphs, and we regret not explaining better the analysis shown. We modified the legends for the apoptosis analysis to better explain the displayed data.

(d) What is the sample size (n) for the genotypes listed in this figure? The authors need to comment on this and explicitly list the sample size in the legend.

We added the n for both conditions to the figure. 

(e) In panels 4L-N, why is the death occurring in the apparent center of the fmiE59>>Myc clone. If these clones are truly losers as the authors claim, then apoptosis should be seen at the boundaries between the fmiE59>>Myc clone and the WT clones. The results in this figure are not compelling, yet this is the critical piece of data to support their claim that fmiE59>>Myc clone are losers. The authors need to comment on this.

The majority of cell death in this example is observed 1-3 cells away from the clone boundary. In some cases, we observe cell death farther from the boundary, but those cells were not counted in our analyses. As described in our methods, we only considered for the analysis cells at the clone boundary or in the vicinity, as those are the ones that most probably have apoptosis triggered by the neighboring clone.

(f) There is no red line in Fig. 4O and 4P, in contrast to what is written in the legend in the revised manuscript. This should be corrected.

We thank the reviewer for catching the error about the line. We have now simplified the graph by removing the line at Y=0 and just leave one dashed line, representing the mean difference between WT and >>Myc cells.

(4) On p. 10, the reference Harvey and Tapon 2007 to support hpo-/- supercompetitor status is incorrect. The references are Ziosi 2010 and Neto-Silva 2010. This should be changed.

We thank the reviewer for the correction. While the review we provided discusses the role of the Hpo pathway in proliferation and cancer, it does not discuss competition. The reference we intended to include here was Ziosi 2010. We now cite both in the revised manuscript.

(5) The legend for Fig. 3A-H is missing from the revised manuscript. This needs to be added.

This was likely a copy-edit glitch. The missing parts of the legend have been restored.

(6) Material and methods is missing details on the hs-induced clones. The authors need to specifically state when the clones were generated and when they were analyzed in hours after egg laying.

The timing of the heat-shock and analysis was described in the methods: “Heat-shock was performed on late first instar and early second instar larvae, 48 hrs after egg laying (AEL). Vials were kept at 25ºC after heat-shock until larvae were dissected”. And additionally, in the dissection methods: “Third instar wandering larvae (120 hrs AEL) were dissected…” We have included in this revision the length of the heat-shock (15 min). 

I have read the rebuttal and some of my concerns are not sufficiently addressed.

(8) I raised the point of continuously-generated clones becoming large enough to evade competition, and I disagree with the authors' reply. I think that competition of RasV12, scrib (or lgl) competition largely depends the size of the clone, which is de facto larger when generated by continuous expression of flp (such as eyeless or tubulin promoters used in this study). I think that at that point, we are at an impasse with respect to this issue, but I wanted to register my disagreement for the record. Related to this, one possible reason for the fragmentation of the fmimutant Myc overexpressing clones in the wing disc is because they were not continuously generated and hence did not merge with other clones.

Please see the discussion above in the public comments. We remain unclear about what, exactly, the reviewer disagrees. As stated above, we think they are correct that the size of the clone is critical in determining winner vs loser status.

Reviewer #2 (Recommendations for the authors):

Although the authors have addressed some of my concerns, I still feel that a detailed mechanistic understanding is essential. I hope the authors will conduct additional experiments to solve this issue.

We also consider the mechanism of interest and will pursue this in the future. To test our hypotheses we require a set of genetic mutants that are still in the making that will help us dissect the function and potential partners of Fmi, and we hope to have these results in a future publication.

Reviewer #3 (Recommendations for the authors):

- There is no clear demonstration that the relative decrease of clone size in UASMyc/Fmi mutant is mostly driven by either a context dependant suppression of growth and/or an increase of apoptosis (the latter being the more classic feature of loser phenotype).

We believe that it is driven by both, and refrain from making assumptions about the magnitude of contribution from each. This question is something that we will be interested to explore in the future.

The distribution of cell death in Fmi/UAS-Myc mutant is somehow surprising and may not fit with most of the competition scenarios where death is mostly restricted to clone periphery (although this may be quite variable and would require much more quantification to be clear).

While we observe some cell death far from clone boundaries, most of the dying cells are a few cells away from a clone boundary. In other publications quantifying cell death, examples of cell death farther from the boundary are not rare (See for example Moreno and Basler 2004 Fig 6, De la Cova et al. Fig 2, Meyer et al 2014 Fig 2). We did not count cells dying far from clone boundaries in our analysis.

I just noticed a few mistakes in the legend :

Figure 3M legend is missing (it would be useful to know at which stage the quantification is performed)

Another reviewer brought to our attention the problems with Fig 3 legend. We restored the missing parts.

It would be good to give an estimate of the number of larvae observed when showing the representative cases in Figure 1 .

This is a good point. We now include these numbers in the figure legend.

Because you use the word hard twice think about changing one of them to another word. I already recommend thesaurus.com

Unclear comma usage - there is a comma after "to some" but not "to others"

Author response:

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

Reviewer #1 (Public Review):

The manuscript by Poltavski and colleagues describes the discovery of previously unreported enteric neural crestderived cells (ENCDC) which are marked by Pax2 and originating from the Placodes. By creating multiple conditional mouse mutants, the authors demonstrate these cells are a distinct population from the previously reported ENCDCs which originate from the Vagal neural crest cells and express Wnt1.

These Pax2-positive ENCDCs are affected due to the loss of both Ret and Ednrb highlighting that these cells are also ultimately part of the canonical processes governing ENCDC and enteric nervous system (ENS) development. The authors also make explant cultures from the mouse GI tract to detect how Ednrb signaling is important for Ret signaling pathways in these cells and rediscovers the interactions between these 2 pathways. One important observation the authors make is that CGRP-positive neurons in the adult distal colon seem to be primarily derived from these Pax2-positive ENCDCs, which are significantly reduced in the Ednrb mutants, thus highlighting the role of Ednrb in maintaining this neuronal type.

I appreciate the amount of work the authors have put into generating the mouse models to detect these cells, but there isn't any new insight on either the nature of ENCDC development or the role of Ret and Ednrb. Also, there are sophisticated single-cell genomics methods to detect rare cell type/states these days and the authors should either employ some of those themselves in these mouse models or look at extensively publicly available single-cell datasets of the developing wildtype and mutant mouse and human ENS to map out the global transcriptional profile of these cells. A more detailed analysis of these Pax2-positive cells would be really helpful to both the ENS community as well as researchers studying gut motility disorders.

We would like to point out that the reviewer’s comments in both Public Review and in some cases reiterated in Recommendations for the Authors are rooted in several misunderstandings. The reviewer writes “Pax2-positive ENCDCs”, as if the Pax2 lineage (properly, the Pax2Cre-labeled lineage) of the ENS is a subset of neural crest, and states that “there isn’t any new insight” from our study on ENS development. Our conclusion is quite different, that the Pax2Cre lineage (placode-derived) is distinct from the neural crest-derived cell lineage. The reviewer may not have appreciated that our study establishes a fundamental reinterpretation of the very long-standing dogma that the ENS is derived solely from neural crest. We believe that finding and characterizing the unique contribution of an independent cell lineage to the ENS provides critical new perspectives into ENS development and the etiology of Hirschsprung disease. One feature of the Pax2Cre (placodal) lineage is as the source of CGRP-positive mechanosensory neurons in the colon (as the reviewer mentioned), but this is one feature of the larger conceptual discovery of the existence of a separate lineage contribution to the ENS, not the most important observation in and of itself.

The reviewer continues by saying that we “rediscovered” the interaction between Ednrb and Ret in ENS development. In our study we show that the two lineages (placode-derived and neural crest-derived) employ Ednrb and Ret signaling in distinct ways. This isn’t simply rediscovery, this is new insight. To the extent that both lineages utilize both signaling axes (albeit with mechanistic differences) is a primary reason why the unique placodal lineage contribution to the ENS remained unsuspected until now. We have revised the text to make these points more clear in our revised manuscript.

The reviewer also suggests single cell genomic methods, which is addressed below in our response to the reviewer’s first recommendation.

Reviewer #2 (Public Review):

This manuscript by Poltavski and colleagues explores the relative contributions of Pax2- and Wnt1- lineagederived cells in the enteric nervous system (ENS) and how they are each affected by disruptions in Ret and Endrb signaling. The current understanding of ENS development in mice is that vagal neural crest progenitors derived from a Wnt1+ lineage migrate into and colonize the developing gut. The sacral neural crest was thought to make a small contribution to the hindgut in addition but recent work has questioned that contribution and shown that the ENS is entirely populated by the vagal crest (PMID: 38452824). GDNF-Ret and Endothelin3-Ednrb signaling are both known to be essential for normal ENS development and loss of function mutations are associated with a congenital disorder called Hirschsprung's disease. The transcription factor Pax2 has been studied in CNS and cranial placode development but has not been previously implicated in ENS development. In this work, the authors begin with the unexpected observation that conditional knockout of Ednrb in Pax2-expressing cells causes a similar aganglionosis, growth retardation, and obstructed defecation as conditional knockout of Ednrb in Wnt1-expressing cells. The investigators then use the Pax2 and Wnt1 Cre transgenic lines to lineage-trace ENS derivatives and assess the effects of loss of Ret or Ednrb during embryonic development in these lineages. Finally, they use explants from the corresponding embryos to examine the effects of GDNF on progenitor outgrowth and differentiation.

Strengths:

-  The manuscript is overall very well illustrated with high-resolution images and figures. Extensive data are presented.

-  The identification of Pax2 expression as a lineage marker that distinguishes a subset of cells in the ENS that may be distinct from cells derived from Wnt1+ progenitors is an interesting new observation that challenges the current understanding of ENS development.

-  Pax2 has not been previously implicated in ENS development - this manuscript does not directly test that role but hints at the possibility.

-  Interrogation of two distinct signaling pathways involved in ENS development and their relative effects on the two purported lineages.

The reviewer provided a succinct and accurate summary of our analysis. We correct just the one statement that the ENS is entirely populated by vagal crest. The paper cited by the reviewer (PMID: 38452824) used Wnt1DreERT2 to lineage label the NC population, so of course only looked at neural crest (comparing vagal vs. sacral NC). The advance in our study is to newly document the independent contribution of the placodal lineage.

Weaknesses:

-  The major challenge with interpreting this work is the use of two transgenic lines, rather than knock-ins, Wnt1Cre and Pax2-Cre, which are not well characterized in terms of fidelity to native gene expression and recombination efficiency in the ENS. If 100% of cells that express Wnt1 do not express this transgene or if the Pax2 transgene is expressed in cells that do not normally express Pax2, then these observations would have very different interpretations and not support the conclusions made. The two lineages are never compared in the same embryo, which also makes it difficult to assess relative contributions and renders the evidence more circumstantial than definitive.

We do not agree that the Cre lines being transgenics rather than knock-ins changes the utility of these reagents or the interpretation of the results; there are also potential problems with knock-in alleles. Wnt1Cre has been in use for 25 years as a pan-neural crest lineage cell marker with exceptional efficiency and specificity (including numerous studies of the ENS), so we disagree that it is not well characterized. Pax2Cre of course has not previously been studied in the ENS, but it has been broadly used in other contexts (e.g., craniofacial, kidney). That said, and as noted in our original manuscript, we are aware that an issue of this study is the uniqueness of the recombination domains of the two Cre lines.  As we wrote, Wnt1Cre and Pax2Cre cannot be combined into the same embryo because they are both Cre lines, and we do not have a suitable nonCre recombinase line to substitute for either. Instead, we demonstrate that the two lines recombine in distinct territories of the early embryonic ectoderm, and that the two lineages thus labeled are distinct in marker expression at the initial onset of their delamination, utilize Edn3-Ednrb and GDNF-Ret in distinct ways during their migration to the hindgut, and contribute to different terminal cell fates in the colon. We think this evidence of the distinct nature of the two lineages from start to finish is compelling rather than merely circumstantial.

-  Visualization of the Pax2-Cre and Wnt-1Cre induced recombination in cross-sections at postnatal ages would help with data interpretation. If there is recombination induced in the mesenchyme, this would particularly alter the interpretation of Ednrb mutant experiments, since that pathway has been shown to alter gut mesenchyme and ECM, which could indirectly alter ENS colonization.

We have several thoughts about this comment. First, we are uncertain why postnatal analysis would be informative, as ENS colonization occurs (or fails to occur in mutants) during embryogenesis. The reviewer might be thinking of a juvenile stage additional contribution to the ENS, which is addressed below (responses to Recommendations for the Authors) but as we discuss there is not relevant to our analysis. Second, we did examine recombination in the distal hindgut at E12.5 during ENS colonization (Fig. 1f and 1h) and did not see overlap between either Cre recombination domain and Edn3 mRNA expression (which is expressed by the nonENS mesenchyme). Furthermore, Ednrb is not expressed in the gut mesenchyme during ENS colonization (Fig. 7figure supplement 1), thus ectopic mesenchymal Cre expression, if any, by either line would have no impact in Cre/Ednrb mutants. Lastly, the reviewer’s idea could have been a plausible hypothesis at the onset of the project, but here we show positive evidence for a different explanation. We do not rigorously exclude the reviewer’s hypothesis, nor other theoretically possible models, but we think we have provided a strong case to support the direct involvement of Ret and Ednrb in ENS progenitors rather than in surrounding non-neural mesenchyme.

-  No consideration of glia - are these derived from both lineages?

To properly address this question would require new reagents and analyses that we have not yet initiated. While an interesting question from a developmental biology standpoint, we don’t think that this investigation would change any of the interpretations that we make in the manuscript.

-  No discussion of how these observations may fit in with recent work that suggests a mesenchymal contribution of enteric neurons (PMID: 38108810).

The recent paper cited by the reviewer is very explicit in describing this mesenchymal contribution to the ENS as occurring after postnatal day P11. Other than the terminal Hirschsprung phenotype, all of our analysis of cell lineage migration and fate and colonic aganglionosis was conducted at embryonic or early (P9) postnatal stages. We therefore do not see a relation of our work to this study. In light of this paper, however, we do agree that it would be worthwhile in a future study to explore Wnt1Cre and Pax2Cre lineage dynamics in the ENS of older mice.

Reviewer #1 (Recommendations For The Authors):

(1) The authors should reanalyze multiple single-cell RNA-seq datasets available now, to see if these cells are detected in those studies and then look at the global transcriptional profile of these Pax2-positive cells compared to the other vagal neural crest-derived ENCDCs. Some of these datasets can be found here - PMIDs: 33288908, 37585461, and https://www.gutcellatlas.org/.

We disagree that the datasets from previous studies provide additional insights that are relevant to the current study. It must be appreciated that Wnt1Cre and Pax2Cre are genetic lineage tracers and that migratory ENS progenitor cells labeled with these reagents do not maintain expression of Wnt1 and Pax2 mRNA or protein. The Wnt1 and Pax2 genes are only transiently expressed within their distinct regions of the ectoderm, and their expression turns off as cells delaminate and begin migration. Thus, Pax2Cre-labeled ENS progenitor cells are not Pax2-positive thereafter. The single cell RNA-Seq studies suggested by the reviewer were collected from older embryos and postnatal mice, and do not represent the E10.5-E11.5 period that accounts for genesis of Ret-mediated and Ednrb-mediated Hirschsprung disease pathology. Even with the most recent work by Zhou et al (Dev Cell, 2024) that included E10.5 cells, this analysis only evaluated neural crest-derived Sox10Cre lineage cells, which does not include the placode-derived Pax2Cre lineage (as we show explicitly in Fig. 2-figure supplement 2).  Consequently, it would not be possible to find the “Pax2-positive cells” in these datasets. Performing a new transcriptomic analysis by isolating Pax2Cre-lineage and Wnt1Cre-lineage cells at the appropriate developmental time points could be the basis of future studies, but we think these are beyond the scope of the present paper. 

(2) Even in their current quantification method of using immunofluorescent cells in a microscopic field, the authors count very few cells. The quantification in Figures 2v-2z is only from 4 embryos and is in the hundreds. This leads to misrepresentation of cell numbers and is best reflected in Figure 2x, where Wnt1Cre/Ret GI tracts have 0 Ret +ve cells, which we now know is not true even in ubiquitous Ret null embryos, where Ret null cells are detected as late as E14.5 (PMID 37585461)

Because of the reviewer’s comment, we recognize that the specific detail about cell numbers wasn’t properly written. We didn’t count a few hundred cells total, it was a few hundred cells per embryo. Exact numbers are provided in the revised figure legend where “cells/embryo” is now explicitly stated. Multiplied by the number of embryos, this means that we evaluated approx. 1000 total cells per genotype and time point in cases where Ret+ and/or GFP+ (lineage+) cells were found. The total absence of such cells in Wnt1Cre/Ret mutants is a rigorous conclusion. Our results do not misrepresent nor contradict the study by Vincent et al (PMID 37585461). Our analyses were performed on gut tissue isolated at E10.5 and E11.5 stages, which is long before Schwann cell precursors (SCPs, the primary focus of the Vincent et al study) colonize the gut (E14.5; Uesaka et al, 2015. PMID: 26156989). Indeed, as the reviewer notes, SCPs migrate into the gut in a Retindependent manner. For being at a much earlier time point, our focus is on the cranial ectoderm sources of ENS progenitors. We have adjusted the text associated with Fig. 2 to make this more clear.

(3) There are multiple sections in the manuscript that rehash already known facts, like the whole section about Wnt1 conditional Ret null mice which show failure of migration of ENCDCs. This has been shown multiple times and doesn't add anything to the author's story.

We think this comment stems from the reviewer’s perception that the Pax2Cre lineage is a subset of neural crest. The Wnt1Cre data (including Ret-deficient and Ednrb-deficient embryos) presented in the manuscript are not intended to rehash what is already known but to establish important similarities and differences between the newly identified placode-derived and the well-established neural crest-derived ENS progenitor cells. In light of the reviewer’s suggestion #8 below, to move the Wnt1Cre lineage analysis to a supplement, this information remains in the main text to provide proper comparison to the Pax2Cre-lineage profile. We think we were fair in the text to the legacy of work on neural crest and ENS development and were explicit in using our Wnt1Cre analysis to compare to the Pax2Cre lineage. Finally, we point out that our analysis was conducted on a different genetic background (outbred ICR) compared to previous studies, and there are strain-specific differences in Hirschsprung-associated lethality between our background and previous studies, so it was not impossible that the behavior of the neural crest cell lineage in the ICR background could be different from past observations on different backgrounds. Although we did not identify any major differences, it is important that the information on NC behavior in this background be presented. 

(4) Also, the conclusion drawn for Figure 5C "this indicates that the Wnt1Cre-derived cells do not harbor a cellautonomous response to GDNF" seems to suggest the authors are not very well versed with the ENS literature. GDNF as well as EDN3 are expressed from surrounding mesenchyme and are cell non-autonomous.

The reviewer seems to have misread or misunderstood the specific statement as well as the more important broader conclusion of the experiment. First, of course the source of GDNF ligand in vivo is the mesenchyme. The explant assay was designed to eliminate this and then to substitute GDNF as provided experimentally. The focus of the experiment was to address the response to GDNF, not the source of GDNF. But more importantly, the experiment revealed a surprising outcome that the reviewer did not appreciate. In Pax2Cre/Ret mutants, the Wnt1Cre lineage still expresses Ret, yet does not grow out from the gut explant when provided with GDNF. This shows that the neural crest lineage requires Ret function in placode-derived cells in order to respond to GDNF. In other words, despite expressing Ret, the NC lineage does not harbor a cellautonomous response to GDNF, as we wrote. Because this might be confusing to some readers, we have revised the description of this analysis to hopefully be more clear.

(5) The fact that Ret and Ednrb signaling pathways interact is not a novel finding and has been reported multiple times in Ret and Ednrb mutant mice and cell lines (PMID: 12355085, 12574515 , 27693352, 31818953), potentially through shared transcription factors (PMID:31313802).It would have been more relevant if the authors could show how the specific tyrosine residue (Y 1015) in Ret is phosphorylated in the presence of Ednrb.

The observation that human mutations in RET and EDNRB both cause Hirschsprung disease is decades old, and of course numerous studies in human, mouse, and cells have addressed the relation between the two signaling pathways. We did not mean to imply that we were the first to discover that Ret and Ednrb signaling pathways interact. The reviewer cites a number of papers all from the Chakravarti lab that address this phenomenon; while these are a valuable contribution to the field, there is still more to be learned. The model elaborated in PMID: 31313802, in which Ret and Ednrb are both enmeshed in a common gene regulatory network, does not readily explain why each has a different phenotypic manifestation and doesn’t take into account the importance of the placodal lineage. The main new contributions of our paper are the existence of a new cell lineage that contributes to the ENS, and that the placodal and neural crest lineages utilize Ret and Ednrb signaling differently. The clarification of how these elements are differentially used by the two lineages explains long-segment and short-segment Hirschsprung disease (Ret and Ednrb mutants, respectively) far better than in past studies. The reviewer unfortunately dismisses these insights and seems to feel that a biochemical exploration of one specific component of the signaling interaction (Y1015 phosphorylation) would be more relevant. This should be the basis of future studies and are beyond the scope of the new findings reported in the present paper. 

(6) What is the mechanism of the presence of Y1015 phosphorylation in 33% of Ednrb deficient Pax2Cre cells? It appears to me what the authors report as absent phosphorylation in the 67% of cells could be just weak staining or cells missing in prep.

The reviewer, referring to Fig. 7q, presumably meant to say Wnt1Cre rather than Pax2Cre. The reviewer overlooked that we provided an explanation for this observation in our original manuscript. This sentence reads “Because Ednrb is expressed only in a subset of Wnt1Cre-derived enteric progenitor cells (Figure 7 – figure supplement 1), the residual Y1015 phosphorylation observed in Wnt1Cre/Ednrb mutant cells is likely to occur in the Ednrb-negative Wnt1Cre-derived cell population”. The sentence is retained unchanged in the revised manuscript. The explanation is not because of weak staining or problems with tissue preparation.

(7) The references the authors cite regarding the previous discovery of Ret expression in the nucleus are incorrect. The review articles the authors cite do not mention anything about Ret expression in the nucleus. The evidence of nuclear localization of Ret previously comes from overexpression studies in HEK293 cells (PMID: 25795775). Such overexpression studies are fraught with generating noisy data for well-documented reasons. But if this observation is correct, the authors miss a great opportunity to identify what the Ret protein is doing in the nucleus. Is it in direct contact with its known transcription factors like Sox10 and Rarb? This would shed a lot of light on the possible mechanism of Ret LoF observed in Ret mutant mice

The reviewer overlooked that the one of the review articles that we cited (Chen, Hsu, & Hung, 2020) has a dedicated paragraph for RET (section 3.14), which summarizes the work by Barheri-Yarmand et al (PMID: 25795775) which is the very paper noted by the reviewer in the comment above. The reviewer also somewhat misstated the results of the Barheri-Yarmand et al study. By immunostaining, this paper showed nuclear localization of endogenous Ret, albeit a version of Ret with a disease-associated mutation that makes it constitutively active by constitutive autophosphorylation. Nonetheless, this was endogenous Ret. The paper also used overexpression of GFP-tagged RET in HEK293 cells to show that wildtype RET can behave in a similar manner, at least under these circumstances. Our point is simply that Ret (and other receptor tyrosine kinases) can be found in the nucleus in certain biological contexts, and our observations are consistent with this precedent.

The reviewer also suggests a biochemical follow-up analysis related to this observation, which we agree would be of interest. Such an investigation however is beyond the scope of the present study.

(8) The manuscript could benefit from a major rewrite by reorganizing sections to make it easy for the readers to follow the narrative.

Many sections about the role of Ret and Ednrb in Wnt1cre-derived ENCDCs can be moved to a supplement. These facts are well-documented and have been proven before.

This was addressed in our response to comment #3 of this reviewer. The figures have been kept as main figures in the revised manuscript to allow side-by-side comparison to parallel analysis of the Pax2Cre lineage.

- The observation that only a handful of Pax2Cre cells at E10.5 express Ret and the observation that conditional Ret null abrogates these cells at E11.5, are not presented together and makes connecting these two facts difficult.

Ret expression at E10.5 and E11.5 are both shown in the same figure (Fig. 2). In the presentation of these results, we first describe in normal development that Ret is expressed differently in E10.5 ENS progenitors between the Pax2Cre and Wnt1Cre lineages. This is additional support for the argument that the two lineages are molecularly distinct. Then comes evaluation of postnatal fates with different markers before we return to embryonic Ret expression. We acknowledge that this can make it difficult to connect these observations. We decided to retain the original organization in order to not lose this important conclusion. However, we have revised the text to hopefully make this connection between the sections more congruent.

Reviewer #2 (Recommendations For The Authors):

- The labeling of some as "figure supplements" is really hard to follow in the text and confusing to interpret when a main figure or supplemental figure is being referenced, and which one.

We understand this comment, but this is journal style and outside of our control. We have kept the journal format in the revised manuscript.

- The data in Figures 3b-c is well established in the field and somewhat misinterpreted. NOS1 neurons in the mouse ENS and their projections have been well described (Sang and Young, 1996, and other studies). CGRP immunoreactivity would reflect both ENS CGRP-expressing neurons and visceral afferents from DRG.

There of course is a history of analysis of NOS1, CGRP, and other markers in the ENS. The focus of the analysis in Fig. 3 is to demonstrate how the cells that express these markers are impacted by gene manipulation in the Wnt1Cre and Pax2Cre lineages. For the giant migrating contractions that are associated with defecation, ample past electrophysiological studies have established that mechanosensory CGRP+ neurons trigger NOS+ inhibitory neurons (and ACh+ excitatory neurons) of the myenteric plexus to propel colonic contents. Thus, these are the relevant markers to explain the lack of colonic peristalsis in Ednrb-deficient mice. To our awareness, our results with NOS1 do not contradict any past study, including the Sang and Young 1996 description. Regarding CGRP, indeed the reviewer is correct that this marker is expressed by both neuronal subtypes. Two arguments support the specific derivation of ENS mechanosensory neurons from the Pax2 lineage. First, the ENS and DRG neurons can be distinguished by the location of their cell bodies and their axon extensions in the gut wall; only the ENS neurons are deficient in Pax2Cre/Ednrb mutants (as documented in Fig. 3). Second, the DRG population is derived from neural crest and is not labeled by Pax2Cre. If this population of CGRP+ neurons had functional relevance to colonic peristalsis, this would not be altered in Pax2Cre/Ednrb mutants. Indeed, the CGRP+ afferent nerve endings of DRG origin in the distal colon are mechanical distension sensors but do not modulate either ENS or autonomic nervous system activity (PMID: 37541195). We believe that our interpretation is correct.

- The evidence in Figure 3 supporting the claim that NOS1 and CGRP-expressing enteric neurons come from distinct lineages is weak. IHC for CGRP is notoriously poor at labeling soma in the ENS. IHC for tdTomato to ensure the detection of low levels of Tomato expression and quantification of observations would strengthen this claim.

CGRP is a vesicular peptide which is stored and transported in vesicles, therefore the antibody against CGRP labels vesicular particles of soma and synaptic vesicles along the axons of those CGRP-producing neurons.

It is not expected to label the entire cytoplasm (or the range of subcellular organelles) as NOS antibody does. We did included quantification of data in Figure 3-figure supplement 1 in the manuscript to support the claim of lineage derivation. As described in the Methods section of the manuscript, we used binary threshold selection for Tomato+ cell count using Fiji-Image J, which detects both TomatoHigh and TomatoLow cells as Tomato+; we feel this is equal to or even superior to IHC for this analysis. 

- IHC panels in Figures 3h-o are largely uninterpretable. Most of the signal seems to be non-specific background staining in the mucosa and quantification of mucosal signal in this context does not seem meaningful.  

We disagree with the reviewer’s comment. As described in the response above, CGRP+ mechanosensory neurons send their peripheral axon projections to innervate mucosa (sensory epithelial cells), and NOS+ inhibitory motor axons innervate the circular muscle. Thus, panels h-o of Fig. 3 focus on the axonal profile and are not intended to visualize soma, which is why sagittal views are presented instead of flatmount views. All of the controls were performed side-by-side to confirm that the signal is real and interpretable.

Note also that the colon does not have villi so this annotation should be revised.

We appreciate that the reviewer brought this misstatement to our attention. We corrected this error in the revised manuscript.

- Phospho-RET staining in Figure 7 is difficult to discern and interpret with high background. Positive and negative controls would strengthen these data.

Fig. 7 shows phospho Ret-Y1015 staining in lineage-labeled Wnt1Cre/Ednrb/R26nTnG mutants. The strength of the signal to noise in the figure is a matter of Ret expression level and the quality of the anti-pY1015 antibody. We are not aware of a meaningful positive control that has been validated in the literature that we could use for comparison. The ideal negative control would be to perform the same analysis in Wnt1Cre/Ret/R26nTnG mutants, but because this manipulation eliminates the entire NC cell lineage from the colon, there would be no NC cells in which to visualize background staining in this lineage with this antibody when Ret protein is not present. We note that anti-pY1096 did not show a difference in staining between control and mutant, which supports the interpretation of a specific impact on pY1015. We also point out here, as in the text, that we do not yet have any validation that phosphorylation of Y1015 is functionally important in NC migration to the distal colon. Clearly, more work to address this role and to demonstrate the mechanism of phosphorylation of this specific residue in response to Edn3-Ednrb signaling will be needed.

The hyperlink to genius is super cool! Depending on how much you want to emphasize it, you could underline or make the blue darker since I didn't realize the hyperlink was there at first

Niches" kelimesi, biyolojide bir organizmanın yaşamını sürdürebilmesi için uygun ortamı ve ekolojik rolünü ifade eder. Bir organizmanın "nişi", onun çevresiyle etkileşimi, beslenme alışkanlıkları, habitatı ve diğer türlerle olan ilişkilerini kapsar. Örneğin, bir böceğin veya bakterinin belirli bir ortamda nasıl hayatta kalıp çoğaldığını belirleyen faktörlerin tümü, o organizmanın ekolojik nişini oluşturur.

Including lyrics from Douglass’s song adds depth to your analysis and illustrates the double meanings behind spirituals in a hands-on manner. This not only enriches your argument but also invites readers to engage more deeply with the music.

Zkusil jsem napsat ještě obecnější úvod, myslím, že může fungovat i bez úpravy následující podkapitoly. Ztratily se tam odkazy, ty jsou zde: https://mmrcz.sharepoint.com/:w:/r/sites/MMR184Oddlenkoncepcedostupnhobydlen/_layouts/15/doc2.aspx?sourcedoc=%7B05AE0777-8EB0-4AB6-9B68-B8FDE54C0D32%7D&file=k%20p%C5%99epracov%C3%A1n%C3%AD_Report%20_data%20brief_draft.docx&action=default&mobileredirect=true

Předkládaný report je první ucelenou iniciativou státu při mapování a hodnocení dostupnosti bydlení v Česku. Ministerstvo pro místní rozvoj v oblasti politiky bydlení vydávalo celkovou analýzu vývoje v oblasti bydlení vždy v rámci Koncepce bydlení České republiky a také každoročně publikovalo Vybrané údaje o bydlení. Tyto analýzy se však specificky hodnocením dostupnosti bydlení nezabývaly, současně periodicita publikování neumožňovala pracovat vždy s nejnovějšími daty. Krize dostupnosti bydlení se mezitím dostala do veřejné i politické debaty a volné místo v hodnocení dostupnosti bydlení zaplnily nestátní subjekty. MMR se rozhodlo tento deficit dohnat, a proto se začalo intenzivně zabývat sběrem a integrací ověřených dat o dostupnosti bydlení a jejich publikováním. Nejnovější data jsou nyní publikována v interaktivním dashboardu, který je pravidelně aktualizovaný. Samotná data a jejich vizualizace však nestačí pro hodnocení vývoje. MMR proto začíná s publikací každoroční Zprávy o dostupnosti bydlení, která bude na základě rigorózní metodologie a s pomocí nejkvalitnějších ověřených dat vývoj v oblasti dostupnosti bydlení hodnotit každý rok a poskytovat tak ověřenou a podloženou evidenci pro státní politiky bydlení.

Since this parenthetical is contained within the same sentence, the "o" should be lowercase.

DOI: 10.1016/j.chom.2024.10.008

Resource: (ATCC Cat# TIB-71, RRID:CVCL_0493)

Curator: @vtello

SciCrunch record: RRID:CVCL_0493

What is this?

DOI: 10.1016/j.isci.2024.111179

Resource: (Abcam Cat# ab3517, RRID:AB_303866)

Curator: @scibot

SciCrunch record: RRID:AB_303866

What is this?

Works as a service to the audience- opportunity to be tactful about misrepresentation - helps the audience help the performer retain their own performance

performer must provide himself an out that is other than the actual reason

example two people having a convo that's private with others around other random unrelated people offer the courtesy or tact of not paying attention two people won't completely cease having conversation or make dramatic show of secrecy but will refrain from discussing all things out right maintain some level of secrecy while respecting or acknowledging other's "tact"

THIISSSSS

If I say that I know that you know I know you know- the whole performance reality breaks - shame or laughter

audience ignoring breaks in the performance- rude to point it out

audience allows nurse to fulfill role by participating in something they didn't want to do

outsiders make performers aware of their own prescence

outsiders themselves work so that the performance can maintain its own impression

Dramaturgical circumspection- investment and therefore foresight in how the performance will play out- anticipation of it

dramaturgical discipline- disassociation from own role as a performance- real or fake intellectual and emotional involvement in activity at stake

example- dramturgical loyalty threatened when clerks say candidly what products are actually worth buying

Dramaturgical loyalty- moral obligation to keep face from individual teammate

sometimes performer pleas to audience for their mercy because of a failure or inability to continue performing.

sometimes audience can't believe impression and confronts performers about it

sometimes this threat of politene appearances is for a certain purpose

facts about performers (of past and present relevance) can pose as threat to illusion

staging talk- talk regarding performance itself

banter = consistent team realignments

this is just a great example

impression of relaxation necessary for superordinate when working with subordinates

we usually think of lower status team as trying to break into higher status of the higher status team- but often this equalizing initiated by higher status team

in this case, specialists endanger not the disclosure of destructive information but a break in the impression of opposing sides

within opposing teams- a specialist may align himself with specialist of other team creating their own perceived team to which the rest of the world stands against.

act like extent of beliefs is minimal of other person's is mini,a;

teams show things about themselves a little bit at a time to gauge other person

unofficial communication- communication that always has potential to be denied, works to communicate and test relations between two teams communicate roles, how the intimacy of relationships should be- communicate boundaries, etc.

sometimes teammates communicate things as a part of performance but only that those "in the know" pick up on

trying to make eachother break or sharing amusement - sideyes - shows more autonomy within interaction or that they are not bound by it

derisive collusion- done by performer and hidden from higher-ups , for the performers own purpose

derisive collusion- in front of audience but not for audience and so they don't pick up on it

i squeezed my dad's hand when his prayer was taking too long

pianist highlights a note that singer should be singing

derogation of audience actually serves a purpose- keeps morale of the team and bonds them in a way that allows them to maintain face during performance

include the rapid swtich in beahviors?

other type of communication referred for backstage- using cynical or strictly technical language to describe performance behaviors

other is downplaying of politeness or name calling

first type- trash talking audience

4 types of communication not for the front stage?

!!! its not a reality and a deviation- there is an active participation in both as forming a sort of whole reality

some colleague groups collective identity more tied than others could racial groups be considered a colleague group?

colleagues let colleagues down when don't retain secretive info

colleagues form mutual agreements based on secretive information

colleagues- put on similar performances for similar audiences but not apart of the same team

confidants- people who performers relay full feelings to- people whom they tell that the performance was a performance and what aspects felt like one.

trainers bring about negative self-image not only have access to backstage but fully aware of the deficiencies within backstage as an imagined audience member

training specialists - role of building up performer requires them to imagine perspective of audeince

what does that have to do with... anything

sometimes acquisition of specialist themself is apart of the performance

why emphasis on discretion so important- that trust is hard to build when there's little incentive for it to be mutual.

difference is the specialist doesn't have the same stake in secret being revealed AND main group doesn't learn any intimacies of the specialist

• no reciprocal breakdown of front stage, specialist maintains front stage the whole time

service specialists attend to front stage but often, must obtain backstage view and destructive information to do their job

ignoring someone is a performance of itself

givens servants functions as almost invisible but visible when necessary, confusion around what behavior acceptable before them

shill, spotters, shoppers, and go-betweens perform role but don't adopt it?

foreman must maintain mindset of audience as well as performance director

go-between example that's having a negative connotation chairman showing active listening skills to encourage audience. Offers direction to audience while providing encouragement to speakers? I kind of don't get how this differs from a regular shill

another type of audience- scoping out the competition

outsiders not given impression provided by performance nor destructive information

audience knows what theyve been allowed to percieved

performers aware of impressions offered by performance + destructive information

three roled- performance, audience, outsiders

insider secrets only real stake is creating air of exclusivity for team if discovered- new secret will arise to create the affect

inside secrets- objective intellectual content

second type of secrets are strategic secrets- things hidden for sake of performance. Still hold a lot of weight and should appear that they don't exist- but in a way could be disclosed eventually.

Strategies against opposition

first type of secrets = dark secrets incompatible with image and it cannot be know that the secret is being kept

team must be able to keep secrets- some information is destructive for audience to learn

• work example- cooking corn dog

this occurs even within the same service- one audience member can't know the service other audience member

• break an illusion of personal intimacy or will let the know that they are getting sold out

makes it easier to put on right performance for each person if different audiences are separated/ don't see other sides

I HATE MIXING FRIEND GROUPS

outsiders cause problem when they are witnessing wrong performance????

one may feel more out of character (or still our of character) backstage in performance to other team members

higher requirement of decorum for higher officials

higher up someone the less the spend backstage

downfall of upward mobility- forget the backstage world that all performances (such as our own) had

"If you like a church don't join it"

frequently- we forgot the backstage of others whilst participating in a backstage environment ourselves

while backstage is more relaxed and informal- doesn't make it more happy or care free - often all enthusiasm goes to what is done in front of audience and backstage is sullen

Three nuances of backstage informality: 1. team still wants to seem trustworthy like they can be trusted with secrets of the team 2. teammate must perform for sake of other teammates moral 3. divides on other factors amongst team members

what if front stage meant to be offensive or provoking- I'm thinking of protests or uprisings

society has front stage and back stage language

back rooms and front rooms can change without even a change in equipment- depends on what room is being used for at one moment and who is present

decorations allow for performance to continue even when performers are not there

biological needs attended to in private for fear of breaking out of roles

performance often relies on the backrooms and the privacy of them

backstage- where performance is openly acknowledged as the performance

decorum is contextual- what you can do depends on where you are

in personal front- - manner = politeness - appearance = decorum

moral requirements of decorum - respecting others space and propriety

politeness- performance while engaged with audience decorum- performance while disengaged with audience

one setting can have many performance interrelations occurring - cocktail party- many conversations

some people in flip flop??- dramatic obligations + directive ones

dramtic dominance different from directive dominance - star of the ballet or sports team isn't stage director or manager

audience holds director to performance more than anyone else responding with higher demands on other performers, sets them apart

gives roles and sets the stage

often requires inciting people to action as opposed to dampening their behavior

while audience and performer can always apply to both teams, many instances where one team has more control, vested interest, and more intimately organizes performance - salesperson

when many different status available- very frequently to performers sort themselves into two performance groups

Anyone stepping out of line poses as performance risk - not just the reckless - drunks become difficult = performance risk - recluse who isn't enthusiastic enough when it requires also = performance risk

solidarity in performance always focused on putting up front in front of subordinates

withholding information from teammate = withholding identity

performers must form unanimous agreements while concealing the fact that such agreements had to be made amongst members

Lol this is so ironic- each member must perform as though they embody a role so wholly that they didn't even need to decide on something

to maintain united front- team members wait until not in public to disagree

huh

performers can be bonded by an informal agreement or not

great example- team members can't always easily be disposed of

individual behavior becomes a reflection of everyone when in this case, they're not even friends or allied

creates a formal intimacy or familiarity

performer rarely performs part in team performance for the sake of his other teammates- define the other as someone " in the know"

performance across social or other divides usually unites people while performance divided leads to divides in teams

teammates need to trust that the other will maintain face

The audience doesn't even need to be other people- it can be an imagined outward judgement or assessment of action that directs a performance when no one is looking

that one Margaret Atwood quote

can look at every coordinated performance as teams if in one-on-one each team only has one member

teamwork- yes everybody puts in their own role and performs at different levels of adequacy but the overall impression or effectiveness in selling their performance can be examined almost independent of each individual performance

like teachers switching up and calling each other "Ms. -----" in front of students

these people called a team

the expression of one role reliant on the proper expression of the other roles in performance

what does it mean to be mentally free?

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost Importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

We thank the Reviewer for their positive evaluation of our manuscript and highlighting the value of obtaining more reproducible and unbiases results, as well as detection of forward and reverse transmigration with UFMTrack.

Weaknesses:

The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.

We thank the Reviewer for this suggestion. In the revised version of our manuscript, we have now emphasized the broader applicability of UFMTrack to analyze the interaction of immune cells with 2dimensional endothelial monolayers in various contexts in the abstract, introduction, and discussion sections.

Reviewer #2 (Public Review):

Summary:

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications.

Strengths:

Algorithm is almost as accurate as manual tracking and importantly saves time for researchers.

We thank the Reviewer for this positive evaluation of our work.

Weaknesses:

Applicability can be questioned because the device used is 2D and physiological biology is in 3D. Comparisons to other automated tools was not performed by the authors.

We thank the Reviewer for pointing our attention to these weaknesses in our manuscript.

We have clarified in the revised manuscript that using 2D endothelial monolayer models in parallel laminar flow chambers is still a state-of-the-art methodology for studying the multi-step extravasation process of immune cells across endothelial monolayers under physiological flow by in vitro live cell imaging. These models provide excellent optical quality that is not yet achieved in 3D models. We have extended the introduction to emphasize the limitations of existing tools that motivated us to establish UFMTrack. We have furthermore extended the discussion section to highlight the features unique to our UFMTrack framework.

Reviewer #3 (Public Review):

Summary:

The authors aimed to establish a faster and more efficient method of tracking steps of T-cell extravasation across the blood brain barrier. The authors developed a framework to visualize, recognize and track the movement of different immune cells across primary human and mouse brain microvascular endothelial cells without the need for fluorescence-based imaging. The authors succinctly describe the basic requirements for tracking in the introduction followed by an in-depth account of the execution.

We thank the Reviewer for their positive evaluation of our manuscript and highlighting the value of label-free analysis of the multistep immune cell extravasation cascade with UFMTrack.

Weaknesses and Strengths:

Materials & methods and results:

(1) The methods section also lacks details of the microfluidic device that the authors talk about in the paper. Under physiological sheer stress, the T-cells detach from the pMBMEC monolayer, and are hence unable to be detected; however, this observation requires an explanation pertaining to the reason of occurrence and potential solutions to circumvent it to ensure physiologically relevant experimental parameters.

We thank the Reviewer for pointing out this oversight. We have used a custom-made microfluidic device that has been published and described in detail before. This information has now been included in the Methods Section under Point 7, and the two references describing the flow chamber in depth are mentioned below and have been included in the manuscript.  

Coisne Caroline, Ruth Lyck and Britta Engelhardt. 2013. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo. Fluids and Barriers of the CNS 10:7 doi:10.1186/20458118-10-7; 21 January 2013

Lyck R, Hideaki Nishihara, Sidar Aydin, Sasha Soldati and Britta Engelhardt. 2022. Modeling brain vasculature immune interactions in vitro. Angogenesis, 2nd edition. Editors PatriciaD’Amore and Diane Bielenberg Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a041185

T cell detachment is a physiologically relevant parameter besides T cell arrest, polarization, crawling, probing, and transmigration during the interaction with an endothelial monolayer. T cell detachment means that post-arrest, the T cell cannot engage adhesion molecules required for subsequent polarization and, eventually, transmigration. 

(2) The author describes a method for debris exclusion using UFMTrack that eliminates objects of <30 pixels in size from analysis based on a mean pixel size of 400 for T lymphocytes. However, this mean pixel size appears to stem from in-vitro activated CD8 T cells, which rapidly grow and proliferate upon stimulation. In line with this, activated lymphocytes exhibit increased cytoplasmic area, making them appear less dense or “brighter” by phase microscopy compared to naïve lymphocytes, which are relatively compact and subsequently appear dimmer. Given this, it is not clear whether UFMTrack is sufficiently trained to identify naïve human lymphocytes in circulating blood, nor smaller, murine lymphocytes. Analysis of each lymphocyte subtype in terms of pixel size and intensity would be beneficial to strengthen the claim that UFMTrack can identify each of these populations. Additionally, demonstrating that UFMTrack can correctly characterize the behavior of naïve versus activated lymphocytes isolated from murine and human sources would strengthen the claim that UFMTrack can be broadly applied to study lymphocyte dynamics in diverse models without additional training

We thank the Reviewer for the suggestion to more precisely evaluate the range of cell sizes that can be analyzed by our framework. We have included a visualization of crawling cell sizes successfully analyzed by the UFMTrack in Supplementary Figure 7. It demonstrates that the human peripheral blood mononuclear cells, that are almost twice as small as the activated mouse CD4 T cells used in these assays, can be successfully segmented, tracked, and analyzed with the UFMTrack framework. Thus, our UFMTrack framework is suitable for a broad application to differentially sized immune cells during their interaction with the endothelial cell monolayer under flow. 

(3) Average precision was compared to the analysis of UFMTrack but it is unclear how average precision was calculated. This information should have been included in the methods section

We thank the Reviewer for pointing our attention to the missing information. We have added a subsection, “Performance Analysis”, to the Materials and Methods section, where we describe the statistical methods and the performance metrics used to evaluate the UFMTrack framework.

(4) CD4 and CD8 T cells exhibit distinct biology and interaction kinetics driven in part by their MHC molecule affinity and distinct receptor expression profiles. Thus, it is unclear why two distinct mechanisms of endothelial cell activation are needed to see differences between the populations.

We thank the Reviewer for pointing out that different cytokine stimulations of endothelial cells were used in the assays used here to test our UFMTrack to analyze CD4 and CD8 T cell interactions with the endothelial monolayer. While the Reviewer is correct that CD4 and CD8 T cells use different mechanism to cross the pMBMEC monolayer as show by us (doi: 10.1002/eji.201546251.) and others and that recognition of cognate antigen on MHC class I on pMBMECs will arrest CD8 T cells and lead to CD8 T-cell mediated apoptosis ( doi: 10.1038/s41467-023-38703-2.) the focus of the present study was not on comparing CD4 and CD8 T cell interactions with the pMBMEC monolayer but rather to test suitability of UFMTrack to study the different multi-step transmigration of these T cell subsets across the endothelial monolayer. 

(5) The BMECs are barrier tissues but were cultured on µdishes in this study. To study the transmigration of T-cells across the endothelium, the model would have been more relevant on a semi-permeable membrane instead of a closed surface.

We understand the critique of the Reviewer, but laminar flow chambers with endothelial monolayers still provide a state-of-the-art and established methodology to study immune cell migration across endothelial monolayers by in vitro live cell imaging including endothelial cells forming the blood-brain barrier.  

(6) Methods are provided for the isolation and expansion of human effector and memory CD4+ T cells. However, there is no mention of specific CD4+ T cell populations used for analysis with UFMTrack, nor a clear breakdown of tracking efficiency for each subpopulation. Further, there is no similar method for the isolation of CD8+ T cell compartments. A clear breakdown of the performance efficiency of UFMTrack with each cell population investigated in this study would provide greater insight into the software’s performance with regard to tracking the behavior and movement of distinct immune populations.

We thank the Reviewer for this comment. Since a fair performance evaluation requires collecting reliable and consistent manual annotations, in this work we have performed such analysis only for the mouse CD8 T-cell population migrating on the pMBMEC monolayer. We have chosen this as a reference since it is a different cell population than the one the segmentation model was trained on. This provides an insight into how high performance is expected when other immune cell types are studied than the ones used for model development.

(7) The results section is quite extensive and discusses details of establishment of the framework while highlighting both the pros and cons of the different aspects of the process, for example the limitation of the two models, 2D and 2D+T were highlighted well. However, the results section includes details which may be more fitting in the methods section.

We thank the Reviewer for highlighting the extensive work carried out in the development of our UFMTrack framework. We decided to include in the results section only the description of key elements and design decisions taken when developing the framework, such as the need to include a time series of images for successful segmentation of the transmigrated cells. At the same time, the majority of implementational details can be found in the Supplementary Material.

(8) A few statements in the results section lacked literary support, which was not provided in the discussion either, such as support for increased variance of T-cell instantaneous speed on stimulated vs non-stimulated pMBMECs. Another example is the enhancement of cytokine stimulation directed T-cell movement on the pMBMECs that the authors observed but failed to relay the physiological relevance of it. The authors don’t provide enough references for developments in the field prior to their work which form the basis and need for this technology.

We thank the Reviewer for this comment and for asking for literature references. However, we cannot provide such references as these are original observations we made by employing the UFMTrack framework.  This shows that UFMTrack observes T-cell behaviors that have previously been overlooked. Their physiological relevance will have to be explored in separate studies. We have extended the introduction section to include the details on the existing methods developed in the field, as well as their weaknesses that motivated the development of the UFMTrack framework.

(9) The rationale for use of OT-1 and 2D2-derived murine lymphocytes is unclear here. The OT-1 model has been generated to study antigen-specific CD8+ T cell responses, while the 2D2 model has been generated to recapitulate CD4 T cell-specific myelin oligodendrocyte glycoprotein (MOG) responses.

To establish and test the UFMTrack framework, we have made use of the specific T-cell subsets and endothelial cell models we generally use within our research context. Especially for animal work, this is according to the 3R rules requesting to reduce animal experimentation.  

Figures and text:

(1) There are certain discrepancies and misarrangement of figures and text. For example, discussion of the effect of sheer flow on T cell attachment as part of the introduction in figure 1 and then mentioning it in the text again in the results section as part of figure 4 is repetitive.

We thank the Reviewer for pointing our attention to this misarrangement. We have adjusted the label of Figure 4 to emphasize that this effect is correctly captured by the UFMTrack.

(2) Section IV, subsection 1 of the results section, refers to ‘data acquisition section above’ in line 279, however the said section is part of materials and methods which is provided towards the end of the manuscript.

We thank the Reviewer for pointing our attention to this misarrangement. We have adjusted the text to reflect the correct chapter order.

(3) There are figures in the manuscript that have not been referenced in the results section, for example, figure 3A and B. Figure 1 hasn’t been addressed until subsection 7 of materials and methods

We thank the Reviewer for pointing our attention to this misarrangement. We have adjusted the text to refer to all figure panels and the clarification of the cell multiplicity estimation in the supplementary information section. References to Figure 1 were added in the introduction section to illustrate the in vitro under flow imaging setup as well as the typical T cell behaviors in such experiments.

(4) A lack of significance but an observed trend of increased variance of T cell instantaneous speed is reported in line 296-298; however, the graph (figure 4G) shows a significant change in instantaneous speed between non-stimulated and TNFα-stimulated systems. This is misleading to the readers.

We thank the Reviewer for pointing our attention to this discrepancy. We have expanded the text to indicate a low statistical significance for the TNF and no significance but just a trend for the IL1-beta conditions.

(5) The authors talk about three beginner experimentors testing the manual T cell tracking process but figure 5 only showcases data from two experimentors without stating the reason for excluding experimentor 1.

We thank the Reviewer for pointing our attention to this ambiguity. While both the migration analysis and the manual cell tracking were performed by all three beginner experimenters, the cell tracking data for the first one was unfortunately lost due to a hardware failure.

Discussion:

(1) While the discussion captures the major takeaways from the paper, it lacks relevant supporting references to relate the observation to physiological conditions and applicability.

This study is not about the physiological relevance of the microfluidic devices and immune cells used but rather about advancing methodology to analyze dynamic immune cell behavior on endothelial monolayers under physiological flow. Therefore, the discussion does not extend to comparing the physiological relevance of the specific in vitro models employed in this study.   

(2) The discussion lacks connection to the results since the figures were not referenced while discussing an observed trend

We thank the Reviewer for pointing our attention to this misarrangement. We have included the references to the relevant figures as well as supporting references.

(3) The authors briefly looked into mouse and human BMECs and their individual interaction with Tcells, but don’t discuss the differences between the two, if any, that challenged their framework.

We thank the Reviewer for pointing our attention to this weakness. We have added to the discussion section clarifications on the challenges of analyzing the T cell interactions with the HBMEC and the BMDM interactions with the pMBMEC monolayer.

(4) Even though though the imaging tool relies on difference in appearance for detection, the authors talk about lack of feasibility in detecting transmigration of BMDMs due to their significantly different appearance. The statement lacks a problem solving approach to discuss how and why this was the case.

We thank the Reviewer for pointing our attention to this weakness and apologize for the misleading explanation of the problem of analyzing the BMDM sample. Since the transmigrated part of the macrophages differs in appearance from a transmigrated part of a T cell, its detection by a Deep Neural Network trained on the T cell data is worse than that for the T cells. At the same time, the detection performance before the transmigration is sufficient for the BMDM migration analysis. The potential approaches to alleviate this are added to the discussion section.

Relevance to the field:

Utilizing the framework provided by the authors, the application can be adapted and/or utilized for visualizing a range of different cell types, provided they are different in appearance. However, this would require extensive changes to the script and won’t be adaptable in its current form.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The authors should announce in the abstract that the software analysis Track is downloadable and free to use for all researchers. They may consider providing some sort of helpdesk, although I realize that that may run into too much time.

As said above, they stress that it can be done in BBB models, but I would argue that it is much more broadly applicable.

We thank the Reviewer for these suggestions. We have emphasized the broader applicability of UFMTrack in the abstract and pointed out the public availability of the code and data.

Can they add an experiment that shows that it also works for neutrophils for example? I understand that on paper yes it should work, but the neutrophils are of course faster etc.

This is an excellent suggestion, but we tested UFMTrack within the current framework of ongoing research, which does not include the investigation of neutrophil transmigration across endothelial monolayers.  

Also, the combination of different leukocytes in one TEM assay would really be a step forward. If the software can detect different-sized leukocytes, then this should be possible.

We thank the Reviewer for this suggestion. We have added Supplementary Figure 7, demonstrating the range of cell sizes that were successfully analyzed by the UFMTrack framework throughout our manuscript. We also added a statement to the discussion that according to this data, “simply by discriminating cells by size, it is possible to extend UFMTrack to study the interaction of several types of immune cells migrating on top of a cellular monolayer under flow.”

Extra challenges: can the method also discriminate between paracellular and transcellular migration modes? In particular for T-cells this is known to happen.

We thank the Reviewer for this suggestion. We have added this to the potential applications of UFMTrack in the discussion section. While this differentiation is not feasible relying solely on the phasecontrast imaging data, UFMTrack can simplify this analysis by providing automatically the predictions of the transmigration locations, for analysis of the fluorescent data of the junctional labels.

Reviewer #2 (Recommendations For The Authors):

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications. There are several points that need to be addressed, particularly about the claims made by the authors.

Please see the comments below for more details:

• Lines 88-92: Add a citation for the characteristics of the BBB as a barrier

We have added two references accordingly.  

• Lines 94-95: Can the authors indicate what models were used for these studies and how those compare to their in vitro model? In addition, can the authors say whether T cells were manually tracked in this study to translate results to the clinic and whether the results were successful when translated to the clinic? This may enhance the argument that automatic trackers are needed if the translation was not 100% successful

This introductory paragraph summarizes in vivo and in vitro observations from several laboratories. Although these studies include manual tracking of T cells, they do not necessarily distinguish all sequential steps of the multi-step T cell transmigration cascade. Thus, automated tracking may provide additional insights, allowing for increased translation of findings to the clinic.  

• Lines 96-98: Citing the work of Roger Kamm and Noo Li Jeon would be helpful here as they pioneered these BBB microfluidic models and have protocol papers on how to build them and how to use them for cancer cell extravasation studies. Roger Kamm has also worked on several extravasation studies with neutrophils, monocytes, and PBMCs from 3D vasculatures in microfluidic devices, under flow using pressurized fluid or recirculating pumps. Mentioning those would be helpful as they are directly related to what the authors are presenting in their paper.

We thank the Reviewer for this comment, and we consider the work of Roger Kamm and Noo Li Jeon as very valuable for the field. However, these authors have focused on developing functional 3D microfluidic devices, including, e.g., all cells of the neurovascular unit which is not the focus of this present study that solely employed parallel flow chamber devices and endothelial monolayers.  

• Lines 110-116: Can the authors comment on the use of ImageJ or similar automatic tracking tools and how these compare to the under-flow migration tracker developed in this paper? Several groups use ImageJ to track cellular migration successfully and in an automatic manner with short intervals between each frame. One paper that comes to mind is Chen et al: DOI: 10.1073/pnas.1715932115 where neutrophil migration in 3D was assessed with ImageJ in microfluidic devices of the vasculature. If the authors can highlight differences between their tool and what is currently available and used for automatic tracking (e.g. ImageJ), this would help in understanding the advantages of the migration tracker developed in this paper.

• Lines 118-121: Add citations for the current state of the art for T cell extravasation tracking

We thank the Reviewer for these suggestions. We have extended the introduction to add more details on the available tools for tracking migrating immune cells and their limitations, as well as the discussion section to emphasize the features unique to the developed UFMTrack framework.

• Figure 1: The device used by the authors is considered to be a 2D microfluidic device with a monolayer of mouse brain endothelial cells. I would recommend the authors to carefully revise the claims made in the paper to mention that this is a 2D device as opposed to a 3D device, in order to not mislead readers who may be expecting these analyses to be performed in 3D vasculatures.

We thank the Reviewer for this suggestion. We have included in the summary the mention of the 2dimensional nature of the employed BBB model.

• Figure 1: The T cells used in this study are not fluorescently-labeled but the authors mention that this is an issue from current state-of-the-art tools. I would recommend that the authors remove this point as being an issue because it is not addressed in their paper. The T cells are also not labeled in this study so this limitation of other systems is not addressed in this paper.

We apologize to the Reviewer as we do not understand this question. There will be many experimental conditions not allowing to study fluorescently tagged T cells. Therefore, UFMTrack is tailored to follow and analyze T cells and other immune cells during their interaction with endothelial monolayers independent of a fluorescence tag.  

• Figure 1: Was the shear stress controlled manually with a syringe? Or with the use of a pressure controller? I would clarify this aspect and discuss human errors that can be introduced from manually controlling the pressure applied to the monolayer.

We thank the Reviewer for pointing our attention to this ambiguity. We have added a mention of the automated syringe pump used to control the shear stress in the text where the values of shear stress applied to the sample are first mentioned.

• Figure 1: Does T cell attachment occur within the first 5 minutes? Can the authors comment on how they chose this timeline and the percentage of T cells that are washed off at the second step at 1.5 dynes/cm^2? Is 30 seconds enough to ensure all the non-adhered T cells are washed off with 1.5 dyns/cm^2?

Superfusion of the T cells over the endothelial monolayer is performed under 0.5 dynes/cm2 to allow the T cells to settle on the endothelial cell monolayer under flow. After increasing to physiological, flow non adherent T cells detach within 30 seconds, as described by the Reviewer. We have included in the Methods Section Point 7 the references describing in depth the design of the flow chamber device and methods used here.  

• Line 154: How many images were used in the training vs. testing dataset for T cell migrations?

We thank the Reviewer for pointing our attention to this missing information. We have added the sizes of the training and validation datasets. Specifically, the 226MPix of available imaging data was split into 154Mpix training and 37 MPix validation sets. The gap in between was introduced to avoid a correlation between validation and training set that would compromise the performance evaluation.

• Are the supplementary videos at real speed or accelerated?

We thank the Reviewer for pointing our attention to this missing information. The videos are sped up by a factor of 96. We have added this information to the Supplementary video descriptions.  

• Lines 208 216: Can the authors comment on how their initial adhesion timeframe of 30sec before starting the recording at 5.5min affects the number of T cells with rapid displacement? 30 seconds may not be enough to ensure T cells have adhered to the endothelium

Please see our comment above. The methodology used in the present assays has been set up and validated in numerous publications. We have included in the Methods Section under Point 7 the references describing in depth the design of the flow chamber device and the methods used here.  

• Lines 275-277: Was the number of testing images 18? Can the authors comment on how this compares to training dataset size and whether these numbers are enough to achieve robust results?

We apologize for this ambiguity in our manuscript. The framework was evaluated on 18 imaging datasets, each corresponding to 32 minutes of recording, not 18 images. We have added this clarification to the “CD4+ T cell analysis” subsection. The total size of these datasets is 18 datasets * 191 timeframe/dataset * 9.9MPix/frame = 34MPix

• Figure 4B: Can the authors add statistics here? Individual datapoints on the error bars would be helpful too. 

We thank the Reviewer for pointing our attention to this weakness. The data corresponds to the statistical errors as evaluated based on all cells in the 18 datasets. We have added the total number of cells in each of the endothelium stimulation conditions to the text.

• Figure 4C-J: Can the authors put individual datapoints here as well and explain whether they considered each T cell to be one datapoint or each endothelium (averaging all T cells) to be one datapoint? 

We thank the Reviewer for this suggestion. However, adding about one thousand points corresponding to each cell would be impractical. We thus present the distributions of the evaluated from the data metrics as a histogram on the violin plot instead of the swarm plot.

• Figure 4: Did the authors wash the monolayers before introducing T cells? Soluble unbound cytokines may still be present and there are two different questions that would be studied here: “Is the inflamed endothelium affecting T cell migration?” (if washing was performed) or “Is T cell and microenvironmental inflammation affecting T cell migration?” (if no washing was performed)

The endothelial monolayers are “washed” by starting the flow in the flow chamber device and this is before superfusing the T cells over the endothelial monolayer. We agree that our flow chamber device combined with UFMTrack will allow to address all these questions.

• Figure 4I: Are all the T cells decelerating? (negative AM speed)

We thank the Reviewer for this question. The cells are moving along the flow, which, in our experiments, is from left to right. The vector of speed is thus pointing against the x-axis, and thus the AM speed is negative.

• Lines 302 306: Please explain how this compares to ImageJ or similar trackers that can achieve similar outputs. 

We thank the Reviewer for this question. We have added a statement in the “T-cell tracking” section emphasizing that standard trackers are incapable of correctly capturing large displacements.

• Lines 306-309: It is not lower for TNF stimulation though. How do the authors address this? TNF is also a pro-inflammatory cytokine.

We have previously shown that stimulation of pMBMECs with IL-1 and TNF-a induces different cell surface levels of ICAM-1 and VCAM-1, which will influence T cell behavior on the pMBMEC monolayer.  

• Lines 313-315: Could this be because the monolayer was not washed and soluble cytokines affected T cell response directly?

Please see our answer to lines 306-309.  

• Lines 319: Please cite Roger Kamm and Noo Li Jeon’s papers on BBB models with human BMECs, pericytes and astrocytes in 3D microfluidic devices.

We thank the Reviewer again for pointing out these studies. As mentioned above, as our present study does not explore 3D models of the BBB, we think it does not fit into the framework of our study to elaborate on 3D models of the BBB. In addition, this would require the inclusion of a discussion of the work of others like, e.g., Peter Searson and others.  

• Figure 5: Several statistics are missing from parts of the figure. Please add those.

We apologize – but we do not understand which statistical analysis the Reviewer is missing from this Figure.  

• Can the authors comment on the number of T cells perfused over the monolayer and if this ratio of T cells to endothelial cells makes physiological sense? Too many T cells may result in endothelium inflammation and increased diapedesis.

The number of T cells used to suprerfuse over the endothelial monolayer is tested to avoid aggregation of T cells in suspension and thus artificial interactions with the endothelial monolayer. T cell behavior on the pMBMEC monolayer remains the same over the dilution of factor 10.  

• Lines 381 383: How does this compare to analyses that look at the cross-section of the endothelium? It is difficult to assess transmigration looking at the top view of the endothelium. Perhaps, cross-section assessments will identify differences in manual vs. automatic tracking.

There is, to the best of our knowledge, no microscopic device that would allow for in vitro live cell imaging of a live endothelial monolayer – this is in the presence of tissue culture medium – from the side at a resolution that would allow to define transmigration. Our current study rather shows the UFMTrack can distinguish cells moving above or below the endothelial monolayer.  

• Figure 5J: This is probably the most important argument of the paper. If the authors can show statistical differences in their graph, this would greatly help convince readers that this tool is necessary and actually computationally efficient compared to manual work by researchers.

We thank the Reviewer for this suggestion. However, comparing a single data point for automated measurement with four manual experimenter analysts is not a statistically sound comparison. We believe that Figure 5K is clearly showing the factor 5 difference in analysis speed as compared to manual analysis. More importantly, though, the automated analysis is taking the machine time, lifting the need for the experimenter to invest even 1/5th of the original analysis time.

• Figure 6: Did the authors use autologous immune cells and endothelial cells? This is particularly relevant with the use of human-derived T cells (line 436) on the BMEC monolayer. Can the authors comment on non-self reactivity by the T cells encountering BMEC from another human subject?

Autologous T cell interaction with BMECs would only be possible when using hiPSC-derived EECM-BMECs and the T cells from the same individual. All other experimental frameworks will not include autologous interactions. This is the experimental framework used by most authors studying immune cell interactions with commercially available donors. We have not studied alloreactive interactions in our assays and thus cannot further comment.  

• Figure 6M,N,O: How does this compare to ImageJ for tracking of fluorescent cells? I recommend the authors to try that, at least for this section, as this may enhance their argument for their tool vs. standard tools like ImageJ if success rates are higher for their tool.

We thank the Reviewer for this suggestion. We included a note on the analysis of the fluorescent datasets using the  TrackMate plugin for imageJ performed previously in our lab in the “Human T cells on immobilized recombinant BBB adhesion molecules” subsection.

• Figure 6: Please put individual datapoints on the bar or violin plots where they are missing.

We thank the Reviewer for this suggestion. However, adding about one thousand points corresponding to each cell would be impractical. We thus present the distributions of the evaluated from the data metrics as a histogram on the violin plot instead of the swarm plot.

• Lines 467-471: This argument is important and should be mentioned earlier in the introduction.

Another point that can be mentioned is the application of this platform to imaging modalities in vivo (mouse or human) given that there is no fluorescent staining in these cases. This review may be relevant: https://doi.org/10.1002/jcb.10454

We thank the Reviewer for this suggestion. We have clarified in the introduction that UFMTrack does not require fluorescent labels of the imaged migrating cells and relies solely on the phase contrast imaging data.

• Discussion: Please address a few more potential applications to this study. One can be cancer and immune infiltration.

We thank the Reviewer for this suggestion. We have elaborated on additional potential applications to the discussion section.

Reviewer #3 (Recommendations For The Authors):

(1) Line 327-328: The authors talk about ‘As we have previously shown…pMBMEC monolayers differs between CD4+ and CD8+ cells…’. Where was this shown? If it was in a previously published article, please provide a reference.

We have added these missing references.  

(2) Line 353: Please provide clear location on where to find the associated information instead of stating ‘see below’.

We thank the Reviewer for pointing our attention to this ambiguity. We have corrected the phrase to “see next paragraph”

(3) Line 439: Please correct the acronym to BMECs

We thank the Reviewer for pointing our attention to this typo. We have corrected it.