2, perhaps we could also include analyze the term:

pulmon/o/logist

WR CV S 3. Define 4. Definition

If so, we would need to then do this for all chapters to be consistent. Would need to add this to the Chapter 3 review.

1. Analyze

bronch/o/scopy

WR CV S

1. Define

2. Definition

Hago aquí mi anotación porque desde un principio me gustó que en la lectura se resuelve esta pregunta, fue una lectura muy interesante porque por lo menos yo desde que entré en la carrera no tenía mucha idea de lo que ser diseñador significaba, pensaba que era que te pagaran por hacer cosas bonitas y sin embargo durante este año he aprendido que el diseño es muchísimo más complejo en muchos aspectos, y creo que es muy importante tener presentes este tipo de lecturas y reflexión, para no solo enfocarnos en lo teórico y tener algo para sustentar nuestro trabajo. En el caso de esta lectura me parece muy interesante el enfoque humanístico que se le puede dar al diseño, es esclarecedor encontrarnos con este tipo de herramientas para solucionar problemas en el diseño y es que hay cosas que uno da por sentando tan cotidianas como el tener un formulario fácil de llenar y leer en un centro de salud, hasta el ser fiel a tus creencias y valores pero ser profesional y poder ayudar a una persona con sus respectivas creencias o valores a comunicarse y resolver algún problema.<br /> Creo que justamente a mi me solía costar mucho trabajo al principio saber que hacer con una página en blanco y me gusta mucho tener este tipo de estructuras para que el diseño sea todo un proceso y no solo intuición. Para finalizar me gustaría concluir que me parece muy valioso y bello este enfoque que se le puede dar al diseño, siendo no solo un logo hueco, sino llegar a empatizar con las personas, lograr justo como nos dice la lectura "escuchar", desarrollar mi trabajo y lograr algo especial, sin duda es algo que me muero por poder lograr en un futuro y me hace sentir orgullo de lo que estoy estudiando.

Esta parte del texto donde dice que para que nuestras investigaciones y nuestros diseños funcionen, hay que tomar en cuenta esta parte del texto, sobre identificar los patrones, temas y las relaciones que tiene la información que recopilamos, ya que gracias a las intuiciones debemos prestar atención y tenemos que explorar todos los puntos de vista de una historia para llegar a una conclusión; y aquí mismo l la lectura nos da consejos importantes: seleccionar información clave, reunir grandes ideas y trabajar siempre al mismo nivel. Lo anterior me parece muy importante porque estoy de acuerdo de siempre conocer los puntos de vista de la mayor parte de personas posibles, ya que si solo nos quedamos con una parte de las historias, nuestra información puede estar incompleta o en el peor de los casos puede ser errónea. Otro método dentro de esta parte del texto que me llamó la atención fue el de encontrar temas, y como si fuera una carpeta debemos organizarlos por categorías y grupos, como se relacionan entre ellos y desmenuzar los datos de formas diferentes para encontrar un significado que ah primera vista uno no puede ver, porque a veces tampoco hay que quedarnos con nuestra opinión y es preferible preguntar a alguien más porque quizás encuentre algo que nosotros no, algo así como cuando vemos una mancha pintura y cada persona que la ve le encuentra una forma diferente

Esta parte de la lectura me hizo tomar con más importancia esta parte del proceso de diseñar. Además de crear y dar a comunicar algún mensaje o ideas; gran parte de nuestro trabajo como diseñadores es el estar en constante investigación, el tener un buen conocimiento y contexto en lo que vamos a trabajar, para así no llegar a cometer errores y que nuestro trabajo de a entender un mensaje negativo o diferente a lo que queremos dar a conocer.

Al trabajar en DCP, es muy importante conocer completamente al público al que irá dirigido nuestro trabajo, saber sus creencias, ideales, opiniones, etc. Esta lectura crea un gran apoyo, ya que nos da diferentes formas de conectar con las personas y así poder trabajar de una manera en la que busquemos satisfacer a la mayoría del público que a pesar de pertenecer a una misma comunidad, existe una diversidad entre ellos. Por ello considero que la investigación es parte fundamental de la creación de un diseño.

Desde un principio conocemos o al menos tenemos entendido qué siempre hay una base ya sea establecida o hecha ppr nosotros para un orden a la hora de hacer o crear algo, siempre existe un manera que nos funciona para resolverlo por qué es un sistema que nos ayuda a la organización y aumenta la efectividad y sí como dice el texto debemos ver desde el punto de las personas qué desean o que buscan, una organización y algo que la haga viable. Es importante saber escuchar, crear y entegar tal cómo lo dice el texto, escuchar nos llevará a entender y crear incluso un vínculo dónde nacen historias qué generan el propósito del proyecto, no es suficiente sí sólo investigamos y nos quedamos con lo que encontramos en Internet es importante saber indagar y conocer más allá de lo vivido de lo que nos rodea, qué es aquello qué el humano necesita, incluso lo qué uno mismo requiere y es aquí de dónde surge la necesidad de plantearse una problemática u objetivo para luego llevarlo acabó crear prototipos y un sin fin de pruebas qué resulten en lo esperado de nuestros datos para finalmente poder ponerlo en marcha y exponerlo ante la gente y qué esté cumpla con lo dicho y esperado en un principio, al hacer proyectos generalmente escolares no sé parece nada la del libró a cómo yo suelo guiarme y es quizás por qué realmente nunca me puse a pensar ni me propuse un método de trabajó solía hacer sólo lo qué me pedían cómo yo viera qué fuera la mejor manera para llevarlo acabó y qué tan sólo cumpliera su finalidad sin siquiera indagar más allá del por qué del proyecto el por qué de lo que hacía para qué me servía o les servía a los demás tan sólo era para mí una actividad más y ya, pero viéndolo desde está lectura desde el comprender el diseño visto desde las personas es un punto de vista muy distinto y me hizo reflexionar en qué a veces sólo hacemos o trabajamos por que no lo piden o por necesitamos o debemos hacerlo sin intentar realmente aprender de la metodología para qué sea un proyecto verdadero uno del qué podamos ver cosas positivas y no una sola calificación o un sólo trabajó sin chiste y sin algo qué decir.

Como profesionales dentro del área del diseño es importante poder hacer análisis e investigaciones profundas mientras se contempla un objetivo y visión claros que puedan ayudar a "solucionar" un problema. Al iniciar la carrera es algo que siempre he contemplado y ahora sé que es lo hace al diseño ser una disciplina en la cual podamos ayudar la sociedad con un producto pensado para la resolución de conflictos a base de necesidades, esto significa que prestar atención y comprender la cotidianidad de nuestros consumidores nos ayuda ampliar una visión factible de las "*necesidades, esperanzas y aspiraciones" que se desean en un producto funcional con el propósito de ser eficiente y eficaz. Esto también se interrelaciona profundamente al poder interactuar, empatizar y corresponder a estos mismos puntos para poder lograr un buen trabajo ayudado con la resolución de problemas con hincapié a las metodologías de innovación para la mejora o producción de un producto con finalidad específica de lograr un acercamiento con nuestros consumidores. Es importante mencionar que el libro habla de la realización de estos proyectos con un amplio equipo que tenga variedad de áreas, conocimientos y especializaciones, por lo que me hace reflexionar que esto hace que el trabajo sea más factible y mejor pensado, ya que podemos rescatar varios puntos de vista en las resoluciones que se quieren llegar a plantear, esto no solo facilita el trabajo de investigación y planeación, sino que también crea nuevos campos de oportunidad y soluciones rápidas e innovadoras por lo que cada integrante forma una parte fundamental de la planeación del diseño.

Me parece muy agradable esta lectura, ya que muchas veces el iniciar un proyecto de diseño no se sabe con exactitud que se debe hacer para poder lograr y crear diseños mejor pensados para el uso del consumidor, creo que es importante una metodología que guíe los pasos a seguir de manera prominente para lograr los objetivos que se requieran teniendo como resultado productos innovadores, funcionales, eficaces, de calidad y sobre todo con justificaciones válidas que comprendan y comprometan la resolución de conflictos determinados.

A lo largo de la lectura me di cuenta que para llegar a tener una resolución del problema no basta con simplemente implementar estrategias para lograr satisfacer las necesidades del usuario. El diseño centrado en las personas me parece un recurso muy valioso y real. Para poder ayudar significativamente a una persona o personas con una problemática hay que conocer a profundidad sobre el tema, no dejándonos guiar por lo que creemos ya saber y acercarnos a la gente, conocerla, comprender su situación, dificultades y contexto pues esto es importante, ya que al ser nuestra fuente primaria es más directo y genuino el contacto. Para poder resolver el problema muchas veces es necesario un equipo conformado por personas distintas, quien además de aportar diferentes puntos de vista y enriquecer más el proyecto, son un apoyo para esta gran labor. Escuchar, crear y entregar me parece un método adecuado aunque complejo en cada una de sus faces, pues puede llegar a ser un trabajo muy intenso pero finalmente gratificante al observar los resultados. Me gustaría llegar a aplicar alguna vez esta metodología, tiene un enfoque humano.

Muchas veces en el diseño nos dejamos llevar por nuestros ideales y solemos basar nuestros diseños en esto mismo. Leer del paso 4 al 5, como una persona que puede ser muy testaruda acerca de mis gustos, me confirma la importancia de mantener una posición neutral acerca del diseño.

No solo eso, sino también considera la importancia de extremos y de un panorama grande a la hora de diseñar; recuerdo muy bien que en mis primeras clases de la carrera me han repetido sin fin de veces sobre que no estoy diseñando para mí, sino que diseño para un cliente con un público objetivo y es por eso mismo que tenemos el deber de estudiar este objetivo para poder lograrlo a su máximo, como el documento menciona ya sea por medio de entrevistas individuales o grupales, todas tienen distintos objetivos para distintas metas, pero eso no reduce su importancia.

Por otro lado, me parece increíble los conejos que nos da el libro acerca de como acercarnos a una comunidad, ya que, muchas veces, las entrevistas pueden llegar a ser hostiles y se enfocan más en tener respuestas que en la persona, cuando estos van de la mano, si sentir a una persona cómoda es cuando vas a conseguir las mejores respuestas.

Esta parte del texto me atrajo desde que la vi en el indice ya que suelo ser una persona metódica a la hora de desarrollar proyectos personales o escolares.

Un proceso de investigación idóneo, es decir, que concluya en soluciones concretas implica varias etapas clave, que son esenciales para transformar la información en oportunidades y finalmente en soluciones coherentes. Por lo que yo considero que nosotros como diseñadores nos es fundamental el poder sintetizar nuestras tareas nos garantiza un proceso creativo correcto y un optimo resultado.

A lo largo de mi vida he tenido la necesidad de crear una guía estructurada que me permita escuchar y entender las necesidades del grupo al que quiero impactar.

De igual forma, considero que es relevante darle valor a nuestra profesión, pues esta puede impactar en todos los aspectos sociales, pues la mayoría del tiempo estamos creando (diseñando) soluciones a problemas cotidianos.

Algo que me ha quedado muy en claro desde que comencé la carrera fue el que al momento de estar diseñando cualquier cosa es importante saber a qué público estoy yendo dirigida y gracias a esta lectura es que puedo tener esta otra herramienta que había pasado por alto, que es la investigación profunda acerca de la problemática específica que tiene el público al que voy dirigida, con eso me refiero a que yo tenía en claro que es importante la investigación antes de, sin embargo el crear no sólo se basa en una investigación supuestamente superficial, se necesita mucho más que eso para poder entender realmente la problemática a resolver. Algo que me gustó mucho de esta lectura fue justamente la primera parte, el escuchar, creo que es algo que las personas lastimosamente hemos dejado muy atrás, y que al momento de diseñar es de las cosas más importantes que se tiene que tener en cuenta, ya que si no se escucha a las personas que tengan cierta problemática no se va a poder llegar a crear algo que realmente mejore o signifique algo dentro de su vida. Uno siempre va a estar buscando el impacto dentro de la vida de las más personas y es por eso que esta frase me quedó tan marcada, porque es real, llegan a ver soluciones vacías, que puede que se hagan con la mejor intención del mundo, pero si no se tiene un método claro de investigación adecuado al tipo de proyecto que tenemos no se va poder crear ni entregar un proyecto bueno y significante. Siempre hay que tratar de conectar, entender y empatizar a las personas con las que estamos trabajando.

check the following 2 terms: should be:

onych/o/phagia WR CV S

xer/o/derm/a WR CV WR S

sclera/o (hardening) should be: scler/o (hardening)

ierhviuerhir e;orfh o;rhu eguh

I really like the second point to make sure to keep students engaged as collaborates and not just as the people getting data collected on themselves. It’s also very true that when we are working on and doing analytics that we see this is just a snapshot in time of the data and the students the data are being collected from. We also want to make sure we can be transparent and open about what we are collecting and why this even needs to be collated. Finally we need to collect this data for some reasons like the ones we talked about which are accreditation, teacher feedback and checking in on student learning.

The bettering of the school is now extremely prioritized

Author Response

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

Reviewer #1

1) Overall, this is a useful tool, the data is well-presented, and the paper is well-written. However, the predictions are only compared with two existing reconstruction tools though more have been recently published

The aim of this work was to facilitate high-throughput generation of strain-specific metabolic models e.g. at the scale of 100s -1000s as indicated throughput the introduction (see lines 74-82, 91-94), and therefore we only compared tools which were capable of high-throughput analysis via command line and excluded others (e.g. merlin). We have now tested this against the other recent command line tool, gapseq which had escaped our gaze. Thank you for bringing this to our attention. Additionally, we have included KBase (ModelSEED, a web-based app that does not support high-throughput analysis) to allow for readers to interpret the results in the context of community standard approaches, since KBase is a popular tool.

We have added an explicit statement about the choice of approaches, now at lines 194-199 as follows:

“We compared the output and performance of Bactabolize to the two previously published tools that can support high-throughput analyses i.e. CarveMe (30) and gapseq (31). To aid interpretation in the context of community standard approaches, we also include a comparison to the popular web-based reconstruction tool, KBase (ModelSEED), and a manually curated metabolic reconstruction of K. pneumoniae strain KPPR1 (also known as VK055 and ATCC 43816, metabolic model named iKp1289) (15).”

The methods section was updated accordingly (now lines 552-558):

“A draft model was generated using gapseq version 1.2 with the ‘doall’ command using the unannotated genome (as gapseq does not take annotated input files). Gap-filling was subsequently performed using the ‘fill’ command and a custom M9 media file to match the nutrient list found in Bactabolize (https://github.com/kelwyres/Bactabolize/blob/main/data/media_definitions/m9 _media.json).

Finally, a draft model was constructed using the annotated genbank K. pneumoniae KPPR1 file and the KBase narrative (15) (https://narrative.kbase.us/narrative/ws.14145.obj.1).”

The results section (now lines 193-308) and Figure 2 have also been updated / restructured to reflect the new analyses, and include a comparison of the relative compute times for the construction of models (lines 281-291) as follows:

“While model features and accuracy are essential metrics for comparison, computation time is also a key consideration for high-throughput analyses. We recorded the time required for each tool to build draft models for 10 of the completed KpSC genomes used in the quality control framework (see below) on a high-performance computing cluster (Intel Xeon Gold 6150 CPU @ 2.70GHz and 155 GB of requested memory on a CentOS Linux release 7.9.2009 environment. CarveMe KpSC pan was the fastest with a mean of 20.04 (range 19.90 - 20.18) seconds, followed by CarveMe universal at 30.28 (range 29.20 - 31.80) seconds, then Bactabolize KpSC pan at 98.05 (range 92.19 - 100.4) seconds. KBase took 183.50 (range 120.00 - 338.00) seconds per genome via batch analysis, including genome upload time and queuing. gapseq took 5.46 (range 4.55 - 6.28) hours to produce draft models (not including the required gap-filling), consistent with previous reports (37).”

Finally, the whole discussion has been updated and substantially restructured (lines 472-474, 475-493, 494-512). Specific mentions to the new analyses are at lines:

472-474: “Consistent with this assertion, our draft KPPR1 model constructed with KBase (without manual curation) was an outlier in terms of the very low number of genes, reactions and metabolites that were included.”

475-493: “CarveMe with universal model (30) and gapseq (31) are the current gold standard automated approaches for model reconstruction, and we show that a draft KpSC model generated by Bactabolize with the KpSC pan v1 reference resulted in similar or better accuracy for phenotype prediction (Figure 2). Both the CarveMe universal and gapseq models resulted in high numbers of true-positive and true-negative growth predictions. However, these were also accompanied by comparatively higher numbers of false-positive predictions that resulted in a lower overall accuracy for substrate usage analysis compared to Bactabolize with the KpSC-pan v1 reference (Figure 2), and comparatively lower precision and specificity for the gene essentiality analysis. False-positive predictions may indicate that the relevant metabolic machinery are present in the cell but were not active during the growth experiments (e.g. due to lack of gene expression). In this regard, false-positives are not always a sign of model inaccuracy. However, false-positive predictions can also occur from incorrect gene annotations e.g. due to reduced specificity of ortholog assignment resulting from the use of the universal model without manual curation. Given a key objective here is to facilitate high-throughput analysis for large numbers of genomes, it is not feasible to expect that all models will be manually curated, and therefore we believe that identifying fewer genes with lower overall error rates provides greater confidence in the resulting draft models. We also note that the BiGG universal reference model which CarveMe leverages is no longer being actively maintained. In contrast, user defined reference models can be iteratively curated and updated to incorporate new knowledge and data as they become available.”

510-512: “However, gapseq’s long compute time makes it inappropriate for application to datasets comprising 100s-1000s of genomes (such as have become increasingly common in the bacterial population biology literature).”

2) My understanding is that the tool requires a set of reference reconstructions for other strains of the target species. If no reference reconstruction is available for another strain of the target species, can this species not be reconstructed?

Any input reference can be used to generate models however, single strain models matching the target species, or ideally a species-specific panreference, are recommended for best results. We have added a discussion on these points at lines 128-133:

“For optimum results we suggest using a pan-model that captures as much diversity as possible for the target species or group of interest, because Bactabolize’s reconstruction method is reductive i.e. each output strainspecific model will include only genes, reactions and metabolites that are present in the reference or a subset thereof (although novel genes, reactions and metabolites can be added via manual curation).”

We expand on these points further in the discussion:

494-512: “Bactabolize’s reference-based reconstruction approach is reductive, meaning the resultant draft models will comprise only the genes, reactions and metabolites present in the reference, or a subset thereof, and will not include novel reactions unless they are manually identified and curated by the user. This is an important caveat that should be considered carefully for application of Bactabolize to large genome data sets, particularly for genetically diverse organisms such as those in the KpSC. For optimum results we suggest using a curated pan-model that captures as much diversity as possible for the target species or group of interest. While we acknowledge that a reasonable resource investment is required to generate a high-quality reference, we have shown that a pan-model derived from just 37 representative strains can be sufficient to support the generation of highlyaccurate draft models (Figure 2 and 5). Additionally, we note that it is possible to use a single strain reference model, which should ideally represent the same or closely related species to that of the input genome assemblies, in order to facilitate accurate identification of gene orthologs. It is technically possible to use an unrelated reference model, but this is expected to result in inaccurate and/or incomplete outputs and has not been tested in this study. In circumstances were no high quality closely-related reference model is available, we recommend alternative reconstruction approaches that leverage universal databases e.g. CarveMe (30) or gapseq (31). However, gapseq’s long compute time makes it inappropriate for application to datasets comprising 100s-1000s of genomes (such as have become increasingly common in the bacterial population biology literature).”

3) How do the reconstructions generated by Bactabolize compare to those generated by other reconstruction tools besides CarveMe and ModelSEED, e.g., gapseq (Zimmermann et al, Genome Biology 2021. 22:81) or merlin (Capela et al, Nucleic Acids Res 2022, 50(11):6052-6066?

See response to rev 1 point 1.

4) How are the accuracy, specificity, and sensitivity of the pan-models calculated? Is the compared experimental data on the species level?

We used the pan-model as a reference from which we generated a strain-specific model for K. pneumoniae KPPR1 (using Bactabolize and CarveMe). This strain-specific metabolic model was then used to simulate growth phenotypes and compared to published experimental data for KPPR1. This was described in the methods section, including the calculations for the metrics (lines 589-593); however, we have also expanded the description within the results section to clarify the approach (lines 201-209):

“De novo draft models for strain KPPR1 were built using; i) Bactabolize with the KpSC pan v1 reference; ii) CarveMe, with its universal reference model (CarveMe universal); iii) CarveMe, with KpSC-pan v1 reference (CarveMe KpSC pan); iv) gapseq; and v) KBase (ModelSEED). ….. Subsequently, each model was used to predict growth phenotypes; i) in M9 minimal media with different sole sources of carbon, nitrogen, phosphorus and sulfur; and ii) for all possible single gene knockouts in LB under aerobic conditions. The predicted phenotypes were compared directly to the published phenotype data.” [Note the published data are cited in the previous manuscript sentence, not shown here].

5) The link https://github.com/rrwick/GFA-dead-end-counter, in line 286 does not work.

Link regenerated – now at line 451-452 and 604

Reviewer #2

1) KpSC pan-metabolic reference model is provided. Are they required as input for Bactabolize? Are the gene, metabolite information open accessible by users? o See response to reviewer 1 point 2 above and;

All data for the KpSC pan-model described in this work are accessible in the model files and amino acid + nucleotide files + data table at https://github.com/kelwyres/KpSC-pan-metabolic-model. This is also linked in the manuscript at line 631 and in the Data availability statement at line 661.

2) In the results section "description of Bactabolize", the authors present technical details on how to generate a metabolic model. For the input and output, please provide concrete examples to show the functionality of Bactabolize.

Detailed instructions, example code and example input/output files are available via the Bactabolize GitHub repository: https://github.com/kelwyres/Bactabolize.<br /> Instructions and example code can be found on the wiki: https://github.com/kelwyres/Bactabolize/wiki Test data and example files are at: https://github.com/kelwyres/Bactabolize/tree/main/data/test_data

The Github repository is linked in the manuscript at lines 95, 124, 552, and 667, and we have added a further reference at line 124, which mentions the example code/data: “Full documentation, including example code and test data are available at the Bactabolize code repository (https://github.com/kelwyres/Bactabolize).”

3) To generate metabolic models, the authors present comparison results with other methods. However, the authors only present the numbers in genes, metabolites and substrates. Since the interactions between gene, metabolite, and substrate are also critical, if possible, please provide the coverage details about these interactions. Venn diagram is recommended to compare these coverage differences.

Two additional supplementary figures have been generated (Figures S5 and 6) showing Venn diagrams of metabolites and reactions for the highthroughput analysis approaches that are most relevant to this work (see also response to rev 1, point 1). These are discussed at lines 224-237:

“Figures S5 and S6 show the overlaps of metabolites and reactions between the high-throughput reconstruction methods after processing with MetaNetX (59) to standardise the reaction and metabolite nomenclatures (excluding CarveMe pan for simplicity and given the likely problems of reaction oversubscription). The majority of the reactions included in the Bactabolize model were conserved in either the CarveMe universal model (n = 1225, 53.2%), gapseq model (n = 54, 2.3%) or both (n = 665, 28.9%). The reaction overlap was skewed to the CarveMe universal model which shared 1225 reactions that were conserved in the Bactabolize model but absent from the gapseq model. Notably, the gapseq model contained a large number (2200) of unique reactions (70.4% of those in the model). Similarly, the vast majority of metabolites in the Bactabolize model were conserved in one or both of the other models (n = 917, 85.6%). However, it is likely that true overlaps between methods are underrepresented due to the different reaction identifiers and chemical synonyms used within the BiGG (Bactabolize, CarveMe) vs ModelSEED nomenclatures (gapseq), which are difficult to harmonise in an automated manner even after the application of MetaNetX.”

Figure 2 shows not only the model numbers but also includes benchmarking to real phenotypic data in 2DEFG as the key mode of comparison between models. This encompasses meaningful interactions between gene, metabolic and substrate. The results are discussed at length in text at lines 253-271:

“We assessed the performance of each model for in silico prediction of growth phenotypes compared to the previously published experimental data (15). Accuracy, sensitivity, specificity, precision and F1 scores were calculated (60). Note that the specific set of growth substrates and gene knockouts that can be simulated is determined by the sets of genes and metabolites captured by each model and is therefore model-dependent (Data S1 and S2). Among those with matched experimental phenotype data, the Bactabolize and CarveMe universal models were able to predict growth for a greater number of carbon, nitrogen, phosphorous and sulfur substrates than gapseq, CarveMe KpSC pan, KBase and iKp1289 models (Figure 2C, Data S1). While the CarveMe universal model had the highest number of truepositive growth predictions overall (n = 132 of 617 total predictions), it also had a comparably high number of false-positive predictions (n = 39 of 617 total predictions, Figure 2D). Similarly, the gapseq and iKp1289 models resulted in 31 (262 total predictions) and 50 (513 total predictions) falsepositive predictions, respectively. In contrast, the Bactabolize model had fewer false-positive predictions (n = 21 of 505 total predictions) alongside a high number of true-positive predictions (n = 117 of 505 total predictions), resulting in the highest overall accuracy metrics (Figure 2E, Data S1). The KBase model was a notable outlier, associated with a high number of falsenegative predictions (n = 31 of 103 total predictions) and low false-positive predictions (n = 3 of 103 total predictions), presumably resulting from the very low number of genes and reactions included in the model, driving low sensitivity and accuracy.”

Lines 272-280:

“The gene essentiality results showed that gapseq produced the highest absolute number of true-positive gene essentiality predictions (n = 79 of), followed by Bactabolize KpSC pan (n = 44 of 1220 total predictions), then CarveMe universal (n = 39 of 1951 total predictions). CarveMe universal had the largest number of true-negatives by a wide margin (n = 1599 of 1951 total predictions), followed by gapseq (n = 1085 of 1403 total predictions), then Bactabolize KpSC pan (n = 939 of 1220 total predictions), driving their high accuracies (83.96%, 82.96% and 80.57%, respectively). The Bactabolize model was associated with the greatest overall precision and specificity (Figures 2F & 2G) while the gapseq model resulted in the highest F1-score and sensitivity.”

4) Are quality control and gap-filling needed to be processed when constructing a new metabolic model?

Our goal here was to implement an approach to support high-throughput analyses (see response to rev 1 point 1), including leveraging draft genome assemblies as the bases for the construction of strain-specific metabolic models. As part of this work, we have described a robust quality control (QC) framework for screening draft K. pneumoniae genomes i.e. to identify genome assemblies that should not be used. We developed this framework by comparison to models generated for matched completed genomes. Our analyses demonstrate the importance of applying QC to the input draft genome assemblies. When appropriate QC is applied to the input genomes, the resultant draft models show a high degree of completeness compared to the matched models derived from complete genomes. The draft models can also be used to simulate growth phenotypes with high accuracy as compared to those simulated for the matched complete genome models.

No specific QC was applied to the draft models themselves, other than confirmation of positive growth prediction in m9 minimal media plus glucose (which is expected to support growth of all K. pneumoniae). In cases where the input assembly passed our QC criteria but the resultant model was unable to simulate growth in m9 minimal media plus glucose, gap-filling may be optionally applied. Again, by comparison to the simulated phenotypes from matched complete genome models, we show that these gap-filled draft models can produce accurate phenotype predictions. See lines 396-404:

“Of the 901 draft genome assemblies which passed our QC criteria (≤200 assembly graph dead ends), 23 of the resulting draft models failed to simulate growth in M9 minimal media with glucose (despite capturing ≥99% of the genes and reactions in the corresponding complete models). It is expected that all KpSC models should be able to simulate growth on M9 media with glucose as a sole carbon source, as this central metabolism is universal amongst KpSC. To replace missing, critical reactions required for growth on M9 with glucose, we investigated model gap-filling using the patch_model command of Bactabolize. We then assessed the accuracy of the gap-filled models for prediction of growth on the full range of substrates, as compared to the predictions from the corresponding complete models.” Lines 409-413: “Substrate usage predictions from the 21 successfully gap-filled models were highly accurate, with 18/21 having a prediction concordance of ≥99% across all 846 growth conditions (12/21 had 100% concordance) (Figure S9). We therefore conclude that models generated for genome assemblies passing our QC criteria, which have been gap-filled to successfully simulate growth on minimal media plus glucose, are suitable for the prediction of growth across a range of substrates.”

5) Are there any visualization results to check the status of the generated draft model?

No. This is a tool for large-scale and rapid production of metabolic models, and phenotype prediction and we have not included visualisation tools. Third party tools are available e.g. https://fluxer.umbc.edu/. We do provide optional generation of MEMOTE reports at lines 136-138:

“Draft genome-scale metabolic models are output in both SMBL v3.1 (41) and JSON formats (one pair of files for each independent strain-specific model), along with an optional MEMOTE quality report (42)”.

Reviewer #3

1) The justification and evaluation of the generated models are inadequate and onedimensional. The authors only focus on statistics such as the number of reactions and genes in the models, which does not accurately depict the completeness of the model.

The reviewer has misunderstood how we have used ‘completeness’ in this manuscript. In the section describing our novel QC framework, we use this term to refer to the relative completeness of draft models generated from draft genome assemblies as compared to curated models generated from complete genome assemblies for the same strains. The latter were considered as the ‘complete’ models for this purpose. We are not referring to any measure of network or metabolic pathway completeness. We specifically refer to gene and reaction capture compared to the ‘complete’ models because these features directly reflect the problem we are trying to address i.e. that draft genome assemblies may not contain the complete set of genes that are truly present in the underlying genome. We have updated the manuscript text to further clarify the problem we aim to address in this section and justify the use of gene and reaction capture metrics:

Lines 310-319: “There are now thousands of bacterial genomes available in public databases, the majority of which are in draft form, comprising 10s to 1000s of assembly contigs. This fragmentation of the genome is caused by repetitive sequences that cannot be resolved by the assembly algorithm and/or sequence drop-out. The latter can result in the loss of genetic information such that some portion of genes present in the underlying genome are lost from the genome assembly (either completely or partially). This in turn, poses a limitation for the reconstruction of metabolic models using these assemblies, since most published approaches use sequence searches to predict the presence/absence of genes and their associated enzymatic reactions. Therefore, if we are to use public genome data for high-throughput metabolic modelling studies, it is essential to evaluate the expected model accuracies and understand the minimum input genome quality requirements.”

The biological accuracy of the curated ‘complete’ models has been described previously, and this is now noted in the text at lines 320-324:

“Here we performed a systematic analysis leveraging our published curated KpSC models (n=37, (14)), which were generated using completed genome sequences and were therefore considered to represent ‘complete’ models for which the underlying genome sequence contains all genes that are truly present in the genome (note the biological accuracy of these models was reported previously (14) and is not the subject of the current study).”

Throughput the manuscript we not only compare models in terms of the numbers of genes and reactions, but through comparison of binary growth predictions. Specifically, in the Performance Comparison section (Bactabolize vs other approaches) we use comparison of predicted to experimental phenotypes for strain KPPR1 (see response to rev 1 point 4 for details). In the QC Framework section we compare the predictions derived from draft models generated from draft genome assemblies to those derived from the matched ‘complete’ models, and report the concordance as a measure of impact of input assembly quality (lines 309-394). In the final results section (Predictive accuracy of draft models), we generate 10 additional models and compare the growth predictions to matched experimental data (lines 414-433). We view these phenotype prediction comparisons as the ultimate measure of ‘completeness’ with which to assess our models, because these data have direct biological meaning.

2) The authors have not provided evidence or discussion on the accuracy of any metabolic fluxes, which are considered to be crucial for reconstructing metabolic models. Additionally, the authors have not mentioned the importance of non-growth associated maintenance and the criticality of biomass composition analysis, both of which significantly determine the fluxes in the system.

We acknowledge the importance of flux calculations and accurate biomass compositions when using genome-scale models to quantitatively predict growth rates. However, at this stage, the reconstructions developed using Bactabolize are intended for binary predictions and comparisons of growth capabilities on various substrates. The accuracies we report are based on measures of network completion (presence/absence of relevant reactions leading to growth or no-growth phenotypes) rather than specific growth rates. Thus, the models generated by Bactabolize can be used to explore diversity at the strain level in terms of growth capabilities and can serve as a scaffold for building detailed (customized biomass), strain-specific models. Measuring biomass composition and metabolic flux analysis require significant experimental comparisons that are outside the scope of the current study but could be performed for target strains based on reconstructions developed using Bactabolize.

3) It would be interesting to compare the accuracy of the models generated using Bactabolize with those manually curated.

We did exactly this. We compared the manually curated model iKp1289 as part of our benchmarking. Lines 194 – 199:

“We compared the output and performance of Bactabolize to the two previously published tools that can support high-throughput analyses i.e. CarveMe (30) and gapseq (31). To aid interpretation in the context of community standard approaches, we also include a comparison to the popular web-based reconstruction tool, KBase (ModelSEED), and a manually curated metabolic reconstruction of K. pneumoniae strain KPPR1 (also known as VK055 and ATCC 43816, metabolic model named iKp1289) (15).”

Unfortunately, as far as we aware there are currently no other published manually curated models for strains with matched phenotype data that are also not included as part of our pan-reference model (the latter is a key point to ensure a fair comparison of models generated using our pan-reference vs those generated with a universal reference).

4) The authors have not provided evidence or discussion on the accuracy of any metabolic fluxes, which are considered to be crucial for reconstructing metabolic models.

See response to rev 3, point 2.

5) The justification regarding the completeness of the models requires further discussion.

See response to rev 3, point 1.

6) A detailed discussion on the importance of manually curated models would significantly enhance the quality of the manuscript.

This has been added at lines 458-474:

“Traditionally, genome-scale metabolic reconstruction approaches have relied upon significant manual curation efforts. While there will always remain a need for high quality curated models, such resource intensive approaches preclude their application at scale, and have therefore limited analyses to small numbers of individual strains (15, 16). However, automated reconstruction approaches can support the generation and comparison of multiple strain-specific draft models from which meaningful biological insights can be derived (61). Additionally, the quality of curated models is likely to vary depending on their age, level and type of curation, as well as the approach used for preliminary drafting. Indeed it is possible for automated approaches to outperform manually curated models; a draft model for K. pneumoniae KPPR1 generated using Bactabolize with the KpSC pan-v1 reference model outperformed the manually curated iKp1289 model representing the same strain (15). iKp1289 was published in 2017 (6 years prior to this study) and was initially drafted via the KBase pipeline (33), which uses RAST to annotate the sequences with Enzyme Commission numbers. It has been demonstrated several times that the Enzyme Commission scheme has systematic errors (62, 63), leading to a loss in accuracy when compared to the ortholog identification methods used by automated approaches. Consistent with this assertion, our draft KPPR1 model constructed with KBase (without manual curation) was an outlier in terms of the very low number of genes, reactions and metabolites that were included.”

it is not your job to babysit them and handle their difficult situations, but you can give them knowledge of how to handle situations

Aplique (formal) o Aplica (imperativo) Misma idea que “mantén”.

[!NOTE] 操作系统有哪些核心内容？

flashcard

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Ação Direta de Inconstitucionalidade parcialmente conhecida e, na parte conhecida, julgados parcialmente procedentes os pedidos para conferir interpretação conforme a Constituição Federal ao art. 1º da Lei 7.960/1989 e fixar o entendimento de que a decretação de prisão temporária autoriza-se quando, cumulativamente:

1) for imprescindível para as investigações do inquérito policial (art. 1º, I, Lei 7.960/1989) (periculum libertatis), constatada a partir de elementos concretos, e não meras conjecturas, vedada a sua utilização como prisão para averiguações, em violação ao direito à não autoincriminação, ou quando fundada no mero fato de o representado não possuir residência fixa (inciso II);

2) houver fundadas razões de autoria ou participação do indiciado nos crimes previstos no art. 1º, III, Lei 7.960/1989 (fumus comissi delicti), vedada a analogia ou a interpretação extensiva do rol previsto no dispositivo;

3) for justificada em fatos novos ou contemporâneos que fundamentem a medida (art. 312, § 2º, CPP); 4) a medida for adequada à gravidade concreta do crime, às circunstâncias do fato e às condições pessoais do indiciado (art. 282, II, CPP);

5) não for suficiente a imposição de medidas cautelares diversas, previstas nos arts. 319 e 320 do CPP (art. 282, § 6º, CPP).

ARGUIÇÃO DE DESCUMPRIMENTO DE PRECEITO FUNDAMENTAL. CONSTITUCIONAL E PROCESSUAL PENAL. ART. 295, INCISO VII, DO CPP. PRISÃO ESPECIAL, DE NATUREZA CAUTELAR, A PORTADORES DE DIPLOMA DE ENSINO SUPERIOR. AUSÊNCIA DE JUSTIFICATIVA RAZOÁVEL PARA O TRATAMENTO DISTINTO ENTRE PRESOS POR PARTE DO PODER PÚBLICO. ELEMENTO DISCRIMINADOR QUE NÃO SE ENCONTRA A SERVIÇO DE UMA FINALIDADE ACOLHIDA PELA CONSTITUIÇÃO. INCOMPATIBILIDADE MATERIAL COM O PRINCÍPIO DA ISONOMIA (ARTS. 3º, IV, E 5º, CAPUT, DA CF). NÃO RECEPÇÃO PELA ORDEM CONSTITUCIONAL ESTABELECIDA EM 1988.

1. Todos os cidadãos têm o direito a tratamento idêntico pela lei, exceto quando presente uma correlação lógica entre a distinção que a norma opera e o fator de discrímen, em consonância com os critérios albergados pela Constituição Federal.

2. O princípio constitucional da igualdade opera em dois planos distintos. De uma parte, frente ao legislador ou ao Executivo, na edição de leis e atos normativos, impedindo que possam criar tratamentos abusivamente diferenciados a pessoas que se encontram em situações idênticas. Em outro plano, na obrigação direcionada ao intérprete de aplicar a lei e atos normativos de maneira igualitária, sem estabelecimento de diferenciações em razão de sexo, religião, convicções filosóficas ou políticas, de raça ou classe social.

3. A prisão especial constitui o recolhimento provisório em local distinto, cuja concessão se admite, à luz da Constituição, quando a segregação do ambiente prisional comum visa a atender a determinadas circunstâncias pessoais que colocam seus beneficiários em situação de maior e mais gravosa exposição ao convívio geral no cárcere. Expô-los ao contato com a população carcerária frustraria a tutela desses interesses constitucionalmente protegidos.

4. Não há amparo constitucional, contudo, para a segregação de presos provisórios com apoio no grau de instrução acadêmica, tratando-se de mera qualificação de ordem estritamente pessoal que contribui para a perpetuação de uma inaceitável seletividade socioeconômica do sistema de justiça criminal, incompatível com o princípio da igualdade e com o Estado democrático de Direito.

5. Ausente qualquer justificativa que empregue sentido válido ao fator de discrímen indicado na norma impugnada, a conclusão é a de que a prisão especial, em relação aos portadores de diploma de nível superior, é inconciliável com o preceito fundamental da isonomia (art. 3º, IV, e art. 5º, caput, CF).

6. Arguição de descumprimento de preceito fundamental conhecida e julgada procedente.

CONSTITUCIONAL E DIREITO PROCESSUAL PENAL. ART. 316, PARÁGRAFO ÚNICO, DO CÓDIGO DE PROCESSO PENAL, COM A REDAÇÃO DADA PELA LEI 13.964/2019. DEVER DO MAGISTRADO DE REVISAR A NECESSIDADE DE MANUTENÇÃO DA PRISÃO PREVENTIVA A CADA NOVENTA DIAS. INOBSERVÂNCIA QUE NÃO ACARRETA A REVOGAÇÃO AUTOMÁTICA DA PRISÃO. PROVOCAÇÃO DO JUÍZO COMPETENTE PARA REAVALIAR A LEGALIDADE E A ATUALIDADE DE SEUS FUNDAMENTOS. OBRIGATORIEDADE DA REAVALIAÇÃO PERIÓDICA QUE SE APLICA ATÉ O ENCERRAMENTO DA COGNIÇÃO PLENA PELO TRIBUNAL DE SEGUNDO GRAU DE JURISDIÇÃO. APLICABILIDADE NAS HIPÓTESES DE PRERROGATIVA DE FORO. INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO. PROCEDÊNCIA PARCIAL.

1. A interpretação da norma penal e processual penal exige que se leve em consideração um dos maiores desafios institucionais do Brasil na atualidade, qual seja, o de evoluir nas formas de combate à criminalidade organizada, na repressão da impunidade, na punição do crime violento e no enfrentamento da corrupção. Para tanto, é preciso estabelecer não só uma legislação eficiente, mas também uma interpretação eficiente dessa mesma legislação, de modo que se garanta a preservação da ordem e da segurança pública, como objetivos constitucionais que não colidem com a defesa dos direitos fundamentais.

2. A introdução do parágrafo único ao art. 316 do Código de Processo Penal, com a redação dada pela Lei 13.964/2019, teve como causa a superlotação em nosso sistema penitenciário, especialmente decorrente do excesso de decretos preventivos decretados. Com a exigência imposta na norma, passa a ser obrigatória uma análise frequente da necessidade de manutenção de tantas prisões provisórias.

3. A inobservância da reavaliação prevista no dispositivo impugnado, após decorrido o prazo legal de 90 (noventa) dias, não implica a revogação automática da prisão preventiva, devendo o juízo competente ser instado a reavaliar a legalidade e a atualidade de seus fundamentos. Precedente.

4. O art. 316, parágrafo único, do Código de Processo Penal aplica-se até o final dos processos de conhecimento, onde há o encerramento da cognição plena pelo Tribunal de segundo grau, não se aplicando às prisões cautelares decorrentes de sentença condenatória de segunda instância ainda não transitada em julgado.

5. o artigo 316, parágrafo único, do Código de Processo Penal aplica-se, igualmente, nos processos em que houver previsão de prerrogativa de foro.

6. Parcial procedência dos pedidos deduzidos nas Ações Diretas (ADI 6581 6582).

Author Response

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

Reviewer #1 (Public Review):

This study provides insights into the early detection of malignancies with noninvasive methods. The study contained a large sample size with external validation cohort, which raises the credibility and universality of this model. The new model achieved high levels of AUC in discriminating malignancies from healthy controls, as well as the ability to distinguish tumor of origin. Based on these findings, prospective studies are needed to further confirm its predictive capacity.

However, there are several concerns about the manuscript, which needs to be clarified or modified.

1) The use of "multimodal model" will definitely increase workload of the testing. From the results of this manuscript, the integration of multimodal data did not significantly outperform the EM-based model. Is this kind of integration necessary? Is that tool really cost-effective? The authors did not convince me of its necessity, advantages, and clinical application.

To provide further evidence supporting the advantages of using multimodal model (stack model) over EM-based model, we performed the DeLong test and provided data in Table S7 and Figure S6. Our data show that the stack model outperformed the EM-based model, with significantly higher AUC (AUC difference = 0.0286, p<0.0001). Moreover, the stack model exhibited significantly higher sensitivity for detecting cancer patients of five cancer types in both discovery (73.8% versus 59.5%, p<0.0001, Figure S6A) and validation cohort (72.4% versus 61.5%, p=0.0002, Figure S6B) at comparable specificity of > 95%. The number of misclassified cases were lower when using stack model as compared to the EM-based model (Figure S6C and S6D). Strikingly, we observed that the stack model significantly improved the sensitivity for detecting lung cancer patients compared to the EM based model in both discovery (78.5% versus 44.1%, Figure S6A) and validation cohort ( 83.7% versus 55.8%, Figure S6B), indicating that other ctDNA signatures are also the important biomarkers for detecting lung cancer. Therefore, we conclude that the combination of multiple signatures of ctDNA, ie. the multimodel approach, could improve the sensitivity of multi-cancer detection.

Given the same wet lab protocol, the difference in computational time between a single EM-based model and the stack model is about 10-11 minutes per sample, but the real difference in analysis time can be reduced to ~1 min/sample by parallelization. With regards to the wet lab protocol, an important novelty of SPOT-MAS technology is its all-in-one approach that enables simultaneous analysis of different ctDNA signatures using a single blood draw and a single library reaction, greatly reducing the experimental cost. Thus, we strongly argue that our approach improves the detection sensitivity by increasing the breadth of ctDNA analysis while achieving cost effectiveness for sample preparation and sequencing with negligible trade-off of analysis time .

We have also added the following sentences in the discussion to clarify this point. (Line 618-625)

“Moreover, this study showed that the feature of EM achieved the highest performance among the five examined ctDNA signatures in discriminating cancer from healthy controls (Figure S6). Importantly, we found that combining EM with other ctDNA signatures in a stack model could further improve the sensitivity for detecting cancer samples, with significant improvement for lung cancer patients (Figure S6A and S6B). These findings highlighted that the multimodal analysis of multiple ctDNA signatures by SPOT-MAS could increase the breadth of ctDNA feature analysis, thus enhancing the detection sensitivity while maintaining the low cost of sample preparation and sequencing.”

2) The baseline characteristics of part of the enrolled patients are not clear. It seems that some of the cancer patients were diagnosed only by imaging examinations. The manuscript described "staging information was not available for 25.7% of cancer patients, who were confirmed by specialized clinicians to have non-metastatic tumors". I have no idea how did this confirmation make? According to clinicians' experience only?

Our study only recruited cancer patients with non-systemic-metastatic stages (Stage I-IIIA) in which cancer is localized to the primary sites and has not spread to other organs. We excluded patients who were diagnosed with metastatic stage IIIB and IV cancer. All healthy subjects were confirmed to have no history of cancer at the time of enrollment. They were followed up at six months and one year after enrollment. The majority of cancer patients (74.3%) were confirmed to have cancer by abnormal imaging examination and subsequent tissue biopsy confirmation of tumor staging and metastasis status. For patients with unavailable staging information (25.7%), they initially went to the study hospitals for imaging examination. Upon receiving positive imaging results (MRI scan or CT scan), they moved to another hospital for surgery, leading to missing tumor staging information at the original study hospitals. The metastasis status of these patients were later obtained via communications between the clinicians at the study hospitals and the clinicians at the surgery hospitals, subject to existing data sharing agreement between the two hospitals. For those with metastatic cancer or unclear metastatic status, they were excluded from our study.

We have added the following sentences in the method (Line 127-135) and discussion section (Line 679-688).

“Cancer patients were confirmed to have cancer by abnormal imaging examination and subsequent tissue biopsy confirmation of malignancy. Cancer stages were determined by the TNM (Tumor, Node, Metastasis) system classification according to the American Joint Committee on Cancer and the International Union for Cancer Control. Our study only recruited cancer patients with non-systemic-metastatic stages (Stage I-IIIA) in which cancer is localized to the primary sites and has not spread to other organs. We excluded patients who were diagnosed with metastatic stage IIIB and IV cancer. All healthy subjects were confirmed to have no history of cancer at the time of enrollment. They were followed up at six months and one year after enrollment to ensure that they did not develop cancer.”

“For patients with unavailable staging information, their initial imaging examinations were conducted at the study hospitals. However, subsequent tests and surgical procedures were performed at a different hospital, as per the patients' preferences. Consequently, the original study hospitals lacked access to comprehensive tumor staging data. To address this limitation, the metastasis status of these patients was obtained via communication channels between the clinicians at the study hospitals and those at the surgery hospitals. This enabled the retrieval of limited information, adhering to an established data-sharing agreement between the two institutions. To maintain the robustness of our analysis, patients diagnosed with metastatic cancer or those with indeterminate metastatic status were subsequently excluded from the study.”

3) It seems that one of the important advantages of this new model is the low depth coverage in comparing to previous screening models for cancer. The authors should discuss more on the reason why the new model could achieve comparable predictive accuracy with an obviously lower sequencing depth.

We thanked the reviewer for the suggestion. We have added the following sentences in the discussion to explain why our assay could achieve good performance at low depth sequencing. (Line 571-584)

“However, the low amount of ctDNA fragments in plasma samples of patients with early-stage cancer as well as the molecular heterogeneity of different cancer types are known as the major challenges for liquid biopsy based multi-cancer detection assays. Thus, sequencing at high depth coverages is required to capture enough informative cancer DNA fragments in the finite plasma sample to achieve early cancer detection. In support to this notion, many groups (1-4) have developed assays that exploited high depth coverage of sequencing to detect ctDNA fragments in plasma of early stage cancer patients. However, this strategy might not be cost effective and feasible for population wide screening in developing countries. Alternatively, we argued that increasing breadth of ctDNA analysis could maximize the ability to detect ctDNA fragments with heterogeneous genetic and epigenetic changes at shallow sequencing depth, thus improving the sensitivity for multicancer detection. To demonstrate the feasibility of this approach, we built a stacking ensemble model to combine nine different ctDNA signatures and demonstrated its superior performance on cancer detection in comparison to single-feature models (Figure 7B and 7C).”

4) The readability of this manuscript needs to be improved. The focus of the background section is not clear, with too much detail of other studies and few purposeful summaries. You need to explain the goals and clinical significance of your study. In addition, the results section is too long, and needs to be shortened and simplified. Move some of the inessential results and sentences to supplementary materials or methods.

We thank the reviewer for these constructive suggestions. Accrodingly, we have reduced the details of other studies (Line 85-91) as follows:

“In recent years, there has been considerable interest in exploring the potential of ctDNA alterations for early detection of cancer (5, 6). One such approach is the PanSeer test, which uses 477 differentially methylated regions (DMRs) in ctDNA to detect five different types of cancer up to four years prior to conventional diagnosis (7). The DELFI assay employs a genome-wide analysis of ctDNA fragment profiles to increase sensitivity in early detection (1). Recently, the Galleri test has emerged as a multi-cancer detection assay that analyses more than 100,000 methylation regions in the genome to detect over 50 cancer types and localize the tumor site (8).”

We have modified the text in the introduction to explain the goals and clinical significance of our study (Line 111-123)

“In this study, we aimed to expand our multimodal approach, SPOT-MAS, to comprehensively analyze methylomics, fragmentomics, DNA copy number and end motifs of cfDNA and evaluate its utility to simultaneously detecting and locating cancer from a single screening test.” “Our findings demonstrate that the multimodal approach of SPOT-MAS enables profiling of multiple ctDNA signatures across the entire genome at low sequencing depth to detect five different cancer types in their early stages. Beyond detecting the presence of cancer signals, our assay was able to predict the tumor location, which is important for clinicians to fast-track the follow-up diagnostic and guide necessary treatment. Thus, SPOT-MAS has the potential to become a universal, simple, and cost-effective approach for early multi-cancer detection in a large population.”

Reviewer #2 (Public Review):

The authors tried to diagnose cancers and pinpoint tissues of origin using cfDNA. To achieve the goal, they developed a framework to assess methylation, CNA, and other genomic features. They established discovery and validation cohorts for systematic assessment and successfully achieved robust prediction power.

1) Still, there are places for improvement. The diagnostic effect can be maximized if their framework works well in early-stage cancer patients. According to Table 1, about 10% of the participants are stage I. Do these cancers also perform well as compared to late stage cancers?

We have performed the comparison of SPOT-MAS performance on different stages and provided the data in Supplementary table S8 and Supplementary Figure S4J and S4L. Our data showed that SPOT-MAS achieved lower sensitivity for detecting stage I and II cancers as compared to stage IIIA cancers in both discovery (61.54% and 69.82% for stage I and II respectively versus 78.67% for stage IIIA, Supplementary table 8) and validation cohort (73.91% and 62.32% for stage I and II, respectively versus 88.31% for stage IIIA, Supplementary table 8). This suggested that cancer stages can influence the performance of our models.

2) Can authors show a systematic comparison of their method to other previous methods to summarize what their algorithm can achieve compared to others.

We have conducted a systematic comparison of our method with others in the Supplementary Table S11.

Reviewer #1 (Recommendations For The Authors):

There are still points for the authors to clarify and consider for incorporation into revision.

• Please first clarify the issues mentioned in "public review". Several complements are needed.

We have addressed all of the reviewer’s comments in “public review”.

1) Line 72-73: Different approaches of early cancer screening assays have different features, application scenarios, and of course, limitations. It's too vague to describe in this way. More importantly, diagnosis of malignancies relies on pathological diagnosis, I don't think the results of unsuccessful screening would be overdiagnosis and overtreatment. That's overstatements.

We have rewritten the statement as follows (Line 72-75)

“Although currently guided screening tests have each been shown to provide better treatment outcomes and reduce cancer mortality, some of them are invasive, thus having low accessibility. Importantly, most of them are single cancer screening tests, which may result in high false positive rates when used sequentially.”

2) Line 115-130: The findings in this study shouldn't be introduced here.

We have removed this section.

3) Line 496-498: It surprised me that the model performed even better in independent validation cohort, which is quite different from the usual situations. Please explain it.

We agree with the reviewer that model performance in independent validation cohort is often lower than in discovery cohort. In our case, we have carefully confirmed our data by utilizing cross-validation (CV). Cross-validation is a widely used process in which the data being used for training the model is separated into folds or partitions and the model is trained and validated for each fold; the performance estimates are then calculated to obtain mean and confidence interval (GraphPad Prism, Wilson/Brown method). To further confirm our findings, we have increased the cross-validation fold into 50, and consistently detected no significant difference in the performance between Discovery and Validation cohorts (p=0.1277, DeLong’s test).

We have added the following sentence in the discussion to explain this (Line 633-635)

“Despite a slightly higher AUC value in the validation cohort compared to the discovery cohort, no significant differences in AUC values were observed between the two cohorts at CV of 10 or 50 (p=0.1277, DeLong’s test).”

4) Line 499-501: For the cut-off value selection, the authors thought that for cancer screening, specificity is more important than sensitivity? It's controversial. The sensitivity is only approximately 70%, I think that a missed diagnosis is even worse.

We agree with the reviewer that both specificity and sensitivity are important metrics of a cancer detection test. However, there is a trade-off between sensitivity and specificity and the preference for either one of them remains a controversial topic. For a screening test, the preference should be determined by considering the prevalence of the disease, in this case - cancer. The low prevalence of cancers indicates that even a small percentage of false-positive test results due to low specificity of the assay, spread across a national population, would hugely increase the demand for confirmatory imaging as well as biopsy sampling of imaging-detected benign abnormalities (9). Thus, false positives have obvious implications for health-care resources as well as patient well-being. Conversely, higher sensitivities will make sure that more cancer cases are detected and avoid delays in diagnosis. To mitigate the impact of insufficient sensitivity of a cancer screening test, it is important to consult the test-takers that current liquid biopsy tests should only be used as a complementary approach to the available diagnosis tests to increase rates of cancer detection. To be used as a stand-alone test, further work is required to improve its performance, with more focus on increasing sensitivity while maintaining high specificity.

We have added the following sentences in the discussion to explain why we set a high threshold of specificity (Line 660-671)

“For an effective screening test, careful consideration of disease prevalence, cancer in this context, is imperative. Given the low prevalence of cancers, even a small proportion of false-positive test results arising from reduced assay specificity, if extrapolated to a national population, could significantly escalate the need for confirmatory imaging and biopsy procedures for benign abnormalities detected during screening. Thus, false-positives can have substantial implications for both healthcare resources and patient well-being. Conversely, a screening test with high sensitivity ensures that most cancer cases are detected and minimizes delays in diagnosis. To address potential limitations posed by low sensitivity in cancer screening tests, we suggest that current liquid biopsy tests should be employed as a complementary approach to existing diagnostic methods to enhance cancer detection rates. To be used a stand-alone test, further work is required to improve its performance, with a particular emphasis on improving sensitivity while preserving high specificity.”

5) The methylation profiles have been used broadly in ctDNA, while your also integrated the fragmentomics, copy number aberration and end motif into the new model. In the discussion section, it would be better to further compare your new model with several previous models based on conventional ctDNA methylation markers (10, 11) for early detection of malignancies. What are the advantages of adding the other two types of data? Why the new model could achieve comparable predictive accuracy with an obviously lower sequencing depth?

We thank the reviewer for the suggestion. We have added the following sentences in the discussion to highlight the novelty of our multimodal approach. (Line 587-610)

“Previous studies have reported that methylation changes at target regions could be exploited for detecting ctDNA in plasma of patients with early-stage cancer (10, 11).”

“In addition to methylation alterations, recent studies have revealed that the DNA copy number, fragmentomics profile (1) and end motif profile (12) at genome wide scales have been shown as useful features for healthy-cancer classification. Therefore, we propose that the combination of these markers might provide added value to increase the performance of liquid biopsy assays. We demonstrated that the same bisulfite sequencing data could be used to identify somatic CNA (Figure 4), cancer-associated fragment length (Figure 5) and end motifs (Figure 6), highlighting the advantage of SPOT-MAS in capturing the broad landscape of ctDNA signatures without high cost deep sequencing. For cancer-associated fragment length, we pre-processed this data into five different feature tables to better reflect the information embedded within the data. Overall, we integrated multiple features of ctDNA including methylation, fragment length, end motif and copy number changes into a multi-cancer detection model and demonstrated that this approach could distinguish healthy individuals with patients from five popular cancer types. This strategy enables increased breadth of ctDNA analysis at shallow sequencing depth to overcome the limitation of low amount of ctDNA fragments in plasma samples as well as molecular heterogeneity of cancers.”

Moreover, we have conducted a systematic comparison of our method with others in the Supplementary Table 11.

6) Line 667-668: The wording should be modest. "Successfully detect and localize" is not appropriate.

We have rewritten the sentence. (Line 713-716)

“Our large-scale case-control study demonstrated that SPOT-MAS, with its unique combination of multimodal analysis of cfDNA signatures and innovative machine-learning algorithms, can detect and localize multiple types of cancer with high accuracy at a low-cost sequencing.”

Reviewer #2 (Recommendations For The Authors):

1) Are the patients and controls all from Vietnam? If I am not mistaken, it is hard to find demographic information for controls. Also it is not clear if samples from controls were processed simultaneously or at a same institution or using the same protocol etc.

We thank the reviewer for asking this question. All cancer patients and controls are from Vietnam, who were recruited from five hospitals including Medic Medical Center, University Medical Center Ho Chi Minh City, Thu Duc City Hospital, National Cancer Hospital and Hanoi Medical University. At each research sites, blood samples from both cancer patients and healthy subjects were collected in in Streck Cell-Free DNA BCT tubes and subsequently transported to a central laboratory located in Medical Genetics Institute for cfDNA isolation, library preparation and sequencing. In a recent publication (10), we have investigated the impact of logistic time and hemolysis rates of blood samples collected from different clinical sites on cfDNA concentration and sequencing quality. We did not observe any noticeable impact of such variations on cfDNA concentrations or sequencing library yields. However, future analytical validation studies are required to evaluate the impact of variation in sampling technique across different clinical sites on the robustness or accuracy of assay results.

We have added the following sentences in the discussion to highlight this important point (Line 696-704)

“At each research sites, blood samples from both cancer patients and healthy subjects were collected in in Streck Cell-Free DNA BCT tubes and subsequently transported to a central laboratory located in Medical Genetics Institute for cfDNA isolation, library preparation and sequencing. In a recent publication (10), we have investigated the impact of logistic time and hemolysis rates of blood samples collected from different clinical sites on cfDNA concentration and sequencing quality. We did not observe any noticeable impact of such variations on cfDNA concentrations or sequencing library yields. However, future analytical validation studies using a larger sample size are required to evaluate the impact of variation in sampling technique across different clinical sites on the robustness or accuracy of assay results.”

References

1. Cristiano S, Leal A, Phallen J, Fiksel J, Adleff V, Bruhm DC, et al. Genome-wide cell-free DNA fragmentation in patients with cancer. Nature. 2019;570(7761):385-9.

2. Cohen JD, Li L, Wang Y, Thoburn C, Afsari B, Danilova L, et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science. 2018;359(6378):926-30.

3. Liu MC, Oxnard GR, Klein EA, Swanton C, Seiden MV. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol. 2020;31(6):745-59.

4. Stackpole ML, Zeng W, Li S, Liu C-C, Zhou Y, He S, et al. Cost-effective methylome sequencing of cell-free DNA for accurately detecting and locating cancer. Nature Communications. 2022;13(1):5566.

5. Constantin N, Sina AA, Korbie D, Trau M. Opportunities for Early Cancer Detection: The Rise of ctDNA Methylation-Based Pan-Cancer Screening Technologies. Epigenomes. 2022;6(1).

6. Phan TH, Chi Nguyen VT, Thi Pham TT, Nguyen VC, Ho TD, Quynh Pham TM, et al. Circulating DNA methylation profile improves the accuracy of serum biomarkers for the detection of nonmetastatic hepatocellular carcinoma. Future Oncol. 2022;18(39):4399-413.

7. Chen X, Gole J, Gore A, He Q, Lu M, Min J, et al. Non-invasive early detection of cancer four years before conventional diagnosis using a blood test. Nature Communications. 2020;11(1):3475.

8. Jamshidi A, Liu MC, Klein EA, Venn O, Hubbell E, Beausang JF, et al. Evaluation of cell-free DNA approaches for multi-cancer early detection. Cancer Cell. 2022;40(12):1537-49.e12.

9. Ignatiadis M, Sledge GW, Jeffrey SS. Liquid biopsy enters the clinic - implementation issues and future challenges. Nat Rev Clin Oncol. 2021;18(5):297-312.

10. Xu RH, Wei W, Krawczyk M, Wang W, Luo H, Flagg K, et al. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat Mater. 2017;16(11):1155-61.

11. Luo H, Zhao Q, Wei W, Zheng L, Yi S, Li G, et al. Circulating tumor DNA methylation profiles enable early diagnosis, prognosis prediction, and screening for colorectal cancer. Sci Transl Med. 2020;12(524).

12. Jiang P, Sun K, Peng W, Cheng SH, Ni M, Yeung PC, et al. Plasma DNA End-Motif Profiling as a Fragmentomic Marker in Cancer, Pregnancy, and Transplantation. Cancer Discovery. 2020;10(5):664-73.

dismissed JA's, war effort

Es la cantidad de activos circulantes requerida para hacer frente a las necesidades mínimas a largo plazo. Podría llamarse también capital de trabajo “puro”.

El capital de trabajo puede ser clasificado de acuerdo con: • Componentes: En efectivo, valores negociables, cuentas por cobrar e inventarios y de, • Tiempo: En permanente o temporal.

change to scler/o (also incorrect in the companion)

• paraphrase

  • A common thread connecting both extremes of dualism and illusionism is that both assume that phenomenal consciousness is an absolute phenomenon,
    • wherein an object O evinces either property P or ¬P.
  • We will show that we need to abandon this assumption.
  • The relativistic principle in modern physics posits a universe in which for many properties an object O evinces either property P or ¬P with respect to some observer X.
  • In such a situation, there is no one answer to the question of whether object O has property P or not.

• question

  • how does this argue against the privacy of phenomena?
  • it argues against it in the sense that it isn't needed. What is interpreted as private is simply the experience from one relative frame of reference.

helped the inhabitants

economic

indian land

17092023 092116 1-260 R15. SL o DOU

I think there might be too much text over top of the picture here. Although it is organized and font sizes vary and some the heading is in bold to stand out, it seems a little cluttered. Too much of an information/visual for one small section.

(VIP) I would never thought this could be an option. It shows how Norma sees people as a connection not assets.

(VIP) Proof that you can change the course of your life anytime you truly want it. The business of your family name is not always what you are created to do. Even if your family doesn't have any family profession or higher education. You can be the one to make difference no one has seen before.

Annotation. E & O: This sentence contradicts itself. The first part basically states that a space is provided for the reader/viewer to think for themself and make their own decisions/interpretations, but the second part of the sentence then says that the reader/viewer does not have the space to make this decision. That doesn't make sense to me.

• Conocimiento claramente que es un creador de guia
• Conocimiento del formato para hacerlo

Here is some reporting on this supplemental reporting request. But I am unable to find the funding request itself.

https://www.politico.com/news/2020/02/24/trump-coronavirus-budget-request-117275

Certainly! Let's break down the sentence:

この店で自分の魔法の杖を見つけるのが待ちきれなかっ (Kono mise de jibun no mahō no tsue o mitsukeru no ga machikirenaka)

• この店で (Kono mise de): In this store
• 自分の (jibun no): One's own
• 魔法の杖 (mahō no tsue): Magic wand
• を見つける (o mitsukeru): To find
• のが (no ga): Subject marker for the clause
• 待ちきれなかっ (machikirenaka): Couldn't wait (incomplete form of "待ちきれなかった," the past tense of "待ちきれない," meaning "can't wait")

Translation: Couldn't wait to find my own magic wand in this store.

The sentence seems to be cut off at the end, but it describes a feeling of impatience or excitement about finding one's own magic wand in a particular store. It suggests that the speaker is eager to find their magic wand and can't wait to do so.

If you have any more questions or need further clarification, feel free to ask!

Certainly! Let's break down the sentence:

ドリスさんが最後に手を振ってきた (Dorisu-san ga saigo ni te o futte kita)

• ドリスさんが (Dorisu-san ga): Ms. Doris (subject marker)
• 最後に (saigo ni): Lastly, finally
• 手を振って (te o futte): Waving hand (te-form of "手を振る" meaning "to wave one's hand")
• きた (kita): Came (past tense of "くる" meaning "to come")

Translation: Ms. Doris finally waved her hand as she came.

This sentence describes a situation where Ms. Doris waves her hand, presumably as a gesture of farewell or acknowledgment, as she approaches or leaves. The word "最後に" (saigo ni) indicates that this action is the final or concluding event in a sequence.

If you have any more questions or need further clarification, feel free to ask!

Certainly! Let's break down the sentence:

ハグリッドは人ごみを押しのけて進んで、ハリーはただついていっただけ (Haguriddo wa hitogomi o oshinokete susunde, Harī wa tada tsuite itta dake)

• ハグリッドは (Haguriddo wa): Hagrid (subject marker, referring to a character, possibly Rubeus Hagrid from the Harry Potter series)
• 人ごみを (hitogomi o): Crowd (object marker)
• 押しのけて (oshinokete): Pushing aside (te-form of "押しのける" meaning "to push aside")
• 進んで (susunde): Advancing (te-form of "進む" meaning "to advance" or "to move forward")
• ハリーは (Harī wa): Harry (subject marker, referring to Harry Potter)
• ただ (tada): Just, merely
• ついていっただけ (tsuite itta dake): Followed along (past tense of "ついていく" meaning "to follow along," with "だけ" emphasizing that it was the only action)

Translation: Hagrid advanced by pushing through the crowd, and Harry merely followed along.

This sentence describes a scene where Hagrid is actively making his way through a crowd, while Harry simply follows him. It paints a picture of Hagrid as the more assertive or dominant figure in this particular situation, with Harry just going along with him.

If you have any more questions or need further clarification, feel free to ask!

説明

このフレーズ「手紙を取るためにみんながもめた」は、「Everyone argued to take the letter」という意味になります。ここで、手紙を取る（てがみをとる）は「to take the letter」、ために（ために）は「in order to」または「for the purpose of」という意味です。もめた（もめた）は、もめる（もめる）の過去形で、「to argue」または「to have a dispute」という意味です。

このフレーズは、手紙を取る行為に関してみんなが異なる意見や願望を持っていたため、議論や対立が生じたことを示しています。

会話の例

状況: あるグループの友達が手紙を受け取り、それを読みたいとみんなが思っています。しかし、誰がそれを開いて読むべきかについて合意できず、結局は互いに議論してしまいます。 - Person A: 手紙を取るためにみんながもめたよ！（Tegami o toru tame ni minna ga mometa yo!） - Person B: え、どうして？（E, doushite?） - Person A: みんなが一番に手紙を読みたかったからだよ。（Minna ga ichiban ni tegami o yomitakatta kara da yo.） - Person B: それはややこしい状況だったね！（Sore wa yayakoshii joukyou datta ne!）

代替表現

1. 「手紙を取り合ってみんながもめた」: カジュアルな表現で、手紙を取ることに関する議論があった状況を説明します。
2. 「手紙の取り合いでみんなが揉めた」: 口語的な表現で、手紙を取ることに関する騒動があった状況を指します。
3. 「手紙を奪い合ってみんなが喧嘩になった」: 強い表現で、手紙を取ることに関する戦いがあった状況を示します。

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security token offering

//

This is a call for the interpersonal indy.net where people are autonomous actors in their ow(ed) local first, private first, offline first, evergreen, interpersonal trust networks

Cabe observar o uso dos vocábulos: ao empregar "privativamente", a Constituição se refere ao poder da União em legislar as matérias ali elencadas sem qualquer participação dos demais entes federados.

Opõe-se ao vocábulo "concorrente" e não à palavra "comum", a qual é destinada a indicar competências administrativas.

Ademais, cabe observar que, ao dizer competência legislativa privativa, pode-se remeter à possibilidade de delegação aos Estados para que legislem sobre questões específicas sobre determinada matéria delegada, devendo-se delegar através de lei complementar. Nesse contexto, o Estado jamais poderá legislar sobre tais questões específicas com abrangência nacional e nem mesmo sobre a integralidade da matéria delegada.

Diferentemente da competência legislativa privativa, a competência exclusiva administrativa da União é indelegável.

O vocábulo "concorrentemente" se refere à competência dos Entes federados indicados em legislar em conjunto.

Opõe-se ao vocábulo "privativamente".

O vocábulo "comum" se refere às competências administrativas a serem exercidos com participação dos entes-federados.

Competência legislativa suplementar supletiva. Isto é, na ausência de lei federal sobre o tema, o Estado exerce a competência legislativa plena sobre o tema até a que sobrevenha lei federal.

Neste caso, o Estado produzirá norma inédita no ordenamento jurídico, não se limitando a apenas especificar as peculiaridades regionais, mas também a editar norma geral sobre o assunto naquele Estado, até à edição de norma geral federal.

Competência legislativa suplementar complementar. Isto é, o Estado edita lei estadual para completar a norma federal, objetivo adequá-la às suas peculiaridades regionais.

Nesse contexto, o Estado produzirá norma mais específica que aquela federal.

Observe que a vedação não alcança a União, sobretudo pelo o que dispõe o art. 151, I.

• 1ª – O termo inicial do prazo da anterioridade anual é a edição da medida provisória, quando o objeto da MP for a instituição ou a majoração de qualquer outra espécie tributária que não impostos (ex. taxas, contribuições de melhoria e contribuições especiais).
• 2ª: O termo inicial do prazo da anterioridade anual é a conversão em lei da medida provisória, quando esta última instituir ou majorar impostos, salvo aqueles ressalvados pelo §2º do artigo 62 – que sequer se submetem à anterioridade anual.
• 3ª: O termo inicial do prazo da anterioridade nonagesimal é a edição da medida provisória.
• 4ª: Em qualquer caso, o termo inicial é a conversão em lei da medida provisória, nos casos em que a instituição ou a majoração do tributo só foi realizada no curso do processo legislativo.

ADI 3768/2007:

• Deve ser, ao final, enfatizado que o direito dos idosos à gratuidade de transporte coletivo urbano não está incluído no rol de benefícios da seguridade social.
• A despeito de estarem dispostas no Título VIII da Constituição da República, que trata da Ordem Social, as disposições relativas à seguridade social (saúde, previdência e assistência social), previstas no Capítulo II, não se confundem com aquelas afeitas aos idosos, situadas no Capítulo VI, sendo correto, por isso mesmo, afirmar que as normas constitucionais atinentes à seguridade social (arts. 194 a 204) não são aplicáveis à específica disciplina do direito dos idosos (art. 230).
• De se concluir que, além de as concessionárias e permissionárias terem a obrigação de cumprir as cláusulas estipuladas para a prestação dos serviços de transporte, devem respeitar a Constituição da República.
• Como membros da sociedade, são elas titulares do dever de contribuir, efetiva e diretamente, para que as pessoas idosas em específico, tenham assegurado o seu direito à gratuidade dos transportes coletivos urbanos por força do princípio da unidade do sistema jurídico republicano.

Empréstimos compulsórios sempre serão instituídos mediante lei complementar.

Somente o empréstimo destinado ao investimento público de caráter urgente e de relevante interesse social será submetido ao, tão somente, o princípio da anterioridade anual,

O Procon pode aplicar multa a fornecedor em razão do repasse aos consumidores, efetivado com base em cláusula contratual, do ônus de arcar com as despesas postais decorrentes do exercício do direito de arrependimento previsto no art. 49 do CDC.

De acordo com o caput do referido dispositivo legal, o consumidor pode desistir do contrato, no prazo de sete dias a contar de sua assinatura ou do ato de recebimento do produto ou serviço, sempre que a contratação de fornecimento de produtos e serviços ocorrer fora do estabelecimento comercial, especialmente por telefone ou a domicílio.

O parágrafo único do art. 49 do CDC, por sua vez, especifica que o consumidor, ao exercer o referido direito de arrependimento, terá de volta, imediatamente e monetariamente atualizados, todos os valores eventualmente pagos, a qualquer título, durante o prazo de reflexão – período de sete dias contido no caput do art. 49 do CDC –, entendendo-se incluídos nestes valores todas as despesas decorrentes da utilização do serviço postal para a devolução do produto, quantia esta que não pode ser repassada ao consumidor.

Aceitar o contrário significaria criar limitação ao direito de arrependimento legalmente não prevista, de modo a desestimular o comércio fora do estabelecimento, tão comum nos dias atuais. Deve-se considerar, ademais, o fato de que eventuais prejuízos enfrentados pelo fornecedor nesse tipo de contratação são inerentes à modalidade de venda agressiva fora do estabelecimento comercial (pela internet, por telefone ou a domicílio)” (STJ, REsp 1.340.604/RJ – Rel. Min. Mauro Campbell Marques – j. 15.08.2013, publicado no seu Informativo n. 528).

I feel this is how learning looks like for some. They are given an idea or a concept which gets their "gears" turning to analyze it and make sense of the new information. I can see this in my son who is 2 as he manipulates his puzzles to make sense. You can see the hears turning in his head as he sticks his tongue out to understand the complexity of the puzzle. Scholars are the same as new information is given to them. I am one who has to process and marinate on new information before it has become part of me. This allows scholars to take it from short term memory to long term memory.

Informativo n. 510

A gastroplastia, indicada para o tratamento da obesidade mórbida, bem como de outras doenças dela derivadas, constitui cirurgia essencial à preservação da vida e da saúde do paciente segurado, não se confundindo com simples tratamento para emagrecimento. Os contratos de seguro-saúde são contratos de consumo submetidos a cláusulas contratuais gerais, ocorrendo a sua aceitação por simples adesão pelo segurado. Nesses contratos, as cláusulas seguem as regras de interpretação dos negócios jurídicos estandardizados, ou seja, existindo cláusulas ambíguas ou contraditórias, deve ser aplicada a interpretação mais favorável ao aderente, conforme o art. 47 do CDC.

Assim, a cláusula contratual de exclusão da cobertura securitária para casos de tratamento estético de emagrecimento prevista no contrato de seguro-saúde não abrange a cirurgia para tratamento de obesidade mórbida. Precedentes citados: REsp 1.175.616/MT, DJe 4/3/2011; AgRg no AREsp 52.420/MG, DJe 12/12/2011; REsp 311.509/SP, DJ 25/6/2001, e REsp 735.750/SP, DJe 16/2/2012” (STJ – REsp 1.249.701/ SC – Rel. Min. Paulo de Tarso Sanseverino – j. 04.12.2012).

Informativo nº 551

DIREITO DO CONSUMIDOR. POSSIBILIDADE DE SEGURADORA OU OPERADORA DE PLANO DE SAÚDE CUSTEAR TRATAMENTO EXPERIMENTAL. A seguradora ou operadora de plano de saúde deve custear tratamento experimental existente no País, em instituição de reputação científica reconhecida, de doença listada na CID-OMS, desde que haja indicação médica para tanto, e os médicos que acompanhem o quadro clínico do paciente atestem a ineficácia ou a insuficiência dos tratamentos indicados convencionalmente para a cura ou controle eficaz da doença.

Cumpre esclarecer que o art. 12 da Lei 9.656/1998 estabelece as coberturas mínimas que devem ser garantidas aos segurados e beneficiários dos planos de saúde. Nesse sentido, as operadoras são obrigadas a cobrir os tratamentos e serviços necessários à busca da cura ou controle da doença apresentada pelo paciente e listada na Classificação Estatística Internacional de Doenças e Problemas Relacionados com a Saúde, da Organização Mundial de Saúde (CID-OMS). Já o art. 10, I, da referida Lei estabelece que as seguradoras ou operadoras de plano de saúde podem excluir da cobertura o tratamento clínico ou cirúrgico experimental. Nessa linha intelectiva, a autorização legal para que um determinado tratamento seja excluído deve ser entendida em confronto com as coberturas mínimas que são garantidas. Tanto é assim que o art. 10 da Lei 9.656/1998 faz menção expressa ao art. 12 do mesmo diploma legal e vice-versa. Desse modo, o tratamento experimental, por força de sua recomendada utilidade, embora eventual, transmuda-se em tratamento mínimo a ser garantido ao paciente, escopo da Lei 9.656/1998, como se vê nos citados arts. 10 e 12.

Isto é, nas situações em que os tratamentos convencionais não forem suficientes ou eficientes - fato atestado pelos médicos que acompanham o quadro clínico do paciente -, existindo no País tratamento experimental, em instituição de reputação científica reconhecida, com indicação para a doença, a seguradora ou operadora deve arcar com os custos do tratamento, na medida em que passa a ser o único de real interesse para o contratante.

Assim, a restrição contida no art. 10, I, da Lei 9.656/1998 somente deve ter aplicação nas hipóteses em que os tratamentos convencionais mínimos garantidos pelo art. 12 da mesma Lei sejam de fato úteis e eficazes para o contratante segurado.

Ou seja, não pode o paciente, à custa da seguradora ou operadora de plano de saúde, optar por tratamento experimental, por considerá-lo mais eficiente ou menos agressivo, pois lhe é disponibilizado tratamento útil, suficiente para atender o mínimo garantido pela Lei. REsp 1.279.241-SP, Rel. Min. Raul Araújo, julgado em 16/9/2014.

bioacumulacao

Anne finds it strange that his absence has such a profound impact on her emotions.

Should IHL have something more closely articulated like this...would this help to limit grey areas?

Hi!

Washington's motive was different o that may be why he was able to make real progress in the minds of those with opposing ideas

I am going to check out her TED talk!

In my opinion, I've never really understood why use chatgpt or any other AI software to cheat when writing about a topic. The writing might be challeging, which is understandable, but everything you're writing is your thoughts and how you feel.Also, you can always draft your ideas, which will significantly improve your thought process on what you are writing about.

This is very important because In most classes once an assignment is late you lose points, but here you have an opportunity to do it as long as it's open. In my opinion, I prefer this compared to other classes because you might need an extra day and if the assignment is still open you can take that chance.

To summarize, This course teaches students to write at a college level in a step-by-step process, so that students are learning at a steady pace and not left behind. Also, students are graded on quality because spending more time on an assignment doesn't necessarily mean it's going to be high quality, It could show students what areas of writing they need improvement on.

I can connect to this because there might be a class I don't enjoy, but at the end of the day, the professors are only there to guide me, not hold my hand all the way through. Sometimes it's hard to find motivation, but It's important to always remember your end goal and what you will achieve.

Jornadas sobre Prevenção e Solução Extrajudicial de Litígios

• ENUNCIADO 114 - O art. 10-A, § 2º, do Decreto-Lei n. 3.365/1941, com redação dada pela Lei n. 13.867/2019, permite o registro de acordo de desapropriação amigável sem necessidade de escritura pública, ainda que de valor superior a 30 salários mínimos.
• Justificativa: A Lei n. 13.867/2019 teve por finalidade estimular a solução amigável para desapropriações, prevendo a obrigatoriedade de apresentação de proposta completa e estímulo à mediação e arbitragem. Nesse sentido, também previu que o acordo assinado constitui "título hábil para transcrição no registro de imóveis". Tal previsão estabelece a possibilidade de registro do acordo sem necessidade de escritura pública.
• Isso colabora para estimular a composição nesses casos, uma vez que torna mais ágil a celebração de acordo, recebimento da indenização pelo expropriado e a conclusão do processo pela administração, enfim, desburocratiza. Tal interpretação encontra-se em harmonia com os demais dispositivos legais, vejamos.
• O art. 108 do Código Civil estabelece expressamente que sua aplicação é subsidiária, pois dispõe que a escritura pública é essencial "dispondo a lei em contrário". No caso, ao estabelecer que o acordo "é título hábil à transcrição", dispensa-se que o acordo seja lavrado em escritura pública. No mesmo sentido, a Lei de Registros Públicos estabelece que podem ser levados a registro "os escritos particulares autorizados em lei", novamente, há autorização expressa.
• Por fim, tal interpretação está em harmonia com os atributos do ato administrativo no que se refere à presunção de legalidade e legitimidade. Isso porque tais atributos proporcionam segurança para o acordo celebrado, de modo que a ausência de escritura pública não representará fator de insegurança jurídica.

Jornadas sobre Prevenção e Solução Extrajudicial de Litígios

• ENUNCIADO 114 - O art. 10-A, § 2º, do Decreto-Lei n. 3.365/1941, com redação dada pela Lei n. 13.867/2019, permite o registro de acordo de desapropriação amigável sem necessidade de escritura pública, ainda que de valor superior a 30 salários mínimos.
• Justificativa: A Lei n. 13.867/2019 teve por finalidade estimular a solução amigável para desapropriações, prevendo a obrigatoriedade de apresentação de proposta completa e estímulo à mediação e arbitragem. Nesse sentido, também previu que o acordo assinado constitui "título hábil para transcrição no registro de imóveis".
• Tal previsão estabelece a possibilidade de registro do acordo sem necessidade de escritura pública. Isso colabora para estimular a composição nesses casos, uma vez que torna mais ágil a celebração de acordo, recebimento da indenização pelo expropriado e a conclusão do processo pela administração, enfim, desburocratiza. Tal interpretação encontra-se em harmonia com os demais dispositivos legais, vejamos.
• O art. 108 do Código Civil estabelece expressamente que sua aplicação é subsidiária, pois dispõe que a escritura pública é essencial "dispondo a lei em contrário". No caso, ao estabelecer que o acordo "é título hábil à transcrição", dispensa-se que o acordo seja lavrado em escritura pública. No mesmo sentido, a Lei de Registros Públicos estabelece que podem ser levados a registro "os escritos particulares autorizados em lei", novamente, há autorização expressa.
• Por fim, tal interpretação está em harmonia com os atributos do ato administrativo no que se refere à presunção de legalidade e legitimidade. Isso porque tais atributos proporcionam segurança para o acordo celebrado, de modo que a ausência de escritura pública não representará fator de insegurança jurídica.

Jornadas sobre Prevenção e Solução Extrajudicial de Litígios

• ENUNCIADO 96 - É válida a inserção da cláusula compromissória em pacto antenupcial e em contrato de união estável.

• Justificativa: É plenamente válida e eficaz a inserção da cláusula compromissória no pacto antenupcial ou no contrato de união estável, desde que tenha por objeto direitos patrimoniais disponíveis, compatibilizando-se, assim, o art. 1º da LArb com o art. 104 do CC, no que tange à licitude do objeto como requisito geral de validade dos negócios jurídicos.

• Nas relações entre os cônjuges ou entre os companheiros, não há que se falar em direitos patrimoniais indisponíveis. Todos os direitos subjetivos que emergem das relações jurídicas patrimoniais entre parceiros conjugais são disponíveis. Assim, v.g., a discussão envolvendo o direito de qualquer dos cônjuges a partilhar bem adquirido antes ou durante o casamento pode ser tranquilamente submetida à jurisdição arbitral, tanto em face de cláusula inserida na convenção matrimonial, quer previamente ao casamento, quer após o casamento, mediante alteração do regime de bens, nos termos do § 2º do art. 1.639 do CCB, como em decorrência de compromisso arbitral que venha a ser celebrado após o surgimento do conflito.

Art. 1.814. São excluídos da sucessão os herdeiros ou legatários:

I - que houverem sido autores, co-autores ou partícipes de homicídio doloso, ou tentativa deste, contra a pessoa de cuja sucessão se tratar, seu cônjuge, companheiro, ascendente ou descendente;

II - que houverem acusado caluniosamente em juízo o autor da herança ou incorrerem em crime contra a sua honra, ou de seu cônjuge ou companheiro;

III - que, por violência ou meios fraudulentos, inibirem ou obstarem o autor da herança de dispor livremente de seus bens por ato de última vontade.

Jornadas sobre Prevenção e Solução Extrajudicial de Litígios

• Enunciado 49. Os Comitês de Resolução de Disputas (Dispute Boards) são um método de solução consensual de conflito, na forma prevista no parágrafo 3º do artigo 3º do CPC.
• Enunciado 76. As decisões proferidas por um Comitê de Resolução de Disputas (Dispute Board), quando os contratantes tiverem acordado pela sua adoção obrigatória, vinculam as partes ao seu cumprimento até que o Poder Judiciário ou o juízo arbitral competente emitam nova decisão ou a confirmem, caso venham a ser provocados pela parte inconformada.
• Enunciado 80. A utilização do Dispute Board, com a inserção da respectiva cláusula contratual, é recomendável para os contratos de construção ou de obras de infraestrutura, como mecanismo voltado para a prevenção de litígios e a redução dos custos correlatos, permitindo a imediata resolução de conflitos surgidos no curso da execução dos contratos.
• Enunciado 131 - As decisões promovidas por Comitês de Resolução de Disputa (Dispute Boards) que sejam vinculantes têm natureza contratual e refletem a vontade das partes que optaram por essa forma de resolução de conflitos, pelo que devem ser cumpridas obrigatória e imediatamente, sem prejuízo de eventual questionamento fundamentado em ação judicial ou procedimento arbitral.
• Enunciado 137 - Na utilização do comitê de resolução de disputas (Dispute Board) como meio alternativo de prevenção e resolução de controvérsias relativas aos contratos administrativos (art. 151 da Lei n. 14.133, de 1º de abril de 2021), deverá ser utilizada, preferencialmente, a modalidade combinada, na qual o comitê pode emitir recomendações e decisões.

sta je o v o

what do you mean by rhetoric, exactly?

It is interesting that how creating new stories today is a way to connect with the past and understand the Native Hawaiian culture and values and how it helps to keep their traditions alive. I am thinking that some of the terms like kaukau ali'i , do not have any use in the modern world. However, when new generations create new mo'olelo they repeat the words and that keeps the culture alive.

Banning Critical Race Theory in Florida classrooms :o

Perfectly valid serving method allowed by law, state has one, both a court e system as well as regular email.

Alinear bullets

Es común que te sientas nervioso o preocupado a medida que se acerca la cirugía. Saber qué esperar antes, durante y después del procedimiento puede ayudarte a prepararte y sentirte más a gusto.

La cirugía para pacientes hospitalizados significa que debes permanecer en el hospital durante la noche o más, para recuperarte después de la cirugía.

La oncología quirúrgica puede ayudarte durante las primeras etapas del cáncer o cuando la afección está más avanzada. Algunas razones comunes por las que podrías necesitarla son

sets: collection of objects ex: A{6,1,2,0....} * set order does not matter * duplicates are not allowed B={2,{3,4},{}} {} empty set (known as 0 with a slash) not actually zero but is only empty union sets: AuB= {2,6,1,{},0,{3,4}} what if A had 3? 3 is not a element of AuB Intersection set: AnB={2} set minus: A-B = A but not B (A\B) Natural numbers: set of even natural numbers= {n \in N| is even(n)} notes:

. = is such that , after for all or exists

A set cannot have itself as an element.

z\in N {1,4,7} not\in N (or sub in not\in fir subet of c with underline) Z={-2,-2,0,1} Q= Rational R= Real C = complex numbers

what does it mean to prove a statement? Prove existence

Thm: exist n \in N, n >=10 and is prime(n) proof: show n=11 satisfies the condition 11>=10 ^ 11 is prime

Thm: exists x \in S. P(x) proof: choose x= ? \in S. P(x) is true because ?

Prove universality or for all cant just say x=11 but all numbers

proof by example is not the way to go for all x \in R . x^2 -6x>-10 proof: suppose x is in R (x^2-6x+9)=(x-3)^2>=0 x^2-6x=(x^2-6x+9)-9>=-9>-10

square is done with proof or QED

thm: for all x \in S. P(x) proof: suppose x is an arbitrary number of S P(x) is true because reasons.

for all or exists needs a period that means such that

proof of implication: for all n \in Z. ( n is mult. of 10)=> is even(n). proof: suppose n \in Z. assume n is mult of 10 ; WTS n is even, n=10k(k is some int) =25K=2some int

WTS - WANT TO SHOW p->q Assume p is true then Q is true EXPLAIN contrapositive: not p -> not p Assume not Q false then not P false EXPLAIN p->q = not p -> not p

proof by contrapositive: introduce variable for "for all" suppose n\in Z. assume n is odd ; WTS n*n is odd odd x odd =odd

Proof by contradiction: counter intuitive to prove P show that not P is false if its not false its true

Thm:

sqrt2 not \in Q Assume sqrt2 is \in Q sqrt2=a/b for some ints a,b ,b cannot be zero assume lowest form and a and b share no common factors (other than + or - 1 ) a^2=2b^2-> a^2 is even -> a is even a=2A for some int A 4A^2=2b^2->2A^2=b^2 b^2 is even-> b is even b=2B for some B \in Z a=2A, b=2B a/b is not reduced because there is a factor of 2 that's common and 2 is not + - 1 .. CONTRADICTION assumption sqrt2 \in Q must be false -> sqrt2 not\in Q QED.

COMMON FACTORS ARE RATIO OF INTS. --?

Review-

INDUCTION:

thm: for all n >=0 , (1+2+3+...+n)=(n(n+1))/2 [sigma k=1 to n (k)] to exlude 0

n=0 an empty sum is 0 (01)/2 <br /> n=1: 1=? (12)/2 add 1 n=2: 1+2 =? (23)/2 add 2 n=3 1+2+3=? (34)/2 add 3 n=4 1+2+3+4=? (4*5)/2 add 4

verify: 1+2+..+(n-1)=(n-1)(n)/2 add n 1+2+..+(n-1)+n=n(n+1)/2

(n^2+n)/2 - (n^2-n)/2 = 2n/2 = n

(1+2+3+...+n)=(n(n+1))/2=: P(n) [sigma k=1 to n (k)] to exlude 0 induction principle: if you prove the implications are true than the rest are true. p(0) true p(1) true p(1)->p(2) (implies) p(2) -> p(3) p(3) -> p(4) ......

Below is all true because the implications are true aswell. p(1) p(2) p(3) p(4)

principle of induction: if [p(0) and for all n >=1. p(n-1)->p(n)] then [ for all n \in N. p(n)]

outline to follow: thm: p(n) is true for all n \in N

first step: define p(n) recall: p(n) ="(1+2+3+...+n)=(n(n+1))/2" proof by induction on n. base case (p(0): is true b/c ( do some work) if [p(0) and for all n >=1. p(n-1)->p(n)] then [ for all n \in N. p(n)] inductive step: assume n is at least 1 and p(n-1) is true and WTS p(n) IS TRUE -------assume left prove right.--------

Ie. assume

1+2+..+(n-1)=(n-1)(n)/2

WTS 1+2+...(n-1)+n=n(n+1)/2

WTS (n-1)(n)/2 + n=n(n+1)/2

true by algebra by induction p(n) is true for all n>=0

16 x 16 grid want to tile w/o middle cell L-trominoes tile once but not covered twice p(n):=2^nx2^n grid w/o one middle cell can be tile w/ L trominoes

p(0), tile |x| missing middle cell QED P(1): 2X2 1 L trominoe P(2): 4X4 start in middle then outward 8x8 example: highlight middle lines x and y start center and observe 4 quads separately cant invoke p(2) twice. if induction is not working try and prove a stronger thm.<br /> remove middle cell restriction less restrictions are stronger thms.

p(2)->p(3) p(3)->p(4) strat: place one L in the middle , hitting the 3 full quadrants now use p(2) on each quad

3 quads: 2^nx2^n -1 cell missing cell in corner 4th 2^nx2^n missing cell wont be in corner

you can always tile no matter where the face is in 2x2 induction

each p(n) is a algorithm not a thm

Existem vários aplicativos que podem te ajudar a fazer exercícios em casa sem a necessidade de equipamentos especiais. Aqui estão alguns dos melhores:

Fitness Point: É um personal trainer virtual que incentiva o usuário a fazer os exercícios. Ele permite incluir a rotina semanal de treino, gerenciar e controlar o progresso em cada série1.

Nike Training Club: Este aplicativo gratuito auxilia na motivação e monitoramento do treino. Ele conta com mais de 100 sequências de exercícios, desenvolvidos em parceria com atletas renomados1.

Fitbit: Este aplicativo permite contar calorias, registrar o peso, gravar pressão arterial e níveis de glicose, compartilhar seu progresso com amigos e várias outras funções1.

iCardio: Trabalha em conjunto com monitor de frequência cardíaca para garantir precisão das taxas durante a realização de exercícios físicos1.

Desafio 30 Dias Fitness: É um aplicativo completo de exercícios que podem ser feitos em casa, sem a necessidade de aparelhos especiais2.

Sworkit – Abdominal & Núcleo Trainer: É um app focado em exercícios abdominais que podem ser feitos em casa e sem aparelhos2.

Todos esses aplicativos estão disponíveis para Android e iOS. Lembre-se sempre de consultar um profissional de saúde antes de iniciar qualquer programa de exercícios.

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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Bideau et al. describe posterior regeneration in the annelid Platynereis. The authors aimed to identify patterns of proliferative cells. Pulse-chase and double labeling by EdU/BrdU was used to track cells. Finally, they attempted to reveal the identity of cycling cells and their contribution to regeneration. Platynereis is a relatively new regeneration model. Understanding the cellular source of regeneration in this annelid would be of considerable interest.

The authors performed EdU labeling of intact worms to find a posterior-anterior decreasing gradient of S-phase cells. Histological sections showed that proliferative cells are located in all tissues in the posterior-most segment, but mostly restricted to the gut epithelium in more anterior segments. Using fluorescent beads that are taken up specifically by gut epithelial cells, they show that gut epithelial cells of the intact animal contribute only to gut regeneration, i.e., they are lineage restricted. The authors also performed immunostaining and mRNA in situ hybridization experiments to better understand the tissue identity of proliferative cells.

The following are my specific comments:

1. I am not sure I understand how the authors identify slow- or fast-cycling cells. EdU gets incorporated in S-phase; the longer the incubation time the more cells will be labeled until saturation is reached. The length of the cell cycle and the number of different populations cannot be directly derived from this experiment. I think it would be fair to conclude that there are more cycling cells in posterior segments and in the gut of anterior segments but the conclusion of two distinct populations is unsupported in my opinion.
2. The Results and Discussion sections will have to be revised to address the above issue. The two supported conclusions are (1) the gradient of proliferative cells (but w/o reference to the number of distinct populations); (2) the fate-restricted nature of gut epithelial cells. The plasticity gradient is unsupported because worms can regenerate if amputated at segment #5. This suggests the presence of either resident stem cells (with broad potential or lineage-specific), cells that can dedifferentiate, or a combination of both. The authors' experiments cannot discriminate between the alternatives.
3. The text would benefit from copy editing to improve the language and making it more accessible. In its current form, it is rather difficult to read, with descriptions of experiments that are not easy to follow.
4. The figures and their legends can be improved. Re the legends, one has to read the full text to understand what each panel shows. The figures are very complex. It would already be easier if usage of A', A', A' was avoided. The figures could also be improved by direct annotation. Finally, consider simplifying the main figures and moving some material to the supplement.
5. How do the authors know that proliferative cells in the gut are "gut progenitors"? They might simply be proliferative gut epithelial cells.
6. The conclusions drawn from the drug experiments are overstated.

Referees cross-commenting

The comments made by the two other reviewers are complementary to mine. Either the authors extensively revise the text to remove unsupported conclusions or they perform additional experiments.

Significance

Little is known about the cellular basis of Platynereis posterior regeneration.

D. Cardon et J. Levrel. «La vigilance participative. Une interprétation de la gouvernance de Wikipédia», Réseaux, n º 154, 2009,

1. J. Jouët et D. Pasquier, «Les jeunes et la culture de l'écran. Enquête nationale auprès des 6-17 ans», Réseaux, n º 92, 1999, p. 25-102.

1. O. Donnat, Les Pratiques culturelles des Français à l'ère numérique. Enquête 2008, Paris, La Découverte, 2009.

1. O. Bomsel, A. Geffroy et G. L. Blanc, Modem le Maudit. Économie de la distribution numérique des contenus, Paris, Presses de l'École des mines, 2006.

G. Poupeau, «L'édition électronique change tout et rien. Dépasser les promesses de l'édition électronique», Le Médiéviste et l'Ordinateur, n º 43, 2004.

H. Le Crosnier, «Désintermédiation et démocratie», Multitudes, vol. 19, n º 5, 2004, p. 143-160.

El pronóstico en el momento de la presentación es peor si hay >50% de semilunas en la biopsia renal con fibrosis avanzada, si la creatinina sérica es >5 a 6 mg/100 ml, si hay oliguria o si hay necesidad de diálisis aguda

as medias lunas , que en algunos casos pueden ser la extensión de las sinequias, se desarrollan cuando colecciones fibrocelulares/fibrina llenan todo o parte del espacio de Bowman

fibroblastos intersticiales y fibrosis en el sitio de la lesión; evidencia integral reciente sugiere que los fibroblastos renales aumentan a través de varios mecanismos: transiciones epiteliales o endotelio-mesenquimales (15%), fibrocitos derivados de la médula ósea (35%) y proliferación de fibroblastos residentes (50%). El factor de crecimiento transformante β (TGF-β), el factor de crecimiento de fibroblastos 2 (FGF-2), el factor inducible por hipoxemia 1α (HIF-1α) y el factor de crecimiento derivado de plaquetas (PDGF)

La inflamación de los capilares glomerulares se llama glomerulonefritis . Se desconocen la mayoría de los antígenos glomerulares o mesangiales implicados en la glomerulonefritis inmunomediada More

USENET: user network

Versión de lo que conocemos como foros o grupos de google.

aqui ele decalra oq estava la em cima como parametro faz outro for que recebe 10 e depois foi para o vetor [i] declarado la em cima que recebe 0, e ali todas as casinhas do vetores recebem 0

aqui ele decalrou um vetor de 10 casas uma variavel int quantidade, depoisfez um for e o vetor [i] = 0 em cada casinha

The author expresses distaste and sorrow over "coloured people" trying to win favor with their oppressors by spreading false info that helps to keep the system of slavery alive. He asks a rhetorical question: What if people of color had opted for unity instead of division?

interesting how cognitive processing can be affected by individualist cultures and collectivist cultures in such niche settings (as mentioned previously) but facial emotions are universally recognized. It would seem like something that is noticeable would have variation in interpretation.

to socrates, educational freedom is imperative

o individuo iria mudar a forma de como ele teve o sonho. Isso acontece por conta da relação óbvia que o medico teria com as características relatadas pelo paciente. Logo a relação que o sonho segue deve seguir sua própria linha para ser capaz de definir qualquer fato relatado.

Who is she referring to in this section? Even though, I believe she is referring to how peoples try to disguise the reality of New York telling people that is the greatest city in the world and that's easy to get money and all of that.

Nesses casos, em que o contrato dura menos de 30 meses, decorrido o prazo, o contrato não se extingue, mas sim se prorroga.

O direito de preferência deve ser exercido em até 30 dias, sob pena de caducidade, após a notificação do locador sobre a venda do imóvel.

Embora ocorra a prorrogação do contrato por prazo indeterminado, ou seja, por prazo superior a 30 meses, o locador somente poderá denunciar o contrato com motivação prevista em lei, não se aplicando a denúncia vazia, via de regra, para os contratos ajustados por prazo superior a trinta meses.

Nos contratos ajustados por prazo inferior a 30 meses, ocorrendo a prorrogação, somente o locador poderá retomar o imóvel e denunciar o contrato com as motivações prevista neste artigo.

Nos contratos ajustados por mais de 30 meses, ocorrendo a prorrogação, o locador poderá denunciar o contrato sem qualquer justificativa.

O locatário não precisará pagar a multa de rescisão se esta for motivada por transferência do locatário a localidade diversa a mando do empregador, devendo o locador ser notificado em 30 dias.

Author Response:

Thank you very much for selecting our paper for peer review and for the thorough evaluation of our manuscript. We appreciate your assessment and the reviewers’ comments that value our work and identify important points that will enable us to improve the paper. We are now working on key experiments to further test the hypothesis that ROCK is essential for the formation, growth, and morphology of the sea urchin larval skeleton. We will address the reviewers’ comments in detail in the revised version of the paper that we will submit after completing the experiments, but for now, there are two points we would like to clarify.

We thank the first reviewer for the appreciation of this paper and of our previous work where we studied calcium vesicle dynamics in whole embryos (Winter et al, Plos Com Biol 2021). In Winter et al 2021, we found that the skeleton (spicules) doesn’t grow when the embryos are immobilized in either control or treated embryos. As a consequence, we cannot determine the role of ROCK in vesicle trafficking and exocytosis based on experiments conducted in whole embryos. We are developing an alternative assay for vesicle tracking using cell cultures, but that is beyond the scope of this current work.

As for the second reviewer’s criticism of the usage of Y-27632 to block ROCK activity: The ROCK inhibitor concentrations we used (30-80µM) are similar the those commonly used in mammalian systems and in Drosophila to block ROCK activity, for example: (Becker et al., 2022; Canellas-Socias et al., 2022; Fischer et al., 2009; Kagawa et al., 2022; Segal et al., 2018; Su et al., 2022). The manufactory datasheet indicates that: “Y-27632 dihydrochloride is a selective ROCK inhibitor (Ki values are 0.14-0.22, 0.3, 25, 26 and > 250 μM for ROCK1 (p160 ROCK), ROCK2, PKA, PKC and MLCK respectively)”. That is, the affinities of Y-27632 for ROCK kinases are at least 100 times higher than those for PKC, PKA, and MLCK. Furthermore, these Ki values are based on biochemistry assays where the activity of the inhibitor is tested in-vitro with the purified protein. Therefore, these concentrations are not relevant to cell or embryo cultures where the inhibitor has to penetrate the cells and affect ROCK activity in-vivo. Y-27632 activity was studied both in-vitro and in-vivo in Narumiya, Ishizaki and Ufhata, Methods in Enzymology 2000 (Narumiya et al., 2000). This paper reports similar concentrations to the ones indicated in the manufactory data sheet for the in-vitro experiments, but shows that 10µM concentration or higher are effective in cell cultures. As stated above, we will add additional experimental verifications to the revised version, but even at this stage, the concentrations we used and the agreement between our pharmacological and genetic perturbations suggests that the affected protein is indeed ROCK.

We share the reviewers and editors wish to identify the molecular targets of ROCK and the specific cellular processes that ROCK is involved in, and we are actively working on achieving this goal. However, we believe that this paper is an important step towards illuminating the cellular components that participate in biomineral construction and the feedback between the cellular machinery and gene expression.

Best,

Smadar, in the name of all co-authors.

References:

• Becker, K.N., Pettee, K.M., Sugrue, A., Reinard, K.A., Schroeder, J.L., Eisenmann, K.M., 2022. The Cytoskeleton Effectors Rho-Kinase (ROCK) and Mammalian Diaphanous-Related (mDia) Formin Have Dynamic Roles in Tumor Microtube Formation in Invasive Glioblastoma Cells. Cells 11.
• Canellas-Socias, A., Cortina, C., Hernando-Momblona, X., Palomo-Ponce, S., Mulholland, E.J., Turon, G., Mateo, L., Conti, S., Roman, O., Sevillano, M., Slebe, F., Stork, D., Caballe-Mestres, A., Berenguer-Llergo, A., Alvarez-Varela, A., Fenderico, N., Novellasdemunt, L., Jimenez-Gracia, L., Sipka, T., Bardia, L., Lorden, P., Colombelli, J., Heyn, H., Trepat, X., Tejpar, S., Sancho, E., Tauriello, D.V.F., Leedham, S., Attolini, C.S., Batlle, E., 2022. Metastatic recurrence in colorectal cancer arises from residual EMP1(+) cells. Nature 611, 603-613.
• Fischer, R.S., Gardel, M., Ma, X., Adelstein, R.S., Waterman, C.M., 2009. Local cortical tension by myosin II guides 3D endothelial cell branching. Curr Biol 19, 260-265.
• Kagawa, H., Javali, A., Khoei, H.H., Sommer, T.M., Sestini, G., Novatchkova, M., Scholte Op Reimer, Y., Castel, G., Bruneau, A., Maenhoudt, N., Lammers, J., Loubersac, S., Freour, T., Vankelecom, H., David, L., Rivron, N., 2022. Human blastoids model blastocyst development and implantation. Nature 601, 600-605.
• Narumiya, S., Ishizaki, T., Uehata, M., 2000. Use and properties of ROCK-specific inhibitor Y-27632. Methods Enzymol 325, 273-284.
• Segal, D., Zaritsky, A., Schejter, E.D., Shilo, B.Z., 2018. Feedback inhibition of actin on Rho mediates content release from large secretory vesicles. J Cell Biol 217, 1815-1826.
• Su, Y., Huang, H., Luo, T., Zheng, Y., Fan, J., Ren, H., Tang, M., Niu, Z., Wang, C., Wang, Y., Zhang, Z., Liang, J., Ruan, B., Gao, L., Chen, Z., Melino, G., Wang, X., Sun, Q., 2022. Cell-in-cell structure mediates in-cell killing suppressed by CD44. Cell Discov 8, 35.

DOI: 10.1016/j.molcel.2023.08.005

Resource: (IMSR Cat# JAX_000664,RRID:IMSR_JAX:000664)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

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

Prazo decadencial para que o Fisco constitua o crédito tributário

Author Response

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

Reviewer #1 (Public Review):

Summary of the major findings -

1) The authors used saturation mutagenesis and directed evolution to mutate the highly conserved fusion loop (98 DRGWGNGCGLFGK 110) of the Envelope (E) glycoprotein of Dengue virus (DENV). They created 2 libraries with parallel mutations at amino acids 101, 103, 105-107, and 101-105 respectively. The in vitro transcribed RNA from the two plasmid libraries was electroporated separately into Vero and C6/36 cells and passaged thrice in each of these cells. They successfully recovered a variant N103S/G106L from Library 1 in C6/36 cells, which represented 95% of the sequence population and contained another mutation in E outside the fusion loop (T171A). Library 2 was unsuccessful in either cell type.

2) The fusion loop mutant virus called D2-FL (N103S/G106L) was created through reverse genetics. Another variant called D2-FLM was also created, which in addition to the fusion loop mutations, also contains a previously published, evolved, and optimized prM-furin cleavage sequence that results in a mature version of the virus (with lower prM content). Both D2-FL and D2-FLM viruses grew comparably to wild type virus in mosquito (C6/36) cells but their infectious titers were 2-2.5 log lower than wild type virus when grown in mammalian (Vero) cells. These viruses were not compromised in thermostability, and the mechanism for attenuation in Vero cells remains unknown.

4) Next, the authors probed the neutralization of these viruses using a panel of monoclonal antibodies (mAbs) against fusion loop and domain I, II and III of E protein, and against prM protein. As intended, neutralization by fusion loop mAbs was reduced or impaired for both D2-FL and D2-FLM, compared to wild type DENV2. D2-FLM virus was equivalent to wild type with respect to neutralization by domain I, II, and III antibodies tested (except domain II-C10 mAb) suggesting an intact global antigenic landscape of the mutant virion. As expected, D2-FLM was also resistant to neutralization by prM mAbs (D2-FL was not tested in this batch of experiments).

5) Finally, the authors evaluated neutralization in the context of polyclonal serum from convalescent humans (n=6) and experimentally infected non-human primates (n=9) at different time points (27 total samples). Homotypic sera (DENV2) neutralized D2-FL, D2-FLM, and wild type DENV similarly, suggesting that the contribution of fusion loop and prM epitopes is insignificant in a serotype-specific neutralization response. However, heterotypic sera (DENV4) neutralized D2-FL and D2-FLM less potently than wild type DENV2, especially at later time points, demonstrating the contribution of fusion loop- and prM-specific antibodies to heterotypic neutralization.

Impact of the study-

1) The engineered D2-FL and D2-FLM viruses are valuable reagents to probe antibodies targeting the fusion loop and prM in the overall polyclonal response to DENV.

2) Though more work is needed, these viruses can facilitate the design of a new generation of DENV vaccine that does not elicit fusion loop- and prM-specific antibodies, which are often poorly neutralizing and lead to antibody-dependent enhancement effect (ADE).

3) This work can be extended to other members of the flavivirus family.

4) A broader impact of their work is a reminder that conserved amino acids may not always be critical for function and therefore should not be immediately dismissed in substitution/mutagenesis/protein design efforts.

Evaluating this study in the context of prior literature -

The authors write "Although the extreme conservation and critical role in entry have led to it being traditionally considered impossible to change the fusion loop, we successfully tested the hypothesis that massively parallel directed evolution could produce viable DENV fusion-loop mutants that were still capable of fusion and entry, while altering the antigenic footprint."

".....Previously, a single study on WNV successfully generated a viable virus with a single mutation at the fusion loop, although it severely attenuated neurovirulence. Otherwise, it has not been generated in DENV or other mosquito-borne flaviviruses"

The above claims are a bit overstated. In the context of other flaviviruses:

• A previous study applied a similar saturation mutagenesis approach to the full length E protein of Zika virus and found that while the conserved fusion loop was mutationally constrained, some mutations, including at amino acid residue 106 were tolerated (PMID 31511387).

• The Japanese encephalitis virus (JEV) SA14-14-2 live vaccine strain contains a L107F mutation in the fusion loop (in addition to other changes elsewhere in the genome) relative to the parental JEV SA14 strain (PMID: 25855730).

• For tickborne encephalitis virus (TBEV-DENV4 chimera), H104G/L107F double mutant has been described (PMID: 8331735)

There have also been previous examples of functionally tolerated mutations within the DENV fusion loop:

• Goncalvez et al., isolated an escape variant of DENV 2 using chimpanzee Fab 1A5, with a mutation in the fusion loop G106V (PMID: 15542644). G106 is also mutated in D2-FL clone (N103S/G106L) described in the current study.

• In the context of single-round infectious DENV, mutation at site 102 within the fusion loop has been shown to retain infectivity (PMID 31820734).

We thank the reviewer for these comments. We have adjusted the text above to better reflect and credit the prior literature. Text is modified as follows in the discussion session.

“Previous reported mutations in the fusion loop are mainly derived from experimental evolution using FL-Ab to select for escape mutant or by deep mutational scanning (DMS) of the Env protein for Ab epitope mapping. Mutations in the FL epitope were observed in a DENV2-NGC-V2 (G106V)39, attenuated JEV vaccine strain SA14-14-2 (L107F)40, attenuated WNV-NY99 (L107F)41. While most of the mutations, including the double mutations reported here lead to attenuation of the virus. A recent DMS study showed that Zika-G106A has no observable impact on viral fitness42. Interestingly, we also recovered a mutation G106L, suggesting position 106 and 107 might be the most tolerable position for mutation in mosquito borne flavivirus FL. On the other hand, tick borne flavivirus as well as vector only flavivirus show a more diverse FL composition. The inflexibility of mosquito borne flavivirus might be due to the evolution constraint of the virus to switch between mosquito and vertebrate hosts.”

Appraisal of the results -

The data largely support the conclusions, but some improvements and extensions can benefit the work.

1) Line 92-93: "This major variant comprised ~95% of the population, while the next most populous variant comprised only 0.25% (Figure 1C)".

What is the sequence of the next most abundant variant?

The sequence of the next most abundant variant has been added to the text.

2) Lines 94-95: "Residues W101, C105, and L107 were preserved in our final sequence, supporting the structural importance of these residues." L107F is viable in other flaviviruses.

We acknowledge that the L107F mutation has been described in other flaviviruses, including the tick-borne flaviviruses DTV and POWV. This mutation in JEV is associated with viral attenuation. This sentence is referring to the fact that, in our libraries, we did not recover variants with mutations at these positions, in contrast to D2-FL with variants at N103 and G106, indicating less mutational tolerance. However, we want to re-direct the focus of this manuscript to engineer a viable DENV that is antigenically different in the FL epitope, but not which residue is more tolerance for mutation.

3) Figure 2c: The FLM sample in the western blot shows hardly any E protein, making E/prM quantitation unreliable.

The samples used in Figure 2C derive from the growth curve endpoint (Figure 2A), in which there is a 1-log difference in viral titer between D2 and D2-FLM. Equivalent volumes of viral supernatant were loaded in the gel, explaining the reduced intensity of the E band in D2-FLM. The higher exposure on the right shows the E band more clearly for D2-FLM. The Western blot assay comparing prM/E ratio as a measure of maturation state was described and validated in our previous study (Tse et al. 2022, mbio). The methods and figure legend have been updated to include greater detail. The polyclonal E antibody was specifically chosen for this study as our previously used monoclonal antibody targeted the fusion loop. The polyclonal antibody was raised against a fragment of E (AA 1-495) and should have minimal effect by the fusion loop mutations.

4) Lines 149 -151: "Importantly, D2-FL and D2-FLM were resistant to antibodies targeting the fusion loop. While neutralization by 1M7 is reduced by ~2-logs, no neutralization was observed for 1N5, 1L6, and 4G2 for either variant (Figure 3 A)".

a) Partial neutralization was observed for 1N5, for D2-FL.

The text has been updated to more accurately describe the 1N5 neutralization data.

b) Do these mAbs cover the full spectrum of fusion loop antibodies identified thus far in the field?

We did not test every known fusion loop antibody that has been described, instead focusing on 1M7, 1N5, 1L6, and 4G2, which were previously described by Smith et al and Crill et al. We also modified the text in discussion to reflect the possibility of other FL-Ab that are not affected by out mutations.

“We have tested a panel of FL-Ab; however, we cannot exclude the possibility that other FL-Abs may not be affected by N103S and G106L. However, we have shown that saturation mutagenesis could generate mutants with multiple amino acid changes, and we are currently using D2-FLM as backbone to iteratively evolve additional mutations in FL to further deviate the FL antigenic epitope.”

c) Are the epitopes known for these mAbs? It would be useful to discuss how the epitope of 1M7 differs from the other mAbs? What are the critical residues?

Critical residues for these antibodies have been described. They are as follows: 1M7: W101R, W101C, G111R; 1N5: W101R, L107P, L107R, G111R; 1L6: G100A, W101A, F108A; 4G2: G104H, G106Q, L107K. The critical residues for 1M7 are slightly different than the others, perhaps explaining the residual binding to D2-FL. Note that the critical residue identified previously for 1M7 and 1N5 do not overlap with D2-FLM mutations, suggesting the FL mutations has extending effect on the antigenic FL epitope.

d) Maybe the D2-FL mutant can be further evolved with selection pressure with fusion loop mAbs 1M7 +/-1N5 and/or other fusion loop mAbs.

We agree that it may be possible to further evolve D2-FL using antibody selection, although we have not yet performed these experiments, we are currently performing iterative saturation mutagenesis and directed evolution to further evolve away from the natural FL.

5) It would have been useful to include D2-M for comparison (with evolved furin cleavage sequence but no fusion loop mutations).

Neutralization data for some of the mAbs against D2-M can be found in our previous study (Tse et al. 2022 mBio), in which no difference in neutralization was observed compared to DV2 wildtype. Given the limited resources of the anti-DENV NHP and human serum, we did not add D2-M for comparison. Although some insight can be deduced from the D2-FL vs D2-FLM comparison, we agree future studies that are designed to delineate CR-Ab population between prM, FL and other CR-epitopes should include D2-M for comparison.

6) Data for polyclonal serum can be better discussed. Table 1 is not discussed much in the text. For the R1160-90dpi-DENV4 sample, D2-FL and D2-FLM are neutralized better than wild type DENV2? The authors' interpretation in lines 181-182 is inconsistent with the data presented in Figure 3C, which suggests that over time, there is INCREASED (not waning) dependence on FL- and prM-specific antibodies for heterotypic neutralization.

We remade Table 1 to show dilution factors instead of dilution factor-1 of FRNT50.

In general, our human convalescent sera from heterotypic infection (DENV1, 3 and 4) showed none to low neutralization against our DENV2. FRNT50s were between 1: 40 – 1:200. Given the weak potency of the antiserum, it is difficult to compare the FRNT50s between DV2-WT and D2-FLM.

Similarly, in a different NHP cohort (2nd NHP cohort shown in Table 1), only one DENV4 infected NHP (R1160) showed a low heterotypic titer against DENV2. The detectable FRNT50s were between 1: 50 – 1:90. The value was extrapolated based on a single data point (1:40) which has above 50% neutralization. Given the Hill slope of all the neutralization curves were below 0.5, the FRNT50 values is should not be

In conclusion, we do not think serum from Table 1 is potent enough to shows difference between the viruses. The intension to show the negative data in Table 1 is to highlight the difference in serum heterogeneity in DENV infected patients and experimental infected NHPs.

As the reviewer pointed out, the dependence of FL-Ab in later time points increased (the difference between DV2 and D2-FL at 20dpi vs 60dpi vs 90dpi), suggesting non-FL CR-Ab is waning but not prM- and FL-Abs. We rewrote the sentence as follow:

“These data suggest that after a single infection, many of the CR Ab responses target prM and the FL and the reliance on these Abs for heterotypic neutralization increase overtime (Figure 3C).”

Suggestions for further experiments-

1) It would be interesting to see the phenotype of single mutants N103S and G106L, relative to double mutant N103S/G106L (D2-FL).

2) The fusion capability of these viruses can be gauged using liposome fusion assay under different pH conditions and different lipids.

3) Correlative antibody binding vs neutralization data would be useful.

We thank the reviewer for the suggestions; we agree these would be of interest and, indeed, these studies are currently underway. In regard to single mutants, these were present in the initial plasmid library but did not enrich after viral production and passage. Two possible explanations can be drawn, 1) The stochastic of directed evolution prevents a single mutant with similar fitness to enriched. 2) The two mutations are compensatory to each other to make a functional mutant. The 2nd hypothesis highlights the difference between saturation mutagenesis (this study) and DMS (in previous studies).

Fusion capability is indeed very interesting, however, the mechanistic difference or not between wildtype FL and the mutated FL in supporting fusion is not the focus of this study. Instead, we are currently working on adapting the D2-FLM in mammalian cells. If successful, the difference in fusion mechanism between the Vero adapted and D2-FLM in different lipid, insect vs mammalian would be of interest.

We are currently developing whole virus ELISA; we avoid using rE monomer for the study as it might neglect the conformation Ab.

Reviewer #2 (Public Review):

Antibody-dependent enhancement (ADE) of Dengue is largely driven by cross-reactive antibodies that target the DENV fusion loop or pre-membrane protein. Screening polyclonal sera for antibodies that bind to these cross-reactive epitopes could increase the successful implementation of a safe DENV vaccine that does not lead to ADE. However, there are few reliable tools to rapidly assess the polyclonal sera for epitope targets and ADE potential. Here the authors develop a live viral tool to rapidly screen polyclonal sera for binding to fusion loop and pre-membrane epitopes. The authors performed a deep mutational scan for viable viruses with mutations in the fusion loop (FL). The authors identified two mutations functionally tolerable in insect C6/36 cells, but lead to defective replication in mammalian Vero cells. These mutant viruses, D2-FL and D2-FLM, were tested for epitope presentation with a panel of monoclonal antibodies and polyclonal sera. The D2-FL and D2-FLM viruses were not neutralized by FL-specific monoclonal antibodies demonstrating that the FL epitope has been ablated. However, neutralization data with polyclonal sera is contradictory to the claim that cross-reactive antibody responses targeting the pre-membrane and the FL epitopes wane over time.

Overall, the central conclusion that the engineered viruses can predict epitopes targeted by antibodies is supported by the data and the D2-FL and D2-FLM viruses represent a valuable tool to the DENV research community.

Reviewer #1 (Recommendations For The Authors):

1) Line 51-52: "Currently, there is a single approved DENV vaccine, Dengvaxia." Line 56-57: "Other DENV vaccines have been tested or are currently undergoing clinical trial, but thus far none have been approved for use."

It should be specified for the global audience that this applies to the United States. Takeda's DENV vaccine, QDENGA is approved in Indonesia, European Union, and Brazil.

The text has been modified to include this information.

2) Line 62-63: - "The core fusion loop-motif DRGWGNGCGLFGK is highly conserved..." Lines 78-80: - We generated two different saturation mutagenesis libraries, each with 5 randomized amino acids: DRGXGXGXXXFGK (Library 1) and 79 DRGXXXXXGLFGK (Library 2).

It may be useful for the readers if the amino acid numbers are stated. The core fusion loop motif DRGWGNGCGLFGK (Eaa98-110) is highly conserved. We generated two different saturation mutagenesis libraries, each with 5 randomized amino acids: DRGXGXGXXXFGK (Library 1; Xaa 101,103, 105-7) and DRGXXXXXGLFGK (Library 2; Xaa 101-105).

This information has been added to the text.

3) Line 91-92: "Bulk Sanger sequencing revealed an additional Env-91 T171A mutation outside of the fusion-loop region."

It looks like the mutation T171A is in domain I of the E protein and does not seem to interface with the fusion loop. Is that why it wasn't pursued further?

The E171A mutation was included in the infectious clone for D2-FL and D2-FLM. The text has been modified to clarify this inclusion.

4) Lines 82-85: "Saturation mutagenesis plasmid libraries were used to produce viral libraries in either C6/36 (Aedes albopictus mosquito) or Vero 81 (African green monkey) cells and passaged three times in their respective cell types."

a) What was the size of the libraries? How does one make sure that the experimental library actually has all the amino acid combinations that were intended?

Each library has 5 randomized amino acids, so there are 205 = 3.2 million combinations. In these experiments, sequencing of the plasmid libraries revealed about 2 million unique amino acid sequences, or approximately 62.5% library coverage. The actual plasmid diversity is expected to be higher than 2 million as our deep sequencing has limited coverage.

b) The wild type sequence was excluded from the libraries, correct?

The wild-type sequence was not specifically excluded from the libraries, as there is no easy method to do so. Wild-type sequence was detected in the plasmid libraries but was not selected in the C6/36 library. However, in the Vero library, we recovered WT virus.

5) Table 1: - Please include in the table description, what the colors indicate.

We remade Table 1 to show dilution factors instead of dilution factor-1 of FRNT50 and removed the unnecessary color code. We also added all relevant information in the table legend.

6) Lines 246-248: "Previously, a single study on WNV successfully generated a viable virus with a single mutation at the fusion loop, although it severely attenuated neurovirulence."

It may be worthwhile to mention the WNV mutation (L107F) as some readers may be curious about where this mutation is relative to the ones described in this study.

This information has been added to the text. We also included the previously described FL mutations in flaviviruses in the text.

Reviewer #2 (Recommendations For The Authors):

Major Critique:

• There is a disconnect between Fig 2A and 2C. FL and FLM viruses have much lower levels of prM-E expression in the viral supernatants based on the western blot in 2C. Why isn't E being detected in the Western? Is the particle-to-pfu ratio skewed in the mutant viruses? Is it possible that the polyclonal is targeting the cross-reactive prM and FL epitopes, and if so would using a monoclonal antibody targeting a known DIII-epitope (2D22) yield a different western result? Also, the legend and methods for Fig 2C are not clear. What is actually being tested in the Western blot? Were equivalent volumes of the different viral preps used?

The samples used in Figure 2C derive from the growth curve endpoint (Figure 2A), in which there is a 1-log difference in viral titer between D2 and D2-FLM. Equivalent volumes of viral supernatant were loaded in the gel, explaining the reduced intensity of the E band in D2-FLM. The higher exposure on the right shows the E band more clearly for D2-FLM. The Western blot assay comparing prM/E ratio as a measure of maturation state was described and validated in our previous study (Tse et al. 2022, mBio) and the methods have been updated to include greater detail. The polyclonal E antibody was specifically chosen for this study as our previously used monoclonal antibody targeted the fusion loop. The polyclonal antibody was raised against a fragment of E (AA 1-495) and should not be affected by the fusion loop mutations. 2D22 is a conformational antibody and does not work in western blot.

• Table 1: The data within Table 1 is ignored in the text, and some of this data contradicts the central conclusions of the manuscript.

o A.) Some of the convalescent data contradicts the hypothesis. DS0275 had an equivalent neut between DV2 and D2-FLM, DS1660, and R1160 (90) had better neut against the D2-FLM than DV2. Discussion of these samples is warranted.

o C.) The description in the legend does not adequately describe the table. What do the colors represent? What are the numerical values being displayed? What is in parentheses, (I assume the challenge strain)? The limit of detection is reported as 1:40; 0.25. 1:40 is 0.025 which matches most of the data? There is inadequate description of these experiments in the materials and methods.

We remade Table 1 to show dilution factors instead of dilution factor-1 of FRNT50 and removed the unnecessary color code. We also added discussion for Table 1 and clarify the difference between the three cohorts of serum in the text with the corresponding references.

In general, our human convalescent sera from heterotypic infection (DENV1, 3 and 4) showed none to low neutralization against our DENV2. FRNT50s were between 1: 40 – 1:200. Given the weak potency of the antiserum, it is difficult to compare the FRNT50s between DV2-WT and D2-FLM.

Similarly, in a different NHP cohort (2nd NHP cohort shown in Table 1), only one DENV4 infected NHP (R1160) showed a low heterotypic titer against DENV2. The detectable FRNT50s were between 1: 50 – 1:90. The value was extrapolated based on a single data point (1:40) which was above 50% neutralization. Given the Hill slope of all the neutralization curves were below 0.5, the FRNT50 values are not reliable.

In conclusion, we do not think sera from Table 1 is potent enough to show difference between the viruses. The intension to show the negative data in Table 1 is to highlight the difference in serum heterogeneity in DENV infected patients and experimental infected NHPs.

Minor critique:

Figure 1C: Legend is not clear for this panel. What is on the x-axis of the bubble plots? Are these mutations across the entire viral genome or is this just the prM-E sequence?

The X-axis is a scatter of all of the sequences contained in the library, similar to graphs used for plotting CRISPR screen results. These represent individual sequences from the saturation mutagenesis libraries in the fusion loop of E as described in Figure 1B.

The wording in Lines 92-94 is not clear. It looks like the T171A mutation was present in 95% of the sequences (Line 92). Yet this sequence was not incorporated into the variant virus. What is the rationale for omitting this mutation in downstream variant virus generation?

The 95% in Line 92 refers to the variant containing N103S/G106L mutations as seen in Figure 1C. The high-throughput sequencing approach did not include residue 171, so the presence of the T171A mutation in combination with fusion loop mutations cannot be determined. However, the E171A mutation was included in the infectious clone for D2-FL and D2-FLM. The text has been modified to clarify this inclusion.

The authors discuss the potential of the D2-FL or D2-FLM virus as a potential vaccine platform in the abstract, introduction, and conclusion. This is a good idea, but the authors provide no evidence of feasibility in this manuscript.

The ultimate goal to engineer a viable DENV with distinct FL antigenic epitope is for it use as live attenuated vaccine. As this is the rationale for the study, we introduce the concept throughout the manuscript. The current study demonstrated the possibility to mutate a novel fusion loop motif in DENV and provided evidence to show the favorable antigenic properties of D2-FLM. We agree with the reviewer that definitive work in animal to show vaccine efficacy need to be done and are currently undergoing. To avoid misleading our audience, we tone down the emphasis of vaccine use in the text.

Line 150-153: Figure 3A demonstrates that the FL-specific antibodies broadly do not neutralize the mutant viruses. However, the conclusions are overstated in the text. 1N5 neutralizes the D2-FL variant.

The text has been updated to more accurately describe the 1N5 neutralization data.

Lines 175-182: The authors make a lot of assumptions about the target of the polyclonal target without any evidence.

These lines reference studies that showed greater enhancement by antibodies targeting the fusion loop and prM as compared to other cross-reacting antibodies. The assumption that both our manuscript and others have drawn was that Abs that are cross-reactive and weakly neutralizing are more prone for ADE. As discussed, other groups have attempted to mutate the FL from recombinant E protein to achieve similar goal to remove the fusion loop epitope to reduce ADE. We have re-written the sentence in the followings:

“As FL and prM targeting Abs are the major species demonstrated to cause ADE in vitro, we and others hypothesized these Abs are responsible for ADE-driven negative outcomes after primary infection and vaccination,10–12,32 we propose that genetic ablation of the FL and prM epitopes in vaccine strains will minimize the production of these subclasses of Abs responsible for undesirable vaccine responses. Indeed, covalently locked E-dimers and E-dimers with FL mutations have been engineered as potential subunit vaccines that reduce the availability of the FL, thereby reducing the production of FL Abs.33–36”

This reminds me of the discussion over how much freedom should be traded for security, it's controversial especially in a post-9/11-PATRIOT act US

that is freedom

It's interesting to see how ideology is diffused through society. Is the role of congress and other officials overstated in this diffusion process if the average citizen isn't aware of current political thought?

Again, reminds me of Plato. Love the form, but not the thing itself.

south

black freedom religion

Poner un link a "Cómo detectar un bulto en tu seno"

Unless there is a need to save space, write full names and maybe inlcude hyperlinks.

https://focus.psychiatryonline.org/doi/10.1176/foc.6.1.foc3 Głównie chodzi o stres i pobudzenie układu współczulnego

para saber la probabilida de que mida mas que el valor dado, ya que siempre pnorm nos da el valor menor o igual

estás calculando la probabilidad de que una variable aleatoria normal estándar sea menor o igual a 1.96 desviaciones estándar por encima de la media.

es para probabilidad bajo la curva ¿cual es la prob de obtener un valor igual o menor a 0 en este caso?

Essa diferenciação: sujeito - objeto

Do we as the audience trust our narrator? She seems to view John with such high regard but also seems to be afraid of him

prueba 2

Fue la mayor transformación de la historia desde el neolítico.

De rural a urbano

Author Response

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

eLife assessment:

This study presents a useful inventory of the joint effects of genetic and environmental factors on psychotic-like experiences, and identifies cognitive ability as a potential underlying mediating pathway. The data were analyzed using solid and validated methodology based on a large, multi-center dataset. However, the claim that these findings are of relevance to psychosis risk and have implications for policy changes are only partially supported by the results.

We appreciate the feedback and insightful suggestions from the editor and reviewers, which aided us to improve the manuscript. We believe the concerns initially raised were mostly due to areas that needed further clarification, which we have now clarified in this revised version. Our primary contribution lies in our meticulous analytical approach aimed at minimizing confounding effects and providing more precise estimates of the genetic and environmental impact on children's cognition and psychology. This method differs from the widely used general linear modeling in the field, which, in our opinion, may not be the optimal strategy for large-scale data analysis. Our comprehensive, tutorial-style description of the methods might serve as a valuable resource for the community.

Regarding the critique that our findings 'partially support the relevance to psychosis risk,' we have updated our manuscript to more accurately reflect this feedback. We have altered the narrative to indicate that psychotic-like experiences (PLE) are associated with the risk for psychosis, a connection substantiated by prior studies cited in our manuscript.

Similarly, in response to the comment that our findings 'partially support implications for policy changes,' we have nuanced our conclusion. However, we would like to emphasize our discovery that a negative genetic predisposition impacting cognitive development (i.e., low polygenic scores for cognitive phenotypes) can be counteracted by a positive school and familial environment. We believe that this finding could have meaningful implication for policy making and is robustly supported by our analyses.

We hope this revised manuscript more accurately reflects our research findings and its significances. Lastly, we would like to express our gratitude for your fair and detailed review process. Our experience working with eLife has been incredibly rewarding, and we commend your dedication to an encouraging and progressive publishing culture.  

Public Reviews:

Reviewer #1

This study by Park et al. describes an interesting approach to disentangle gene-environment pathways to cognitive development and psychotic-like experiences in children. They have used data from the ABCD study and have included PGS of EA and cognition, environmental exposure data, cognitive performance data and self-reported PLEs. Although the study has several strengths, including its large sample size, interesting approach and comprehensive statistical model, I have several concerns:

• The authors have included follow-up data from the ABCD Study. However, it is not very clear from the beginning that longitudinal paths are being explored. It would be very helpful if the authors would make their (analysis) approach clearer from the introduction. Now, they describe many different things, which makes the paper more difficult to read. It would be of great help to see the proposed path model in a Figure and refer to that in the Method.

We clarified the longitudinal paths tested in this study in Intro [line 149~159]. We also added a figure of the proposed path model (Figure 1) [Methods: line 231~238].

• There is quite a lot of causal language in the paper, particularly in the Discussion. My advice would be to tone this down.

We adjusted and moderated the use of causal languages throughout the manuscript.

• I feel that the limitation section is a bit brief, and can be developed further.

We clearly specified the limitations of our study. These included concerns about the representativeness of the ABCD samples, of the limited scope of longitudinal data, and the use of non-randomized, observational data [line 524~544].

• I like that the assessment of CP and self-reports PEs is of good quality. However, I was wondering which 4 items from the parent-reported CBCL were used and how did they correlate with the child-reported PEs? And how was distress taken into account in the child self-reported PEs measurement? Which PEs measures were used?

Thanks for the clarification question. We report the Pearson’s correlation coefficients between the PLEs [line 198~200]. (The Reviewer #1 may have referred to the prior version of our manuscript submitted elsewhere, for this point has been already addressed in our initial submission to eLife).

• What was the correlation between CP and EA PGSs?

The Pearson’s correlation between CP and EA PGS was 0.4331 (p<0.0001). We added the statistics to the manuscript. [line 214]

• Regarding the PGS: why focus on cognitive performance and EA? It should be made clearer from the introduction that EA is not only measuring cognitive ability, but is also a (genetic) marker of social factors/inequalities. I'm guessing this is one of the reasons why the EA PGS was so much more strongly correlated with PEs than the CP PGS. See the work bij Abdellaoui and the work by Nivard.

We appreciate the reviewer’s insightful feedback. Acknowledging the role of both CP and EA PGSs in our study, we agree with the observation that EA PGS goes beyond gauging cognitive aptitude—it also serves as an indicator of societal influences and inequalities. The multifaceted nature of EA PGS could be the reason underlying the stronger correlation with PLEs compared to CP PGS. In response to this feedback, we revised our introduction to articulate the multifaceted role of EA PGS in more precise terms. For supporting our assertions, we have included references to prior studies (Abdellaoui et al., 2022) [line 131~142].

Abdellaoui, A., Dolan, C. V., Verweij, K. J. H., & Nivard, M. G. (2022). Gene–environment correlations across geographic regions affect genome-wide association studies. Nature Genetics. doi:10.1038/s41588-022-01158-0

• Considering previous work on this topic, including analyses in the ABCD Study, I'm not surprised that the correlation was not very high. Therefore, I don't think it makes a whole of sense to adjust for the schizophrenia PGS in the sensitivity analyses, in other words, it's not really 'a more direct genetic predictor of PLEs'.

We thank the reviewer for the thoughtful comments. We acknowledge that the correlation between schizophrenia PGS and PLE may not be exceedingly high, as evidenced by previous work, including analyses from the ABCD study. However, we would like to emphasize our rationale for adjusting schizophrenia PGS in the sensitivity analyses. Our study design stemmed from the established associations between PLEs and increased risk for schizophrenia. Existing studies have reported significant associations between schizophrenia PGS and cognitive deficits in both psychosis patients (Shafee et al., 2018) and people at risk for psychosis (He et al., 2021). Notable, the PGS for schizophrenia has shown significant associations with PLEs, arguably more so than PGS for PLEs itself (Karcher et al., 2018). Our updated manuscript has incorporated these references to improve clarity. [line 307~309]. By adding this layer of adjustment, we believe that our mixed linear model more precisely examines the relationship between the cognitive phenotype PGS and PLEs, in terms of both sensitivity and specificity.

He, Q., Jantac Mam-Lam-Fook, C., Chaignaud, J., Danset-Alexandre, C., Iftimovici, A., Gradels Hauguel, J., . . . Chaumette, B. (2021). Influence of polygenic risk scores for schizophrenia and resilience on the cognition of individuals at-risk for psychosis. Translational Psychiatry, 11(1). doi:10.1038/s41398-021-01624-z

Karcher, N. R., Paul, S. E., Johnson, E. C., Hatoum, A. S., Baranger, D. A. A., Agrawal, A., . . . Bogdan, R. (2021). Psychotic-like Experiences and Polygenic Liability in the Adolescent Brain Cognitive Development Study. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging. doi:https://doi.org/10.1016/j.bpsc.2021.06.012

Shafee, R., Nanda, P., Padmanabhan, J. L., Tandon, N., Alliey-Rodriguez, N., Kalapurakkel, S., . . . Robinson, E. B. (2018). Polygenic risk for schizophrenia and measured domains of cognition in individuals with psychosis and controls. Translational Psychiatry, 8(1). doi:10.1038/s41398-018-0124-8

• How did the FDR correction for multiple testing affect the results?

Please note that we have clarified our FDR correction in the methods

As detailed in the method section [line 254~255], we applied False Discovery Rate (FDR) correction for multiple testing across nine key variables in the study: PGS (CP or EA), family income, parental education, family’s financial adversity, Area Deprivation Index, years of residence, proportion of population below -125% of the poverty line, positive parenting behavior, and positive school environment. An exception was made in our additional sensitivity analysis, where we included schizophrenia PGS in the linear mixed model for adjustment, thus the FDR correction was applied across ten key variables instead. Overall, the application of FDR correction had minimal impact on our findings. Most associations between the key variables and the outcomes that were originally marked as highly significant sustained their significance after the FDR correction.

Overall, I feel that this paper has the potential to present some very interesting findings. However, at the moment the paper misses direction and a clear focus. It would be a great improvement if the readers would be guided through the steps and approach, as I think the authors have undertaken important work and conducted relevant analyses.

We express our appreciation to the reviewer for the positive feedback and constructive suggestions, which only serve to improve and strengthen our manuscript. We have incorporated the suggested corrections and clarifications in response to the reviewer's suggestions. We believe that these changes will not only enhance the overall readability but also more effectively emphasize the significance and implication of our work.

Reviewer #2 (Public Review):

This paper tried to assess the link between genetic and environmental factors on psychotic-like experiences, and the potential mediation through cognitive ability. This study was based on data from the ABCD cohort, including 6,602 children aged 9-10y. The authors report a mediating effect, suggesting that cognitive ability is a key mediating pathway in the link between several genetic and environmental (risk and protective) factors on psychotic-like experiences.

While these findings could be potentially significant, a range of methodological unclarities and ambiguities make it difficult to assess the strength of evidence provided.

Strengths of the methods:

The authors use a wide range of validated (genetic, self- and parent-reported, as well as cognitive) measures in a large dataset with a 2-year follow-up period. The statistical methods have the potential to address key limitations of previous research.

Weaknesses of the methods:

The rationale for the study is not completely clear. Cognitive ability is probably a more likely mediator of traits related to negative symptoms in schizophrenia, rather than positive symptoms (e.g., psychosis, psychotic-like symptom). The suggestion that cognitive ability might lead to psychotic-like symptoms in the general population needs further justification.

We appreciate the reviewer’s concern regarding the role of cognitive ability in relation to schizophrenia symptoms. We are aware that cognitive ability often serves as a mediator of psychotic-like experiences. However, to our best knowledge, a growing body of research has proposed that cognitive ability can mediate positive symptoms in schizophrenia including psychotic-like experiences. The studies by Howes & Murray (2014) and Garety et al. (2001) suggested that deficits in cognitive ability can potentially contribute to the manifestation of positive symptoms such as psychotic-like experiences. We have elaborated on this aspect in the Introduction section [line 104-115].

Howes, O. D., & Murray, R. M. (2014). Schizophrenia: an integrated sociodevelopmental-cognitive model. The Lancet, 383(9929), 1677-1687. doi:https://doi.org/10.1016/S0140-6736(13)62036-X

Garety, P. A., Kuipers, E., Fowler, D., Freeman, D., & Bebbington, P. E. (2001). A cognitive model of the positive symptoms of psychosis. Psychological Medicine, 31(2), 189-195. doi:10.1017/S0033291701003312

Terms are used inconsistently throughout (e.g., cognitive development, cognitive capacity, cognitive intelligence, intelligence, educational attainment...). It is overall not clear what construct exactly the authors investigated.

We thank the reviewer’s feedback regarding the consistency of terminology in our manuscript. Per the suggestion, we standardized the use of ‘cognitive capacity’ and now consistently refer to it as ‘cognitive phenotypes’ throughout our manuscript. Furthermore, we explicitly stated in the Introduction section that our two PGSs of focus will be termed ‘cognitive phenotypes PGSs’, aligning with terminology used in prior studies (Joo et al., 2022; Okbay et al., 2022; Selzam et al., 2019) [line 140~142].

Joo, Y. Y., Cha, J., Freese, J., & Hayes, M. G. (2022). Cognitive Capacity Genome-Wide Polygenic Scores Identify Individuals with Slower Cognitive Decline in Aging. Genes, 13(8), 1320. doi:10.3390/genes13081320

Okbay, A., Wu, Y., Wang, N., Jayashankar, H., Bennett, M., Nehzati, S. M., . . . Young, A. I. (2022). Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individuals. Nature Genetics, 54(4), 437-449. doi:10.1038/s41588-022-01016-z

Selzam, S., Ritchie, S. J., Pingault, J.-B., Reynolds, C. A., O’Reilly, P. F., & Plomin, R. (2019). Comparing Within- and Between-Family Polygenic Score Prediction. The American Journal of Human Genetics, 105(2), 351-363. doi:https://doi.org/10.1016/j.ajhg.2019.06.006

Not the largest or most recent GWASes were used to generate PGSes.

We appreciate the reviewer’s observation. Indeed, we were unable to utilize the most recent or the largest GWAS for cognitive performance, educational attainment, and schizophrenia due to the timeline of our study. Regrettably, the commencement of our study preceded the publication of the ‘currently’ the largest or most recent GWAS studies by Okbay et al. (2022) and Trubetskoy et al. (2022). Our research was conducted with the best available data at that time, which was the GWAS of European-descent individuals for educational attainment and cognitive performance (Lee et al, 2018). To eliminate any potential confusion, we adjusted the text to specify that our study used 'a GWAS of European-descent individuals for educational attainment and cognitive performance' rather than the largest GWAS [line 206~208].

It is not fully clear how neighbourhood SES was coded (higher or lower values = risk?). The rationale, strengths, and assumptions of the applied methods are not fully clear. It is also not clear how/if variables were combined into latent factors or summed (weighted by what). It is not always clear when genetic and when self-reported ethnicity was used. Some statements might be overly optimistic (e.g., providing unbiased estimates, free even of unmeasured confounding; use of representative data).

Thank you for pointing this out. Consistent with the illustration of neighborhood SES in the Methods, higher values of neighborhood SES indicate risk [line 217~228]. In the original Figure 2, higher value of neighborhood SES links to lower intelligence (direct effects: β=-0.1121) and higher PLEs (indirect effects: β=-0.0126~ -0.0162). We think such confusion might have been caused by the difference between family SES (higher values = lower risk) neighborhood SES (higher values = higher risk). Thus, we changed the terms to ‘High Family SES’ and ‘Low Neighborhood SES’ in the corrected figure (Figure 3) for clarification.

Considering that shorter duration of residence may be associated with instability of residency, it may indicate neighborhood adversity (i.e., higher risk). This definition of the ‘years of residence’ variable is in line with the previous study by Karcher et al. (2021).

During estimation, the IGSCA determines weights of each observed variable in such a way as to maximize the variances of all endogenous indicators and components. We added this explanation in the description about the IGSCA method [line 266~268].

We deleted overly optimistic statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead, throughout our manuscript.

Karcher, N. R., Schiffman, J., & Barch, D. M. (2021). Environmental Risk Factors and Psychotic-like Experiences in Children Aged 9–10. Journal of the American Academy of Child & Adolescent Psychiatry, 60(4), 490-500. doi:10.1016/j.jaac.2020.07.003

It appears that citations and references are not always used correctly.

We thoroughly checked all citations and specified the references for each statement: We deleted Plomin & von Stumm (2018) and Harden & Koellinger (2020) and cited relevant primary studies (e.g., Lee et al., 2018; Okbay et al., 2022; Abdellaoui et al., 2022) instead. We also specified the references supporting the statement that educational attainment PGS links to brain morphometry (Judd et al., 2020; Karcher et al., 2021). As Okbay et al. (2022) use PGS of cognitive intelligence (which mentions the analyses results in their supplementary materials) as well as educational attainment, we decided to continue citing this reference [line 131~141].

Strengths of the results:

The authors included a comprehensive array of analyses.

We thank the reviewer for the positive comment.

Weaknesses of the results:

Many results, which are presented in the supplemental materials, are not referenced in the main text and are so comprehensive that it can be difficult to match tables to results. Some of the methodological questions make it challenging to assess the strength of the evidence provided in the results.

As you rightly identified, we inadvertently failed to reference Table S2 in the main text. We have since corrected this omission in the Results section for the IGSCA (SEM) analysis [line 376]. The remainder of the supplementary tables (Table S1, S3~S7) have been appropriately cited in the main manuscript. We recognize that the quantity of tables provided in the supplementary materials is substantial. However, given the comprehensiveness and complexity of our analyses, which encompass a wide array of study variables, these tables offer intricate results from each analysis. We deem these results, which include valuable findings from sensitivity analyses and confound testing, too significant to exclude from the supplementary materials. That said, we are open to, and would greatly welcome, any further suggestions on how to present our supplementary results in a more clear and digestible format. Your guidance in this matter is highly valued.

Appraisal:

The authors suggest that their findings provide evidence for policy reforms (e.g., targeting residential environment, family SES, parenting, and schooling). While this is probably correct, a range of methodological unclarities and ambiguities make it difficult to assess whether the current study provides evidence for that claim.

We believe that with the improvement we made in this revised manuscript, this concern may have been successfully mitigated.

Impact:

The immediate impact is limited given the short follow-up period (2y), possibly concerns for selection bias and attrition in the data, and some methodological concerns.

We appreciate the feedback provided in the reviewer's impact statement. We added as study limitations [line 524~544] that the impact of our findings may be limited due to the relatively short follow-up period, the possibility of sample selection bias, and the problems of interpreting results from an observational study as causality (despite the novel causal inference methods, designed for non-randomized, observational data, that we used).

As responded above (and also in more detail in the Reviewer #2’s Recommendations For The Authors section below), we made necessary corrections and clarifications for the points suggested by the reviewer. As we are willing to make additional revisions, please feel free to give comments if you feel that our corrections are insufficient or inappropriate.

Nevertheless, we would like to discuss some points. We sincerely hope this following response does not come across as argumentative to the reviewer and the editor. We fully understand the reviewer's perspective on this matter, and we agree that the issues raised about the ABCD study are absolutely valid. However, when evaluating the overall impact of a study, other factors, such as how the field has been assessing the impact of similar studies, should also be considered.

Firstly, the potential selection bias and attrition in the ABCD data may not necessarily limit the conclusions of this study. While recognizing the potential issues with the ABCD data is important, we feel that judging the impact of our findings as "limited" based on these issues may not be entirely fair. This is because no study, particularly those of a nationwide scale such as the UK Biobank, IMAGEN, HEAL, HBCD, etc., is completely free of limitations. Typically, the potential limitations of the data don't undermine the impact of individual studies' findings. Numerous studies using ABCD data have been published in top-tier journals—despite the limitations of the ABCD study—underscoring the scientific merit of the findings. For example, the study by Tomasi, D., & Volkow, N. D. (2021), entitled "Associations of family income with cognition and brain structure in USA children: prevention implications," published in Molecular Psychiatry, might be highly relevant to the limitations of the ABCD study raised by the reviewer. The scientific community, including editors, reviewers, and readers, may have appreciated the impact of this study despite the acknowledged limitations of the ABCD data.

Secondly, the two-year time window of our longitudinal analysis might not impact the aim of this study—an iterative assessment of the associations between genetic and environmental variables with cognitive intelligence and mental health, with a focus on PLE, in preadolescents. Had we aimed to test the developmental trajectory from childhood to adolescence, perhaps a longer timeframe would have made more sense. So, we do not agree with the reviewer’s assessment that the short time window limits the impact of our study.

Suggested revisions based on the combined reviewer feedback:

1) The terminology used should be carefully reviewed and revised

• Please use the correct terminology for the key concepts assessed in this study. For example, authors sometimes conflate PLEs and psychosis, two related but separate constructs. Furthermore, the terms 'good parenting' and 'good schooling' are vague and subjective.

• The authors use multiple terms to refer to cognitive ability (cognitive capacity, intelligence, cognitive intelligence, etc). The term 'cognitive development' in the title and manuscript does not seem to be justified given the focus on different measures of cognitive ability at a single time point (i.e. baseline).

• Please avoid causal language and using statements that cannot be entirely substantiated (e.g. unbiased estimates, free from unmeasured confounding)

Thank you for suggesting this point. We revised all key terminologies used throughout our manuscript.

Per your suggestion, we specified that PLEs indicate the risk of psychosis and often precede schizophrenia. We checked all misused cases of the term ‘psychosis’ and corrected them as ‘PLEs’. We also changed the terms 'good parenting' and 'good schooling' to ‘positive parenting behavior’ and ‘positive school environment’.

We changed the term ‘cognitive development’ to ‘cognitive ability’ throughout our manuscript. We also changed the title to ‘Gene-Environment Pathways to Cognitive Intelligence and Psychotic-Like Experiences in Children’ because we used ‘cognitive intelligence’ for NIH toolbox variable in the text.

We corrected and tone-downed all causal languages used in our manuscript. As mentioned by the reviewers, we deleted statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead.

2) A stronger rationale for the focus on PLEs, and the potential mediating role of cognitive ability in genetic and environmental effects on PLES, should be provided

We appreciate the raised concerns that cognitive ability may serve as a mediator of psychotic-like experiences. To our best knowledge, it has been proposed that cognitive ability can be a mediator of positive symptoms in schizophrenia (including psychotic-like experiences), as well as negative symptoms. This mediating role of cognitive ability was proposed in several prior studies on cognitive model of schizophrenia/psychosis. Per your suggestion, we included an additional justification in Intro [line 104~115] where we highlighted that cognitive ability has been proposed as a potential mediator of genetic and environmental influence on positive symptoms of schizophrenia such as psychotic-like experiences. We refer to studies conducted by Howes & Murray (2014) and Garety et al. (2001).

Howes, O. D., & Murray, R. M. (2014). Schizophrenia: an integrated sociodevelopmental-cognitive model. The Lancet, 383(9929), 1677-1687. doi:https://doi.org/10.1016/S0140-6736(13)62036-X

Garety, P. A., Kuipers, E., Fowler, D., Freeman, D., & Bebbington, P. E. (2001). A cognitive model of the positive symptoms of psychosis. Psychological Medicine, 31(2), 189-195. doi:10.1017/S0033291701003312

3) As described in more detail by the reviewers, more information should be provided about the measures used in the study and how they relate to one another (e.g. correlations between PQ-BC and CBCL; PGS-CA and PGS-EA).

Thank you for your suggestion. Although this information was already provided in our initial submission, it appears that the Reviewer #1’s might have referred to the prior version of our manuscript submitted elsewhere before eLife.

To clarify, our findings reveal significant Pearson’s correlation coefficients between PLEs across all time-points (baseline year: r=0.095~0.0989, p<0.0001; 1-year follow-up: r=0.1322~0.1327, p<0.0001; 2-year follow-up: r= 0.1569~0.1632, p<0.0001) and we added this information in the Method section [line 198~200]. We also added the Pearson’s correlation between the two PGSs (r=0.4331, p<0.0001) in the Methods for PGS [line 214].

4) More details are needed regarding the analytical strategies used (e.g. how imputation was performed, why PGS were not based on the largest and most recent GWASes, whether latent or observed variables were examined, what exactly the supplementary materials show and how they relate to information provided in the main text).

We appreciate your feedback. We acknowledge the concerns about the GWAS sources utilized for the study. Unfortunately, our study commenced prior to the publication of the ‘currently’ most recent or largest GWAS by Okbay et al. (2022) and Trubetskoy et al. (2022). Our research was conducted with the best available data at that time, which was the largest GWAS of European-descent individuals for educational attainment and cognitive performance (Lee et al, 2018). We have now clarified this point in the manuscript. [line 206~208]

Also, we specified the use of composite indicators for the PGS, family SES, neighborhood SES, positive family and school environment, and PLEs, while latent factors were used for cognitive intelligence [line 269~285].

We highly appreciate the reviewer’s comments regarding the supplementary materials. We regret overlooking the citation of Table S2 in the main manuscript, and this has now been rectified in the Results section for the IGSCA (SEM) analysis [line 376]. The remaining supplementary tables (Table S1, S3~S7) have been correctly referenced within the manuscript. We acknowledge that the supplementary materials are extensive due to the comprehensive array of study variables and intricate results from each analysis. However, given that our analyses encompass a wide array of study variables, these tables offer intricate results from each analysis. We deem these results, which include valuable findings from sensitivity analyses and confound testing, too crucial to exclude from the supplementary materials. That said, we are open to any further suggestions to make our supplementary results more accessible and digestible. In order to improve the accessibility and clarity of our presentation, we are fully committed to making any necessary changes and look forward to any further recommendations.

5) The limitation section should be expanded and statements regarding the implications of the study findings should be qualified accordingly (e.g. short follow-up period, potential for attrition and selection bias, reverse causation, etc)

We specified additional potential constraints of our study, including limited representativeness, limited periods of follow-up data (baseline year, 1-year, and 2-year follow-up), possible sample selection bias, and the use of non-randomized, observational data [line 524~544].

6) Please ensure that the references provided support the statements in the text to which they are linked to.

Thank you for pointing this out. We thoroughly went over all citations and corrected the inaccurately or vaguely cited references for each statement.

Reviewer #2 (Recommendations For The Authors):

1) Please use terms consistently and correctly. E.g., 'cognitive capacity' is not the same as 'educational attainment'.

We thank the reviewer’s feedback regarding the consistency of terminology in our manuscript. Per the suggestion, we standardized the use of ‘cognitive capacity’ and now consistently refer to it as ‘cognitive phenotypes’ throughout our manuscript. Furthermore, we explicitly stated in the Introduction section that our two PGSs of focus will be termed ‘cognitive phenotypes PGSs’, aligning with terminology used in prior studies (Joo et al., 2022; Okbay et al., 2022; Selzam et al., 2019) [line 140~142].

Joo, Y. Y., Cha, J., Freese, J., & Hayes, M. G. (2022). Cognitive Capacity Genome-Wide Polygenic Scores Identify Individuals with Slower Cognitive Decline in Aging. Genes, 13(8), 1320. doi:10.3390/genes13081320

Okbay, A., Wu, Y., Wang, N., Jayashankar, H., Bennett, M., Nehzati, S. M., . . . Young, A. I. (2022). Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individuals. Nature Genetics, 54(4), 437-449. doi:10.1038/s41588-022-01016-z

Selzam, S., Ritchie, S. J., Pingault, J.-B., Reynolds, C. A., O’Reilly, P. F., & Plomin, R. (2019). Comparing Within- and Between-Family Polygenic Score Prediction. The American Journal of Human Genetics, 105(2), 351-363. doi:https://doi.org/10.1016/j.ajhg.2019.06.006

2) The authors study 'cognitive performance using seven instruments', but it is not clear how fluid and crystalline intelligence was defined/operationalized.

Thank you for pointing this out. We specified the NIH Toolbox tests used for composite scores of fluid and crystallized intelligence, respectively. “We utilized baseline observations of uncorrected composite scores of fluid intelligence (Dimensional Change Card Sort Task, Flanker Test, Picture Sequence Memory Test, List Sorting Working Memory Test), crystallized intelligence (Picture Vocabulary Task and Oral Reading Recognition Test), and total intelligence (all seven instruments) provided in the ABCD Study dataset” [line 180~187].

3) I don't think Lee 2018 is the largest GWAS for educational attainment. That would be Okbay 2022. It needs to be described how cognitive performance was defined in Lee 2018. Why did the authors not use the Trubetskoy 2022 schizophrenia GWAS?

Thank you for mentioning this point. The reason why we were not able to use the largest GWAS for CP, EA and schizophrenia is because (unfortunately) our study started earlier than the point when the GWAS studies by Okbay et al. (2022) and Trubetskoy et al. (2022) were published. We corrected that our study used ‘a GWAS of European-descent individuals for educational attainment and cognitive performance’ instead of the largest GWAS [line 206~208].

4) It is unclear how neighbourhood SES was coded. The authors seem to suggest that higher values indicate risk, but Figure 2 suggests that higher values links to higher intelligence and lower PLE.

Thank you very much for pointing this out. Consistent with the illustration of neighborhood SES in the Methods section, higher values of neighborhood SES indicate risk. In the original Figure 2, higher values of neighborhood SES links to lower intelligence (direct effects: β=-0.1121) and higher PLEs (indirect effects: β=-0.0126~-0.0162). We think such confusion might have been caused by the difference between family SES (higher values = lower risk) neighborhood SES (higher values = higher risk). Thus, we changed the terms to ‘High Family SES’ and ‘Low Neighborhood SES’ in the corrected figure (Figure 3) for clarification.

5) Also, the 'year of residence' variable is unclearly defined. Does this mean that a shorter duration of residency (even in a good neighbourhood) indicate risk?

Thank you for mentioning this point. Considering that shorter duration of residence may be associated with instability of residency, it may indicate neighborhood adversity (i.e., higher risk). This definition of the ‘years of residence’ variable is in line with the previous study by Karcher et al. (2021).

Karcher, N. R., Schiffman, J., & Barch, D. M. (2021). Environmental Risk Factors and Psychotic-like Experiences in Children Aged 9–10. Journal of the American Academy of Child & Adolescent Psychiatry, 60(4), 490-500. doi:10.1016/j.jaac.2020.07.003

6) Please provide information on how correlated the two PGSes were.

Thank you for your suggestion. We added the Pearson’s correlation between the two PGSs (r=0.4331, p<0.0001) in the Methods section for PGS [line 214].

7) Information on the outcome variable in the 'linear mixed models' section is missing. I assumed it was PLE.

Thank you for notifying us of this point. We added the information on the outcome variables in the section for linear mixed models [line 242~244].

8) In the 'Path Modeling' section, please explain what 'factors and components' concretely refer to. How is this different from a standard SEM with latent factors?

Thank you for your comment on the need to elaborate the IGSCA method. We added that different from standard SEM methods which only uses latent factors, the IGSCA method can use components as well as latent factors as constructs in model estimation. This allows the IGSCA method to control bias more effectively in estimation compared to the standard SEM [line 261~268].

9) The sentence starting line 229 is unclear. Does this mean variables were not used to generate latent factors. And if not, what weights were used to create a 'weighted sum'?

Thank you for mentioning this point. The sentence means that we treated PGSs, family SES, neighborhood SES, positive family and school environment, and PLEs as composite indicators (derived from a weighted sum of relevant observed variables), while general intelligence was represented as a latent factor.

It has been suggested from prior studies that these variables (PGSs, family SES, neighborhood SES, positive family and school environment, and PLEs) are less likely to share a common factor and were assessed as a composite index during analyses. For instance, Judd et al. (2020) and Martin et al. (2015) analyze genetic influence of educational attainment and ADHD as composite indicators. Also, as mentioned in Judd et al. (2020), socioenvironmental influences are often analyzed as composite indicators. Studies on psychosis continuum (e.g., van Os et al., 2009) suggest that psychotic disorders are likely to have multiple background factors instead of having a common factor, and notes that numerous prior research uses composite indices to measure psychotic symptoms. Based on this literature, we used components for these variables.

The IGSCA determines weights of each observed variable to maximize the variances of the endogenous indicators and components [added in line 265~268].

On the other hand, we treated general intelligence as a latent factor/variable underlying fluid and crystallized intelligence. This is based on the extensive literature of classical g theory of intelligence [added in line 269~284].

Judd, N., Sauce, B., Wiedenhoeft, J., Tromp, J., Chaarani, B., Schliep, A., ... & Klingberg, T. (2020). Cognitive and brain development is independently influenced by socioeconomic status and polygenic scores for educational attainment. Proceedings of the National Academy of Sciences, 117(22), 12411-12418.

Martin, J., Hamshere, M. L., Stergiakouli, E., O'Donovan, M. C., & Thapar, A. (2015). Neurocognitive abilities in the general population and composite genetic risk scores for attention‐deficit hyperactivity disorder. Journal of Child Psychology and Psychiatry, 56(6), 648-656.

van Os, J., Linscott, R., Myin-Germeys, I., Delespaul, P., & Krabbendam, L. (2009). A systematic review and meta-analysis of the psychosis continuum: Evidence for a psychosis proneness–persistence–impairment model of psychotic disorder. Psychological Medicine, 39(2), 179-195. doi:10.1017/S0033291708003814

10) It is overall not clear when genetically and when self-reported information of ethnicity was used. This needs to be clearer throughout.

Thank you for mentioning this point. We only used genetically defined ethnicity, and we have not mentioned that we used self-reported ethnicity. Per your suggestion, we clarified that we used ‘genetic ethnicity’ throughout the paper.

11) The sentence starting line 253 is also unclear. How is schizophrenia PGS a 'more direct genetic predictor of PLE' and compared to what other measure?

Thank you for pointing this out. Please note that our adjustment (or sensitivity analyses) was based on the reported associations between PLEs and the risk for schizophrenia: schizophrenia PGS is associated with a cognitive deficit in psychosis patients (Shafee et al., 2018) and individuals at-risk of psychosis (He et al., 2021), and psychotic-like experiences (more so than PGS for psychotic-like experiences) (Karcher et al., 2018). We added these references for clarification [line 307~309]. We believe that because of the adjustment our results from the mixed linear model show the sensitivity and specificity of the association between cognitive phenotype PGS and PLEs.

He, Q., Jantac Mam-Lam-Fook, C., Chaignaud, J., Danset-Alexandre, C., Iftimovici, A., Gradels Hauguel, J., . . . Chaumette, B. (2021). Influence of polygenic risk scores for schizophrenia and resilience on the cognition of individuals at-risk for psychosis. Translational Psychiatry, 11(1). doi:10.1038/s41398-021-01624-z

Karcher, N. R., Paul, S. E., Johnson, E. C., Hatoum, A. S., Baranger, D. A. A., Agrawal, A., . . . Bogdan, R. (2021). Psychotic-like Experiences and Polygenic Liability in the Adolescent Brain Cognitive Development Study. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging. doi:https://doi.org/10.1016/j.bpsc.2021.06.012

Shafee, R., Nanda, P., Padmanabhan, J. L., Tandon, N., Alliey-Rodriguez, N., Kalapurakkel, S., . . . Robinson, E. B. (2018). Polygenic risk for schizophrenia and measured domains of cognition in individuals with psychosis and controls. Translational Psychiatry, 8(1). doi:10.1038/s41398-018-0124-8

12) Please include a statement on the assumptions made when using the method used in this study and developed by Miao 2022, explain what evidence you have to support these assumptions and how this method, which I believe was developed for RCTs, can be applied to observational data.

We specified the assumptions for the causal inference method proposed by Miao et al. (2022) and why it is applicable to our study. Also, we noted that this novel method was developed to identify the causal effects of multiple treatment variables within non-randomized, observational data [line 309~319].

13) Some of the statements are potentially misleading. E.g., I would be very cautious to claim that the methods applied allowed the authors to estimate 'unbiased associations again potential (even unobserved) confounding variables'. There are many concerns such as selection bias, attrition, reverse causation, genetic confounding, etc that cannot be addressed satisfactorily using these data and methods.

Thank you for pointing this out. We deleted statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead.

Nevertheless, please note that due to some limitations in the data (e.g., confounders), an analytic approach should be robust enough to handle potential violations of assumptions. This was the point we wanted to emphasize--In contrast to the majority of studies using the ABCD study, which employ simplistic GLM or conventional SEM with only latent variable modeling, our study provides less biased, thus more accurate, estimates through the use of sophisticated modeling for confounding effects (instead of simplistic GLM) and IGSCA (instead of conventional simplistic SEM). We hope our study may help improve our analytical approach in this field.

14) I would be equally cautious to claim that the ABCD study is representative. Please add information on the whole ABCD cohort to Table 1 and describe any relevance with respect to attrition effects or representativeness.

Thank you for highlighting this issue. We previously characterized the ABCD Study as representative of the US population, given its aim to ensure representativeness by recruiting from a broad range of school systems located near each of its 21 research sites, chosen for their geographic, demographic, and socioeconomic diversity. Using epidemiological strategies, a stratified probability sample of schools was selected for each site. This procedure took into account sex, race/ethnicity, socioeconomic status, and urbanicity to reduce potential sampling biases at the school level. Based on these strategies, previous research (e.g., Thompson et al., 2019; Zucker et al., 2018) has referred to the ABCD Study as ‘representative.’ However, we overlooked the fact that “not all 9-year-old and 10-year-old children in the United States had an equal chance of being invited to participate in the study,” and therefore, it should not be deemed fully representative of the US population (Compton et al., 2019). Heeding your suggestion, we have removed all descriptions of the ABCD Study being representative.

Compton, W. M., Dowling, G. J., & Garavan, H. (2019). Ensuring the Best Use of Data: The Adolescent Brain Cognitive Development Study. JAMA Pediatrics, 173(9), 809-810. doi:10.1001/jamapediatrics.2019.2081

Thompson, W. K., Barch, D. M., Bjork, J. M., Gonzalez, R., Nagel, B. J., Nixon, S. J., & Luciana, M. (2019). The structure of cognition in 9 and 10 year-old children and associations with problem behaviors: Findings from the ABCD study’s baseline neurocognitive battery. Developmental Cognitive Neuroscience, 36, 100606. doi:10.1016/j.dcn.2018.12.004

Zucker, R. A., Gonzalez, R., Feldstein Ewing, S. W., Paulus, M. P., Arroyo, J., Fuligni, A., . . . Wills, T. (2018). Assessment of culture and environment in the Adolescent Brain and Cognitive Development Study: Rationale, description of measures, and early data. Developmental Cognitive Neuroscience, 32, 107-120. doi:https://doi.org/10.1016/j.dcn.2018.03.004

15) The imputation methods need to be explained in more detail / more clearly. What concrete variables were included? Why was 50% of the sample excluded despite imputation? How similar is the study sample to the overall ABCD cohort - and to the US population in general (i.e., is this a representative dataset)?

Thank you for mentioning this point. We clarified the method and detailed processes of the imputation (e.g., R package VIM, number of missing observations for each study variables such as genotypes, follow-up observations, and positive environment) [Methods; line 167~176].

The final samples had significantly higher cognitive intelligence, parental education, family income, and family history of psychiatric disorders, lower Area Deprivation Index, percentage of individuals below -125% of the poverty level, and family’s financial adversity (p<0.05). As you have noted above, these results also show the limited representativeness of the data used in our study. We fully acknowledge that our study sample, as well as the overall ABCD cohort, is not representative of the US population in general.

16) There are a range of unclear statements (e.g., 'Supportive parenting and a positive school environment had the largest total impact on PLEs than genetic or environmental factors' - isn't parenting an environmental factor?).

Thank you for mentioning this point. We clarified seemingly vague expressions and unclear statements. We corrected the sentence you noted as ‘Supportive parenting and a positive school environment had the largest total impact on PLEs than any other genetic or environmental factors’ [line 57~58].

17) The authors' conclusion (that these findings have policy implications for improving school and family environmental) are not fully supported by the evidence. E.g., genetic effects were equally large.

Thank you for pointing this out. Our description should be clearer. Our models consistently show that the combined environmental effects of positive family/school environment, and family/neighborhood SES exceeds the genetic effects. We suggest that these findings may have policy implications for “improving the school and family environment and promoting local economic development” [line 62~64].

To clarify, we newly added “Despite the undeniable genetic influence on PLEs, when we combine the total effect sizes of neighborhood and family SES, as well as positive school environment and parenting behavior (∑▒〖|β|〗=0.2718~0.3242), they considerably surpass the total effect sizes of cognitive phenotypes PGSs (|β|=0.0359~0.0502)” [line 510~513]. Based on these results, we suggest that our findings hold potential policy implications for “preventative strategies that target residential environment, family SES, parenting, and schooling—a comprehensive approach that considers the entire ecosystem of children's lives—to enhance children's cognitive ability and mental health” in the Discussion [line 507~510].

Admittedly, our results do not directly demonstrate a causal effect wherein an intervention in the school or family environmental variables would necessarily lead to a significantly meaningful positive impact on a child's cognitive intelligence and mental health. We do not make such a claim in this paper. However, we anticipate that further integrative analyses akin to ours might help identify potential causal or prescriptive effects. We hope this perspective will be recognized as one of the contributions of our study. We leave the final decision to the discerning judgment of the editors and reviewers.

18) Many citations do not support the statements made and are sometimes used rather vaguely. For example, I believe Judd 2020 and Okbay 2022 did not use a PGS of cognitive capacity, but of educational attainment. Plomin 2018 and Harden 2020 are reviews, but the primary studies should be cited instead. Which reference exactly is supporting the statement that cognitive capacity PGS links to brain morphometry?

Thank you very much for your precise observations. We thoroughly checked all citations and updated the references for each statement.

We deleted Plomin & von Stumm (2018) and Harden & Koellinger (2020) and cited relevant original research articles (e.g., Lee et al., 2018; Okbay et al., 2022; Abdellaoui et al., 2022) instead. We also specified the references supporting the statement that educational attainment PGS links to brain morphometry (Judd et al., 2020; Karcher et al., 2021). As Okbay et al. (2022) used the PGS of cognitive intelligence (which presented the analyses results in their supplementary materials) as well as educational attainment, we decided to continue citing this reference [line 131~141].

19) Citations are formatted inconsistently.

We apologize for the inconsistency of the citation formatting. We formatted all citations in APA 7th style, using EndNote v20. We checked that all citations maintain consistency according to the reference style.

20) Re line 281, I believe effect sizes are 'up to twice as large', but not consistently twice as large as suggested in the text.

Thank you for mentioning this point. We corrected the sentence as ‘The effect sizes of EA PGS on children's PLEs were larger than those of CP PGS’ [line 342~343].

21) Please add to the results a short statement on what covariates these analyses were controlled for.

Thank you for giving us this comment. We added that we used sex, age, marital status, BMI, family history of psychiatric disorders, and ABCD research sites as covariates in the Results section [line 329~331].

22) Cho 2020 does not provide recommendations on FIT values (line 315). Please provide another reference and explain how these FIT values should be interpreted.

Thank you for mentioning this point. We added the correct reference for FIT values (Hwang, Cho, & Choo, 2021). We also added that the FIT values range from 0 to 1, and a larger FIT value indicates more variance of all variables is explained by the specified model (e.g., FIT=0.50 denotes that the model explains 50% of the total variance of all variables) [line 291~293].

23) Regarding Figure 2, please add factor loadings to this figure and explain what the difference between the hexagon and circular shapes are. Please also add the autocorrelations between the 3 PLE measures. I assume these were also modelled statistically, given the strong correlations between time points?

Figure 2B needs reworking.

It is unclear what the x-axis of Figure 2C represents. Proportion of R2 or effect size? SM table 2 provides key information, which should be added to Figure 2.

Thank you for pointing this out. We added factor loadings to the corrected figure (Figure 3A and 3B). We also added that the X-axis of Figure 3C represents standardized effect sizes.

24) I suggest adding units directly to Table 1, not in the legend. Was genetic or self-reported ethnicity used in this table? List age in years, not months?

Thank you for your suggestion. We added the units of age and family history of psychiatric disorders directly inside Table 1. We used genetic ethnicity in Table 1, as we only used genetic ethnicity (but not self-reported ethnicity) throughout our study. This is noted on the last row of Table 1. We listed age in chronological months, which is how each child’s age at each point of data collection is coded in the ABCD Study.

25) Please include exact p-values in Table 2.

Thank you for your suggestion. We highly appreciate the reviewer’s comment on the importance of showing exact p-values in the analysis results. Unfortunately, we cannot estimate the standard errors based on normal-theory approximations to obtain the exact p-values of our IGSCA model results. This is described in detail in the original paper of the IGSCA method (Hwang et al., 2021): “Like GSCA and GSCAM, IGSCA is also a nonparametric or distribution-free approach in the sense that it estimates parameters without recourse to distributional assumptions such as multivariate normality of indicators. As a trade-off of no reliance on distributional assumptions, it cannot estimate the standard errors of parameter estimates based on asymptotic (normal-theory) approximations. Instead, it utilizes the bootstrap method (Efron, 1979, 1982) to obtain the standard errors or confidence intervals of parameter estimates nonparametrically.”

Efron, B. (1979). Bootstrap methods: Another look at the jackknife. Annals of Statistics, 7, 1–26. http://dx.doi.org/10.1214/aos/1176344552

Efron, B. (1982). The jackknife, the bootstrap and other resampling plans. Philadelphia, PA: SIAM. http://dx.doi.org/10.1137/1.9781611970319

Hwang, H., Cho, G., Jung, K., Falk, C. F., Flake, J. K., Jin, M. J., & Lee, S. H. (2021). An approach to structural equation modeling with both factors and components: Integrated generalized structured component analysis. Psychological Methods, 26(3), 273-294. doi:10.1037/met0000336

26) There are way too many indigestible tables presented in the supplementary materials, which are also not referenced in the main manuscript.

We appreciate your insightful observation. As you rightly identified, we inadvertently failed to reference Table S2 in the main text. We have since corrected this omission in the Results section for the IGSCA (SEM) analysis [line 376]. The remainder of the supplementary tables (Table S1, S3~S7) have been appropriately cited in the main manuscript. We recognize that the quantity of tables provided in the supplementary materials is substantial. However, given the comprehensiveness and complexity of our analyses, these tables offer intricate results from each analysis. We deem these results, which include valuable findings from sensitivity analyses and confound testing, too significant to exclude from the supplementary materials. That said, we are open to, and would greatly welcome, any further suggestions to ensure clarity and ease of comprehension. Your guidance in this matter is highly valued.

27) Figure S1 is unclear, possibly due to the journal formatting. Is this one figure presented on two pages? Clarify which PGS is listed in Figure S1 and in any case, please add both PGSs.

Thank you for mentioning this point. Figure S1 presents two correlation matrices: the first one is the correlation matrix of component / factor variables in the IGSCA model and the second one is the that of observed variables used to construct the relevant component / factor variables in the IGSCA model. We noted each matrix as Figure S1-A and Figure S1-B. We also corrected the figure legend as “A. Correlation between all component / factor variables of the IGSCA model. B. Correlation between all observed variables used to construct the relevant component / factor variables in the IGSCA model.” Since Figure S1-A presents correlations between the components and latent factors, it lists a single PGS variable constructed from the CP PGS and EA PGS. On the other hand, Figure S1-B presents correlations between the observed variables. Thus, both CP PGS and EA PGS are listed in this correlation matrix.

I like that they gave the definition of each of these terms because I had never heard of them before. This helped to further my understanding of the text drastically.

It is interesting to know how humidity can have an effect on marathon performance. After doing some research, I found out that when we run, our body produces sweat to reduce the core temperature of our body. Through evaporation, our body cools off by releasing heat energy. But when the humidity is high, evaporation gets affected (it lessens), resulting in the heat energy not being released from our body. This leads to dehydration, fatigue, and more. Thus affecting the marathon performance.

Another fact toward the idea that freedom is used, in America, only in regard to the "majority" Anything that is not the "majority" is to be changed or gotten rid of, regardless of method. This extends to many societies, in my opinion, not just America.

Water is often associated with images of cleansing. In this case, the soldiers are reinforcing that image and projecting it as the water being able to cleanse the Filipinos for their non-America views. So, it is less about liberty and more the enforcement of what Americans believe people should believe and do. All the American ideologies are encompassed as liberty. The soldiers view anything that is not apart of the American ideology as something that must be cleansed.

In this instance, I believe that the concept of freedom is being used as a scapegoat to justify immoral actions. Even in today's world, I think the ideology of freedom is too often misconstrued in order to fit the mold created by those of power (on any level). Often people believe they can do anything as long as they bury their real goal in figurative good intent. In short, here, freedom is being used to justify action regardless of rationality.

This sentence reminds me of the ideologies expressed in Langston Hughes Freedom. In the poem, Langston expresses his belief that freedom cannot be founded on the premise of force, the method in which the soldiers and US are employing. The word "compel" insinuates acceptance under threat, which has historically led to rebellion.

Why? Freedom, at least in my view, must be taken willingly, for anything that is forced is inherently the opposite of free.

Em um sentido figurado, o diapasão é considerado um modelo de condição utilizado para comparar algo.

Em seu uso popular, a palavra diapasão também pode significar o "ritmo acelerado da vida", a agitação da rotina pessoal ou profissional.

O termo "diapasão" ainda é bastante utilizado na literatura jurídica, nas expressões "nesse diapasão" ou "nesse mesmo diapasão", significando "nessa mesma linha de pensamento". Representa uma "afinação" dos argumentos apresentados pelo autor do texto.

O Estado se alimenta do território e por isso, tal expansão é uma necessidade e não um desejo específico.

The global landscape is witnessing significant macro-level changes as numerous social movements mobilize extensive segments of society. These movements are reshaping the language of protest and advocating for more inclusive politics. While traditional political power structures remain important, social movements are increasingly expanding their focus beyond the state. Nonetheless, there's growing recognition that social movements must assert their presence within the political sphere, collaborating with states and governments.

Pablo Solon emphasizes the importance of social forces seizing and transforming state power for deglobalization to succeed. This entails democratizing the management of state-owned public enterprises, strengthening existing communes, and developing new ones to shift consumers into producers, promoting self-organization and self-management in society, and combating corruption and nepotism to prevent regression in the transition process.

The alter-globalist movement exhibits remarkable strengths, including its ability to forge immediate coalitions to resist policies and propose alternatives, sometimes even leading to changes in political leadership. However, it also exhibits weaknesses, such as dispersion, fragmentation, and difficulty in developing coherent and long-term programs due to the constraints of the alliances formed.

While the old adage "think globally, act locally" may no longer fully apply, alter-globalists are actively engaging on a global scale. An illustrative example is their response to the HIV/AIDS epidemic, initially addressed by gay communities in California, then affecting impoverished communities, predominantly women, in sub-Saharan Africa. This led to the formation of extensive coalitions in places like South Africa and intervention in international processes, including the United Nations and influential agencies like the World Bank, along with direct engagement with major pharmaceutical corporations. This collective pressure compelled affluent nations to grant affected countries access to generic medication, bypassing the conventional patent and protection systems for pharmaceutical companies. While not a complete victory, it provided some access to treatment for disadvantaged populations. These grassroots efforts to combat AIDS are emblematic of various global movements arising from the bottom up, challenging governments and agencies with well-organized, transnational structures capable of sharing information and developing strategies across the globe.

For over seven decades, the development discourse has revolved around the notion of economic growth. However, critics of the current model of globalization argue that it is unsustainable. They view "hyper-growth" and the unbridled exploitation of the Earth's resources as pressing challenges that must be confronted and overcome. Organizations like the International Forum on Globalization stress that the economy should be oriented towards satisfying genuine human needs in the present without compromising the ability of future generations to meet their own needs, all while preserving the planet's natural diversity.

Since the Earth Summit in Rio de Janeiro in 1992, warnings have been issued regarding the excessive exploitation of resources, which poses an unprecedented threat to humanity. Despite more than two decades of talks and negotiations, this problem persists. The world is facing multiple threats, including the substantial increase in emissions from fossil fuel combustion. Based on scientific evidence, there is a real possibility that the Earth's climate could undergo significant changes in the next few decades, jeopardizing many life forms and human settlements, particularly in the Global South. Despite efforts to reach a consensus, some governments, including the United States, struggle to implement affirmative policies to address the climate challenge, such as setting firm emissions reduction targets and establishing legal enforcement mechanisms. Consequently, various social movements and NGOs are taking on the responsibility of addressing this critical issue.

We are currently at a crucial turning point where both neoliberalism and globalization have lost credibility, facing opposition from social movements as well as conservatives. Their return in their traditional forms seems unlikely. Some argue that persisting with neoliberal policies in the face of social protests can lead to "de-democratization" as governments resort to coercive measures to quell dissent. However, there is a growing consensus among ordinary people, social movements, and scholars that the existing system needs a comprehensive overhaul, a concept provocatively termed "de-globalization" by Walden Bello.

De-globalization does not advocate isolation or autarchy but envisions an alternative form of global integration that is not dominated by capital. It rejects the idea that interests, competition, and efficiency are inherently problematic but calls for their subordination to values such as equity, justice, and community. The goal of de-globalization, according to Bello, is to promote "effective economics" that prioritize social solidarity and broaden democratic decision-making within the economic sphere. This concept has emerged from the realization, shared by various social movements and intellectuals, that systemic alternatives are needed to address complex and interconnected challenges like inequality, widespread poverty, economic devastation, and authoritarian regimes. To work on these systemic alternatives, it is essential to move beyond mere opposition and embrace transformative practices.

Part of the discontent surrounding globalization found resonance with right-wing reactionary forces who attributed blame to progressive policies for being too accommodating towards migrants and refugees. In the United States, the Tea Party movement emerged as the voice of a substantial segment of the underprivileged population that had suffered job losses and social status declines due to neoliberal austerity measures. Similar sentiments were echoed by populist movements and political entities in Europe, India, the Philippines, Brazil, and other regions.

The election of Donald Trump as President of the United States was a pivotal moment in this context. Trump positioned himself as a "disruptor" of the established way of conducting politics, which included the United States' support for the international order that had been constructed with its allies since the end of World War II. He challenged policies that had dominated during the "golden era" of globalization, such as free trade agreements, the reduction of trade barriers, and multilateral accords on various issues. The United States experienced an increase in economic and commercial conflicts with most nations, including longstanding allies like Canada, Mexico, and the European Union. Concurrently, Trump questioned or rejected military alliances like NATO and multinational cooperation efforts aimed at countering groups like ISIS in the Middle East and the Taliban in Afghanistan. The most significant trade conflict arose with China, the world's second-largest economy. Trump's foreign policy shifted away from conventional American concerns like promoting democracy and human rights abroad, favoring personal relationships and negotiations with autocratic leaders and dictators, such as Russia's Vladimir Putin, Brazil's Jair Bolsonaro, and North Korea's Kim Jong-un.

Trump's "America first" approach, which emphasized bilateral negotiations to secure more favorable deals for the United States, ultimately eroded the international order it had helped establish. Many saw this as symptomatic of the United States' economic and military decline, especially in the face of rising powers like China, which, despite its authoritarian governance, remained committed to advancing global free trade and investment liberalization under President Xi Jinping's leadership.

However, it is crucial to acknowledge that Donald Trump's election also reflected the electoral voice of those who felt neglected by globalization in the United States. People who considered themselves victims of free trade and globalization and held deep attachments to traditional interpretations of American identity and exceptionalism were drawn to Trump's promise to disrupt the existing order.

In October 2011, a predominantly young group of individuals initiated an occupation of Zuccoti Park, located in the heart of New York City's famous financial district, Wall Street. Over the course of several weeks, Zuccoti Park became a focal point for demonstrations, cultural events, and workshops, drawing hundreds of participants. The protesters aimed to voice their grievances against corporate influence on the political system, political institutions, and the privileges enjoyed by the wealthiest 1% of Americans.

The Occupy movement gained global attention due to its location in New York City and continued until May 2012 when a massive demonstration of 50,000 people took to the streets. One of the key issues that fueled the protest was the increasing income inequality in the United States, where those with higher incomes had claimed a disproportionately larger share of the national income over the preceding decades.

Notable characteristics of the Occupy movement included its decentralized nature, apparent lack of hierarchy, and the absence of a single leader or governing body. Decision-making occurred through daily general assemblies in which the participants collectively played a pivotal role. The Occupy movement was widely seen as a spontaneous and determined citizen initiative, capturing the attention of the public, political figures, including then-US President Barack Obama.

Similar protests sprang up in various American cities such as Toronto, Los Angeles, Chicago, Seattle, San Francisco, Boston, and Houston, among others. The Occupy movement also extended to several Canadian cities, as well as countries in Europe, Asia, and South America. However, with time, many of the encampments were dismantled, and the Occupy movement gradually faded from the public spotlight.

Occupy demonstrated that the concept of citizen mobilization was gaining credibility worldwide. As economic hardships and political gridlocks persisted, many individuals felt that these issues could not be effectively addressed within existing state and international institutions with their current structures.

The demand for an alternative form of globalization has given rise to various national and international movements and social forces. Since 2001, many of these movements and initiatives have engaged in a global dialogue to create what they refer to as a "counter-hegemonic project." One significant manifestation of this collective effort is the World Social Forum (WSF), which originated from Latin American activism and held its first annual conference in Brazil in 2001 as a counterpoint to the World Economic Forum in Davos, Switzerland.

The WSF has evolved into a worldwide process led by civil society groups. Its purpose extends beyond protesting against the neoliberal aspects of globalization; it aims to define alternatives to the current global system. Presently, more than 500,000 social movements, both large and small, participate in the WSF process, which is decentralized into numerous local, national, and thematic forums. These forums utilize advanced information technologies to facilitate ongoing and complex discussions.

In 2013 and 2015, after years in South America, the WSF moved to Tunis, Tunisia, the epicenter of the Arab Spring. During these events, attended by 65,000 delegates, social movements agreed on a program of social action. They emphasized the need to challenge the current capitalist system, which they viewed as causing global suffering. They criticized transnational corporations, banks, media conglomerates, and international financial institutions for pursuing profit through interventionist and neocolonial policies, often with the cooperation of neoliberal governments. The participants in these forums also asserted their commitment to people's sovereignty, self-determination, and the rejection of false discourse around human rights that sometimes justified military occupations. Instead, they advocated for a socially just development that respects nature.

In North America, the "Occupy Wall Street" movement spread to more than 70 cities in the United States and Canada. It was primarily led by a new generation of educated youth who were disillusioned by the lack of suitable job opportunities. Additionally, Mexico experienced its share of protests and mobilizations against state violence, gang violence, and corruption during this period.

Many anti-globalization or alter-globalization movements aim not to replace one system with another but to articulate a new perspective that seeks to dismantle structures of domination and exclusion that marginalize the poor. This approach was particularly evident in South America, where progressive governments came to power following the initial wave of anti-globalization protests. Countries like Venezuela, Brazil, Argentina, Ecuador, Bolivia, and Uruguay saw the rise of these governments. However, after more than a decade in power, these people's movements continue to face challenges, including unfulfilled promises and manipulative rules that hinder their effective participation in the political process.

In 2008, the world entered a new phase marked by the global financial crisis. The collapse of major financial institutions on Wall Street and worldwide resulted in widespread social and economic upheaval, reminiscent of the Great Depression of 1929. Millions of people lost their jobs and homes, leading to a growing sense that the existing status quo was unsustainable.

A few years later, a wave of popular uprisings erupted globally. The "Arab Spring" in 2011 saw the overthrow of long-standing dictatorships in Tunisia and Egypt, sparked by the self-immolation of a young unemployed street vendor, Mohamed Bouazizi. Tunisians took to the streets, peacefully confronting security forces and ultimately leading to the regime's collapse. Following the Arab Spring, protests against economic austerity swept through southern Europe, particularly in Spain and Greece.

These movements were characterized by the occupation of public spaces, the takeover of closed factories, and the extensive use of social media to mobilize and expose the negative consequences of neoliberal policies, widespread corruption, and elite complicity, transcending national borders. These protests and movements persisted for several years, reflecting a global discontent with the existing socioeconomic order.

In 1994, Indigenous communities represented by the Zapatista Army of National Liberation, known as the Zapatistas, emerged in southern Mexico to voice their opposition to NAFTA (North American Free Trade Agreement) and the neoliberal globalization policies associated with it. They believed that these policies threatened the livelihoods of the local peasant and Indigenous populations. The Zapatistas gained global attention due to their symbolic association with Emiliano Zapata, a leader of the Mexican revolution, and their unique approach to activism.

The Zapatistas utilized modern communications, including the Internet, in innovative ways, setting a new standard for social movements. They emphasized that their rebellion aimed to "change" power rather than "take" it, reshaping the paradigm for many social movements.

In 1999, the spirit of the Zapatistas resonated with a diverse coalition of US and international NGOs and social movements in Seattle during the ministerial meeting of the World Trade Organization. This marked the beginning of a broader "movement of movements," which brought together various groups, including trade unions ("teamsters") and environmentalists ("turtles"). This movement gained momentum, with demonstrations occurring in cities worldwide, including Europe, Asia, and Africa, as it opposed and critiqued globalization and its effects.

In addition to the recent trend of states challenging neoliberal globalization, there is the emergence of a global civil society that voices its concerns through various demands, protests, movements, and networks (as discussed in Chapter 13). Beyond the imagery of anti-globalization protests and riots, there is a growing "movement of movements" that is becoming a significant player in global politics. Radical thinkers like Michael Hardt and Antonio Negri (2000) and Naomi Klein (2015) believe that new alternative movements and demands from the "alter-globalist" perspective have the potential to fundamentally transform globalization. While the empire of globalization continues to expand, an alternative political organization for global flows and exchanges is developing alongside it, suggesting the potential for significant changes in the future.

The Global South is undergoing a complex set of processes characterized by both reconfiguration and disintegration. Amidst this chaos, various proposals have emerged over the years. In the early 2000s, influenced by a new generation of center-left South American governments and China, there were initiatives to restructure the global economy away from the traditional dominance of the United States and its G7 allies. These proposals included reforms for international institutions like the United Nations, the World Bank, the IMF, and even the World Trade Organization. There was also a focus on strengthening regional organizations like Mercosur in South America and the Association of Southeast Asian Nations in Asia. Additionally, some "emerging" countries with robust economic growth formed the informal BRICS alliance (Brazil, Russia, India, China, South Africa) with the goal of accessing the global market on equal terms, promoting a more stable and equitable form of globalization.

However, by the end of the first decade of the millennium, these reform efforts had stalled. China's economy began to experience a period of slower growth, and countries like Brazil and Argentina, heavily reliant on commodity exports, were hit hard by declining commodity prices. The 2020 pandemic further complicated the situation, and China faced challenges as the "workshop of the world" in a constrained international market. In many of these countries, slowing economic growth led to significant political and social conflicts, with right-wing elements often playing a prominent role in toppling reformist governments, as seen in the case of Brazil.

The 2007-2008 financial crisis had its roots in the growing dominance of the finance sector, a phenomenon known as "financialization." This shift towards financialization was particularly successful in the wake of the neoliberal policies of the 1980s. Financialization involved the massive creation of indebtedness to compensate for stagnant incomes, thereby stimulating demand for goods and services. Large financial institutions, such as JP Morgan Chase, Citigroup, Wells Fargo, Bank of America, Goldman Sachs, and Morgan Stanley, amassed an estimated $100 trillion from wealthy individuals seeking tax shelters.

By 2010, the global recession began affecting the Global South, including rapidly growing economies in Asia and Latin America. Other regions like North Africa and the Middle East also experienced rising unemployment and reductions in essential services, leading to political and social upheavals. The economic conditions of the middle class became increasingly precarious in many countries. Even nations previously considered part of the "emerging economies" group, like South Africa and Brazil, faced challenges such as high unemployment, declining wages, increased indebtedness, reduced social safety nets, and cuts in public services. Similar issues were observed in the economies of the Global North.

The COVID-19 pandemic's economic impact has the potential to be even more severe for developing countries, although the full extent of the recession is uncertain at the time of writing. In the short term, estimated losses for 2020 exceeded 10 percent of total global GDP. Many African countries, which heavily rely on income generated by resource exports, could lose tens of billions of dollars. Some experts believe a complete recovery may not occur before 2023. The International Monetary Fund (IMF) characterized the consequences of the pandemic as a "crisis like no other," involving multiple crises - a health crisis, a financial crisis, and a collapse in commodity prices, all interacting in complex ways. Global growth in 2020 was projected to fall to -3 percent, making it the worst recession since the Great Depression and more severe than the Global Financial Crisis of 2008.

While it's unlikely that the world economy will undergo "deglobalization" due to the pandemic crisis, there may be increased constraints on the global flow of goods imposed by major economic powers like China, the United States, and the European Union. Conflicts related to border controls, tariffs, and trade measures are expected to rise in the context of heightened competition. The poorest countries will be more vulnerable to these changes, and international institutions like the UN and the World Bank may face limitations in their actions due to budget cuts and political disagreements among their members.

Before the global financial crisis of 2007-2008, there was a prevailing belief that globalization was an unstoppable and inevitable force. However, a series of crises since then has eroded the global consensus on the benefits of globalization.

The 2007-2008 financial crisis was a pivotal moment when significant financial institutions in the United States suffered substantial losses. Due to the interconnectedness of global financial systems, this crisis quickly spread to affect many other countries.

Globalization has a profound impact on politics, particularly the sovereignty of nation-states. Large multinational corporations (MNCs) and financial institutions operate freely across borders, leading to a gradual erosion of the nation-state's authority. Kenichi Ohmae, in his book "The End of the Nation State," argues that global capital markets have become so dominant that nation-states struggle to control exchange rates or protect their currencies. As a result, they become vulnerable to economic decisions made elsewhere by entities they cannot practically control.

Many experts believe that political structures inherited from the nation-state are becoming outdated. Critical macroeconomic policies are formulated and decided by entities removed from the public sphere. Developing countries often find themselves excluded from these processes, with powerful states and institutions like the IMF and World Bank, governed by a small group of influential countries due to their greater financial contributions, having a significant say.

This loss of influence affects not only smaller or weaker states on the international stage but also their domestic political landscapes. In some cases, it can lead to political breakdowns, as witnessed in several sub-Saharan countries and other regions. The disconnect between the economic/private and political/public realms creates a void, raising questions about the role and future of institutions like the United Nations, which may struggle to regain prominence given the reluctance of rich and powerful nations to support its resurgence.

The bleak picture of Africa's development needs some nuance. Over the past decade, African social indicators have shown improvement, largely due to the strong economic performance of select countries. Oil-rich nations like Angola and Nigeria have experienced high growth rates, and some countries have made progress in providing access to basic social services, working towards achieving the Millennium Development Goals (UN Department of Economic and Social Affairs 2010) and their successor, the Sustainable Development Goals (SDGs).

However, more critical perspectives argue that economic growth in Africa has not translated into significant social improvements and remains fragile. Much of this growth is tied to the exploitation of African natural resources by investors from the US, the European Union, and China. Africa's recent economic growth has been driven by commodity exports, which haven't prompted substantial structural changes and have reinforced the continent's narrow export base. Additionally, the value of many commodities has decreased since 2013, and external markets have been impacted by economic downturns in Europe and Asia. Despite Africa's growth, it still accounts for less than 1 percent of the world's GDP. Moreover, Africa's external debt has surged from $89 billion in 1980 to $149 billion in 2010. In 2014, while aid and investment inflows to Africa amounted to $134 billion, financial outflows from Africa reached nearly $192 billion, creating a net deficit of nearly $60 billion for the African economy.

Political economist Walden Bello from the Philippines highlights that income disparities continue to widen between "rich" and "poor" countries, even though similar patterns of inequality persist within these countries. He notes that neoliberal policies associated with globalization and free trade have often led to more poverty, inequality, and stagnation. Bello points out that China is one of the few places where poverty has decreased in the last 15 years, but this was achieved through interventionist state policies rather than neoliberal prescriptions. Moreover, the advocates of eliminating capital controls have witnessed the economic collapse of countries that embraced this policy. The globalization of finance has outpaced the globalization of production and has proven to be a source of chaos rather than prosperity.

In the late 1990s, it became evident that the promises of inclusive growth and improved living conditions for all through globalization were falling short. While there was a notable reduction in global poverty levels (from 43 percent of the world's population in 1990 to 17 percent in 2011), the majority of people in deprived countries still faced significant challenges. This mixed record of poverty reduction raised doubts about the effectiveness of conventional approaches involving economic liberalization and privatization. Economist Amartya Sen, hailing from India, argued that the main issue was not globalization itself but the unequal distribution of its benefits.

The traditional North/South divide persisted, with advanced capitalist countries maintaining control over more than 90 percent of financial assets, 85 percent of global foreign direct investments, over 65 percent of the world's GDP, and 70 percent of industrial exports. Additionally, many East Asian "tiger" economies continued to rely on the Global North in crucial sectors like finance and high technology.

Even staunch advocates of globalization admitted that Africa faced significant challenges. The poorest countries, designated by the UN as the "least developed countries" (LDCs), remained excluded from the benefits of globalization. Numerous African nations found themselves trapped in a harmful cycle of interconnected challenges, including poverty, illiteracy, internal conflicts, environmental pressures, governance issues, and inflexible economies heavily reliant on the export of a single commodity. For instance, income from African exports declined from $255 billion in 2014 to $190 billion in 2016. Sub-Saharan Africa still lagged far behind in development despite the globalizing trends.

The question arises: what is truly new about globalization, or are we witnessing a continuation of the same process that has been reshaping the world since the expansion of Western capitalism? Could it be a pattern similar to what Karl Marx and Friedrich Engels observed over 160 years ago?

Marx and Engels noted that the bourgeoisie, through its exploitation of the world market, had given production and consumption a cosmopolitan character in every country. This had replaced the old local and national isolation and self-sufficiency with global interactions and interdependence among nations. They even foresaw the emergence of a world literature.

What does appear to be genuinely novel in today's globalization is the speed and intensity of connections among entities worldwide. Financial markets operate 24/7, influencing immediate economic decisions globally. New technologies have played a role in creating what can be called the "world factory," managed by global corporations under a unified brand, where production, marketing, and design are seamlessly integrated across continents and communities. According to scholars like Luis Hebron and John F. Stack, globalization is powerful because it encompasses politics, economics, law, social structures, media, and information technologies, all interconnected on a global scale.

Globalization fundamentally involves new economic relationships and activities that occur regardless of geographical boundaries. It transforms the way human affairs are organized by connecting and expanding human interactions across regions and continents. Territory, as traditionally understood, no longer encompasses the entirety of the social space where human activity unfolds.

According to social geographer David Harvey, modern capitalism has integrated the world more profoundly than ever before, rendering time and space no longer insurmountable obstacles. Advances in communication and transportation mean that everything, including goods, services, and people, can move freely across the globe. Consequently, the once clear geographic divide between the Global North and the Global South has become less distinct, though it hasn't entirely disappeared. Instead, globalization is creating a new pattern where poverty and wealth are redistributed through a restructured system of exclusion.

In this context, current globalization-induced development patterns result in shifts in the distribution of uneven development, both among economic sectors and across geographical regions. This suggests that globalization is reshaping the landscape of economic inequality and development disparities.

In the early days of globalization, leaders like former President Cardoso of developing countries believed there were no viable alternatives. In the 1990s, this development model seemed successful, exemplified by the "East Asian miracle." Countries in East Asia, such as China and South Korea, found competitive advantages, attracted foreign investment, and experienced economic growth, significantly reducing poverty rates. For instance, China lifted over 600 million people out of poverty during this period. Meanwhile, North America and Western Europe struggled with economic challenges following the 2008 financial crisis, while China emerged as a global industrial powerhouse, significantly increasing its share of world industrial production and merchandise trade.

Despite concerns about outsourcing and authoritarian governance in some developing nations, globalization was generally viewed as contributing to economic growth and prosperity in the Global North. This export-driven growth in developing countries was encouraged by the IMF and World Bank, which promoted economic liberalization, trade openness, and export expansion. The prevailing theory was that the market's invisible hand, as described by Adam Smith, would help countries find their comparative advantages in trading goods and services.

The World Trade Organization (WTO), a strong advocate of globalization, liberalization, and economic integration, now boasts membership from most countries worldwide. The World Bank also maintains the belief that globalization and integration into the global market benefit the poor and developing countries. According to David Dollar, an economist at the World Bank, evidence of globalization's success can be seen in the fact that, for the first time in modern history, growth rates in poor countries outpaced those in rich countries. These positive trends, including faster growth and poverty reduction, are particularly noticeable in developing countries that have rapidly integrated into the global economy. The World Bank is further encouraged by the significant growth of exports from developing countries, especially in manufactured products, and the substantial foreign direct investment flowing into these nations.

In the early 1990s, there was a prevailing belief that capitalism had emerged as the global victor following the collapse of the Soviet Union. Developing countries were expected to adopt policies advocated by the advanced capitalist nations at the core of the global economy. The World Bank's outlook was optimistic, foreseeing widespread prosperity and progress through full integration into the global economy, a concept termed "globalization." This idea was defined by former World Bank official John Williamson as encompassing the adoption of "Washington Consensus" policies.

The term "globalization" became closely associated with neoliberal policies initially implemented by the United States and the United Kingdom in the early 1980s, which were later endorsed by the World Bank and the International Monetary Fund (IMF) in developing countries. The "consensus" entailed recommendations for liberalizing capital flows and trade, often through free trade agreements, privatizing the public sector, and eliminating regulations that constrained market activities. It became a central component of structural adjustment programs imposed by the World Bank and the IMF on countries seeking financial assistance.

In the early 1990s, as the concept of globalization gained prominence, it surprised many scholars and policymakers in the field of development, including Fernando Henrique Cardoso, a prominent figure from the dependency school of thought that had a significant influence on development studies in the 1970s. Cardoso, who later became the president of Brazil in 1994, notably shifted his previous views on the causes of underdevelopment in a provocative manner. He declared that past development theories were obsolete and that everything he had previously believed about development was incorrect.

Cardoso's new perspective advocated for complete integration into the global economic system. This meant accepting the macroeconomic policies outlined a decade earlier in the Washington Consensus. During his presidency, Brazil redirected its social and economic priorities to align with the needs and demands of international markets. This was a significant departure from his earlier stance, where he and his colleagues in development studies had argued for "delinking" from international capitalism as the only path to development.

The advent of globalization led many developing countries, including Brazil, to shift their course. They started pursuing greater integration into the capitalist system, often adopting policies aligned with the principles of the Washington Consensus. These policies were promoted by the G7, an informal association of the world's wealthiest countries, which included Canada, France, Germany, Italy, Japan, the United Kingdom, and the United States.

Since the 1980s, "globalization" has been a prominent term in international development. The "Washington Consensus," which emphasized trade and investment liberalization, reducing the public sector in favor of private capital, and cutting back public services and institutions, greatly influenced development theory and practice during this period. It led to the erosion of national borders and the creation of a single global economic entity due to technological advancements. However, the anticipated benefits of globalization did not always materialize as expected. It led to rising poverty and political instability in many countries, prompting social movements to call for an alternative approach known as "alter-globalization."

Globalization faced additional challenges in the form of the 2007-2008 financial crisis, which destabilized many nations and exacerbated income inequalities while contributing to environmental degradation. This crisis also fueled nationalist sentiments and political rivalries among superpowers. The election of Donald Trump as the U.S. president in 2016 both reflected and accelerated these trends. The subsequent COVID-19 pandemic, starting in China in early 2020, further threatened globalization. It spread rapidly worldwide through international production chains and mass tourism, infecting millions and causing significant loss of life. Although efforts are ongoing to develop coping strategies, the pandemic's long-term impact on globalization remains uncertain.

In light of the challenges and uncertainties surrounding globalization, this chapter focuses on its effects on development and developing countries. It aims to stimulate new discussions and reflections, particularly among those engaged in development work and study, as globalization faces potential threats and transformations.

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