Research target

O texto sugere que um decreto judicial de "inconstitucionalidade" representa um desafio significativo ao poder do governo, enquanto um veredito de "constitucionalidade" é uma ferramenta poderosa para aumentar a aceitação pública do crescente poder governamental.

Definición

LEAD Scholars Academy

Capitalize and add UCF, change FALL to Fall

move to next line

typo--I'll highlight any others I notice.

servant

love this! I'll be praying for you.

sector

awesome!! doing what?

I've

physique

remove underline

growth

I think the expression is "nerve wracking"

don't change your career plans based on one assessment. It's food for thought but you do what you feel called to do!

interesting to keep in mind

APA style-numbers under 10 are written out.

• Summer reading program
• Gamer club
• Battle of the Books march madness
• Read Across America
• Makerspace
• Destress activities
• Hosted college representatives and representatives from the branches of the military
• Partnership between public and school library
• Bookmark decoration

• Photography club

• Get them in and establish relationships so that they can understand what the Library can do for them

• Adding to the student experience beyond the classroom
• Library as a third place

I looked up this word and it means "noble generosity' and I thought that was very cool

These people were treated unflairy and it pushed them to the point of escapism which lead to war

This was an unfiar and unjust way of living

I like how they take responsibility to fix the actions of their elders because they know if they don't nobody will.

I like the use of the word "unalienable".

I like this whole section because they're just naming all the things that are wrong with the current government and it goes on for a really long time which makes it almost comical.

purposely-impared languages

cause they do not really want to empower people

but machines and only the people behind the machines

We the people should fight the buttleriabn jihad now before the machines get all the firepower

yep

This could definitely be a theorem that you state...but will not be proving as the proof is like 60 pages long and involves the Gaussian integral and shit ai yai yai

Hopefully we do not need to use vectors for this...

This could be useful for examples / figures

I guess this makes sense because you're viewing DNA from different projections and it seems different - like knots/links can even have different numbers of crossings

Again this is confusing...like what are "all possible views" isn't this infinite

twist

writhe

This may have the proof of twist + writhe = linking number

Can give these as examples...with pictures!

knot invariant

Ok so this is actually something we've done we won

So this is like just a link projection

I feel like this one doesn't have to do with DNA as much but maybe you should do this instead idk...

There are no in-text citations (something you must have every time you reference specialized knowledge. See my announcement from 5/1.

Why is this a good thing?

?

sound quality? What are these realms you keep speaking of? Do they have geographic, cultural, political, even spiritual borders?

Not necessary

I guess I don't have to read any more of the essay? Don't give away your argument so completely and so early.

This thesis statement is for a different persuasive/argumentative writing assignment than the one you have been assigned.

You describe a relatable situation well, but try and rephase without using "you"

is does?

even for people who are hard or hearing? What about people who can't afford personal audio? What about people who don't know what you mean when you say, "personal audio?"

Is this the problem you are trying to solve? The problem posed by the assignment is that readers may not care to critically study an object like headphones. Your solution is how you convey the important of studying headphones.

good intro!!

excellent!

yay!

can you remove the white highlighting?

эта линия отражает потерю права собственности или суверенитета над землей из-за участия в войне.

this line reflects on the loss of ownership or sovereignty over the land due to the involvement in warfare

Предполагает, что дети достигли точки, когда у них отсутствуют какие-либо значимые желания или стремления.

Suggests that the children have reached a point where they lack any meaningful desires or aspirations.

na

comment

for - comparison - kinship in digital vs nation state

comparison - between - digital or network state - nation state - comparison statement - kinship is key to forming digital / network states, but are impossible in nation states - nation states are far too large for any real intimacy - The raison d'etre of network states is strong kinship, it's what defines them - Indyweb is designed to catalyze network states - @GyuriLajos

for - answer - size of a digital nation - definition - economy of scope

answer - size of a digital nation - In contrast to nation states with the concept of economy of scale, - in Network states, we have the concept of economy of scope

definition - economy of scope - for small group through strong alignment of interests and values, to foster close kinship - then expand to other similarly aligned groups with synergies between groups

for - question - Is there a limit to the size of a digital / network nation or state?

question - Is there a limit to the size of a digital nation or network state?

for - network states - coordi-Nation - Primavera De Filippi - from - Michel Bauwens article

from - Michel Bauwens Substack article - The Age of Trans-Local Self-Organized CoordiNations: What will it do to our Empire of Nation-States ? - https://hyp.is/wBH0tAi4Ee-xIpco8ZVZLg/4thgenerationcivilization.substack.com/p/the-age-of-trans-local-self-organized

I chose this section because it emphasizes the need for systemic change in educational/ professional environments to be openly inclusive. Designing for equity and inclusion involves the recognition and integration of diverse types of cognitive abilities into the organizational structure of schools/industries. In healthcare, where I have a particular interest, these inclusions not only enhance the quality of work but also support the mental health/well-being of those who just think/feel differently than others.

It looks like the figures are mislabeled in this chapter. They are numbered 14 instead of 15 and don't match the text. In this case, the text says the table below should be 15.4, but it is 14.2.

Chapters 23 and 24 are out of order in the table of contents.

you can always work on this!!

APA writing style, numbers under 10 are spelled out.

great goal!

helpful intro

you will be surprised how your interest in history will enhance your perspective on many things in life

Seven

delete

remove the white behind the lettering.

for - definition - Coordi-Nation

definition - Coordi-Nation - virtual, digital, non-territorial groups of Network States with strong degress of interdependency and kinship and that digitally coordinate their mutual sovereignty - author - Primavera de Filippi

to - Primavera De Filippi Edcon 2023 talk - The Rise of the Network State and Coordi-Nations - https://hyp.is/3etQygi4Ee-17K-Lej3Fzg/docdrop.org/video/F-ckcvpSttA/

for - question - missing ingredient of global digital productive network

question - what is missing ingredient in a global, digital productive network? - fusion of - productive ecosystems - crypto-coordination infrastructure

for - book - cosmo-local reader - https://clreader.net/

for - adjacency - open source - copyleft - Achilles Heel - unpaid workers - predatory capitalism

adjacency - between - open source - copyleft - Achilles Heel - predatory capitalism - unpaid workers - adjacency statement - The Achilles Heel of the open source copyleft system is that it allows everyone to participate. Everyone can look at the innovation, including corporate raiders in it for their own self-interest. - This enables predatory capitalism. The well-capitalized corporations take the best open source ideas and integrate them into their own private systems. With their abundant capitalization, they can maintain the existent structural inequality - Meanwhile, most open source software is maintained by underpaid programmers

I chose this section because these statistics really show the disparity that exists when it comes to medicine and how bias can affect it. You have this majority who are more likely to contract this disease and are much more likely to die from it, yet the minority are used to test the treatment. It doesn’t make sense, and the difference is too wide for it to be a case of normal probability.

Like Dr. Coney states in the article, it’s important to become aware of existing biases, racial or otherwise, and work to reduce them as much as possible. This is the only way we can truly design systems for equity and inclusion. As a Black woman, reading and hearing about the disparities in health care that exist both for Black people and women of all races legitimately scares me sometimes. I’ve seen videos about people bringing awareness to incidents like symptoms being relegated as “normal” for Black people when they’d raise flags when found in a White person or pain for cis-gendered women being minimized by doctors while it’s taken much more seriously for cis-gendered men. This article is primarily about racial bias in clinical trials, but it makes it clear that bias is found all over the healthcare field, whether in the clinic, medical school, or a lab.

The article overall really resonated with me. I find it very valuable when people write and publish about bias-caused disparities in their fields, particularly when it affects nearly every person in the country. This topic is important and awareness is the first step to solving the problem.

true--and when the seas are rough, you will want to have some unwavering values, morals or ethical commitments that will guide you...

be careful not to fall into "people pleasing"

delete

A real estate lease contract, often called a lease agreement or rental agreement, is a legally binding document outlining the terms and conditions under which a property is rented. Here's an example of a general real estate lease contract:

Real Estate Lease Contract

Parties: This Lease Contract ("Lease") is made and entered into on [Date], by and between [Landlord's Name], whose address is [Landlord's Address] ("Landlord"), and [Tenant's Name], whose address is [Tenant's Address] ("Tenant").

Property: The Landlord hereby leases to the Tenant, and the Tenant leases from the Landlord, the following property ("Premises"): [Address of Property, including apartment/unit number, if applicable].

Term: The term of this Lease shall begin on [Start Date] and end on [End Date]. After the initial term, the Lease will automatically renew on a [monthly/annual] basis unless terminated by either party with at least [number of days] days' notice.

Rent: The Tenant agrees to pay the Landlord a monthly rent of [amount] on or before the [day of the month] of each month. Rent shall be paid by [cash, check, electronic transfer, etc.], and any late payment will incur a penalty of [late fee amount]. If rent is more than [number of days] days late, the Landlord may initiate eviction proceedings.

Security Deposit: The Tenant agrees to pay a security deposit of [amount] upon signing this Lease. The security deposit will be returned to the Tenant within [number of days] after the end of the Lease, less any deductions for damages or unpaid rent. A written itemized statement of deductions, if any, will be provided to the Tenant.

Use of Premises: The Premises shall be used solely for residential purposes. The Tenant agrees not to engage in any illegal activities on the Premises and to comply with all applicable laws, regulations, and rules set by the Landlord or homeowners' association (if applicable).

Maintenance and Repairs: The Landlord is responsible for major repairs to the property, such as structural repairs, plumbing, and electrical systems. The Tenant is responsible for routine maintenance, such as cleaning and replacing light bulbs. The Tenant must notify the Landlord of any necessary repairs in a timely manner.

Utilities: The Tenant is responsible for payment of utilities, including [list specific utilities, e.g., electricity, water, gas, internet, etc.], unless otherwise specified in this Lease.

Pets: Pets are [allowed/not allowed] on the Premises. If pets are allowed, the Tenant must pay a non-refundable pet deposit of [amount]. The Tenant is responsible for any damage caused by pets.

Termination and Eviction: The Landlord may terminate this Lease for breach of terms, non-payment of rent, or other specified reasons. The Tenant may terminate the Lease by providing written notice to the Landlord at least [number of days] days in advance.

Governing Law: This Lease shall be governed by and construed in accordance with the laws of [State/Province/Country].

Additional Provisions: [List any additional terms or conditions, such as parking arrangements, storage space, or specific rules for common areas.]

Signatures: This Lease represents the entire agreement between the parties. No other terms or conditions shall apply unless expressly stated in this document. By signing below, both parties agree to abide by the terms of this Lease.

[Landlord's Signature] ____ [Date]

[Tenant's Signature] ____ [Date]

This example serves as a general template. When creating or reviewing a real estate lease contract, it's important to consider state and local laws, specific property requirements, and the unique terms agreed upon by the landlord and tenant. Legal counsel can help ensure compliance with applicable laws and resolve any questions about lease terms.

Does this kind of lysis bias the community to easy lysers? - I'm currently just heating 95C, 10 m for lysis and worry about the same. Will switch to not pelleting cells, adding a lysis buffer and proceeding into a mag bead based DNARNA extraction kit (Maxwell)

The more recent method used by Klumper paper, 2022 uses a proper extraction kit but also acquires more cells -

A minimum of 50,000 cells were acquired in all sorting runs.The sorted transconjugant and recipient cells were lysed and DNA extractions were performed using the DNeasy Powersoil Pro Kit. Source: Wang, Yue, et al. "Non-antibiotic pharmaceuticals promote conjugative plasmid transfer at a community-wide level." Microbiome 10.1 (2022): 124.

Thermo discontinued lyse n go. alternatives? - check Microlysis from gelcompany

That's quite long..

new paragraph

can you make the board bigger? it's hard to see...

personality

new paragraph

hmmmm....

Customer relations management: "CRM higher education technology enables institutions to manage relationships with all of their customers (including students, alumni, faculty, staff, and corporate partners) and connect insights from those interactions in a unified view. " https://www.salesforce.org/resources/article/crm-higher-education/#:~:text=First%2C%20let's%20define%20what%20CRM,interactions%20in%20a%20unified%20view.

How do you feel about the student as customer? How does this map onto the issues raised in this course?

• Como Ditador, concedeu cidadania aos povos conquistados e promoveu a construção de obras públicas
• O senado, que se desagradou com os feitos do césar, matam ele a facadas

É formada a Tribuna da Plebe - assembleia de plebeus que tinha o poder de barra as leis do senado - Criação das leis da 12 tábuas - garantia a isonomia (igualdade) na justiça

A monarquia tem seu fim apos disputas entre latinos e estrucos - Tarquino, o soberbo que era estruco ultimo rei, tenta governar acima do senado é deposto. - o senado então proclama uma república

this is something to work on Noah. Your "lack of enthusiasm" can be misread by potential employers or co-workers.

begs the question: what are they??

glad you make note of this!!

can you make the board image larger? It's a ittle hard to seee

you

Thinking no FAQ, but a link to reach out with questions.

I think here we can link to a CETL Bearcat landing page, which I will maintain with current workshop offerings that are relevant.

Apply for Certificate?

Could be expanded on.

Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives

the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the adsorbate on the surface of the adsorbent

everywhere

that's a big deal at that age! :)

Captitalize

you probably don't need to take it again!

capitalize

you

The presence of family photographs, children's artwork, and other personal items further underscores the importance of creating an environment that fosters literacy and reflects the family's identity and values. Additionally, the fact that both homes have crayons, markers, and pencils available suggests an emphasis on hands-on learning and creative expression.

The fact that more than half of the residents are Caucasian, with significant African American and Latino populations, reflects a certain degree of racial and ethnic diversity. However, the poverty level of 44% is strikingly higher than the city average, indicating significant economic challenges within the community. Additionally, the low level of educational attainment, with 32% lacking a high school diploma, suggests systemic issues that may hinder upward mobility and opportunities for residents.

The study focuses on an urban emergent setting situated within a midsize city, sharing some similarities and challenges with larger urban intensive schools and districts in terms of resources, teacher qualifications, and student academic development. While the term "urban declining" might be more fitting for the current state of the Rust Belt city, characterized by population decline and a dwindling industrial base, the district is identified as one in transition, reflecting characteristics of its past as a larger city and its aspirations for redevelopment.

The study aims to develop a culturally relevant curriculum by recognizing and leveraging the wealth of knowledge and skills present within households and communities. It explores children's out-of-school literacy experiences, encompassing formal learning related to school curriculum as well as informal learning involving popular culture and new technologies. Additionally, it examines specific contexts such as religious settings or community schools, emphasizing the role of language and culture in children's learning experiences.

By examining these factors across different neighborhoods, Neuman and Celano aim to shed light on the disparities in access to literacy resources and the impact of neighborhood environments on children's literacy development. Their findings challenge the simplistic view that literacy outcomes are solely determined by family characteristics, emphasizing the importance of considering broader ecological factors in understanding children's literacy experiences.

definition of state capture, only used historically for malignant and corrupted groups. Bagg reformulates the applicability of this term

see below re: your source

Komives, S. R., & Wagner, W. (Eds.). (2017). Leadership for a better world: Understanding the social change model of leadership development. John Wiley & Sons.

resilience

true!!

new paragraph

say more about this...

Moving clockwise, ....

upper right corner

can you make your board bigger? it's hard to see

did you copy this from somewhere?

typo

did you copy this from somewhere?

This approach not only fosters Yamaira's passion for history but also fosters a broader understanding among the class that translanguaging is a valuable resource for deepening comprehension and critical thinking in any subject. Ms. López's stance on translanguaging not only empowers Yamaira to engage with her passion for history but also creates a more inclusive and supportive learning environment for all students, regardless of their linguistic or cultural background.

Ms. López empowers Yamaira by affirming her expertise in history, despite her struggles with reading the textbook in English. By providing Yamaira with primary documents in Spanish and emphasizing that she will be evaluated based on her historical knowledge rather than her English proficiency, Ms. López creates an environment where Yamaira feels valued and capable of excelling.

Ms. López, drawing from her own experiences as a Latinx bilingual, actively incorporates translanguaging into the classroom dynamics. She ensures that Yamaira's discussion group includes other Spanish-speaking students, allowing Yamaira to comfortably express herself and share her insights in Spanish. By encouraging Yamaira to draw from her Spanish-language readings and primary documents, Ms. López recognizes and validates the richness of Yamaira's linguistic repertoire.

Paco's pre-reading experience in his bilingual home offers a fascinating glimpse into the dynamic and fluid nature of translanguaging in early literacy development. Paco, a 3-year-old bilingual child, grows up in a household where both English and Spanish are seamlessly integrated into daily communication. His mother, a U.S.-born Latina woman from a bilingual background, and his father, a U.S.-born white man with some knowledge of Spanish, create a nurturing environment where Paco's bilingualism is celebrated and nurtured.

It critiques the prevalent monolingual and monoglossic views of literacy held by many teachers in U.S. schools. It suggests that most teachers perceive literacy instruction as primarily occurring in English, with occasional inclusion of Spanish or other languages. This perspective reflects a monolingual view of literacy, which posits that literacy practices should be confined to one language or another, rather than recognizing the dynamic and fluid nature of bilingualism.

The unequal distribution and valuation of these resources within society and schools. English tends to be privileged over other languages, such as Spanish, in U.S. schools, where English is predominantly the language of instruction, tests, and texts. Additionally, within meaning-making systems, verbal communication, particularly in its written form, is often prioritized over visual or gestural modes.

Author response:

We thank eLife and the reviewers for the thoughtful summary and valuable review of our manuscript. We largely agree with the summary and review and have provided our responses to the comments below. We believe BADGER is a significant new tool for identifying associated risk factors for complex diseases, and the associations we observed in the analysis provide insights into the genetic basis of Alzheimer's disease.

Reviewer #1 (Public Review):

The major aim of the paper was a method for determining genetic associations between two traits using common variants tested in genome-wide association studies. The work includes a software implementation and application of their approach. The results of the application of their method generally agree with what others have seen using similar AD and UKB data.

The paper has several distinct portions. The first is a method for testing genetic associations between two or more traits using genome-wide association tests statistics. The second is a python implementation of the method. The last portion is the results of their method using GWAS from AD and UK Biobank.

We thank the reviewer for the conclusion and positive comments.

Regarding the method, it seems like it has similarities to LDSC, and it is not clear how it differs from LDSC or other similar methods. The implementation of the method used python 2.7 (or at least was reportedly tested using that version) that was retired in 2020. The implementation was committed between Wed Oct 3 15:21:49 2018 to Mon Jan 28 09:18:09 2019 using data that existed at the time so it was a bit surprising it used python 2.7 since it was initially going to be set for end-of-life in 2015. Anyway, trying to run the package resulted in unmet dependency errors, which I think are related to an internal package not getting installed. I would expect that published software could be installed using standard tooling for the language, and, ideally, software should have automated testing of key portions.

We thank the reviewer for their comments. To clarify, the primary difference between our proposed method, BADGERS, and LDSC lies in their respective objectives and applications. LDSC is designed to estimate heritability and genetic correlations between traits by utilizing GWAS summary statistics, thereby aiding in the elucidation of the genetic architecture of complex traits and diseases. Conversely, BADGERS is specifically developed to explore causal relationships between risk factors, such as biomarkers, and diseases of interest. It employs genetic variants as variables to deduce causality, thereby addressing the challenges of confounding and reverse causation that are common in observational studies. Although BADGERS utilizes the LD reference panel derived from LDSC, the LD reference panel is used to obtain the predicted trait expression. The ultimate goal is to focus on linking biobank traits with Alzheimer’s disease and building causal relationships instead of identifying genetic architecture.

Regarding the technical aspects mentioned, we acknowledge the concerns about the use of Python 2.7 and the issues encountered during the package installation. We are in the process of updating the software to ensure compatibility with current versions of Python and to enhance the installation process with standard tooling and automated testing for a more user-friendly experience. We have provided tests for each portion of the software so the user can test if the software is working properly.

Regarding the main results, they find what has largely been shown by others using the same data or similar data, which add prima facie validity to the work The portions of the work dealing with AD subgroups, pathology, biomarkers, and cognitive traits of interest. I was puzzled why the authors suggested surprise regarding parental history and high cholesterol not associated with MCI or cognitive composite scores since the this would seem like the likely fallout of selection of the WRAP cohort. The discussion paragraph that started "What's more, environmental factors may play a big role in the identified associations." confused me. I think what the authors are referring to are how selection, especially in a biobank dataset, can induce correlations, which is not what I think of as an environmental effect.

We thank the reviewer very much for their comment. We're glad that our findings align with existing research using similar data, increasing the validity of our work and the proposed BADGER algorithm. Your point about the lack of association between parental history, high cholesterol, and mild cognitive impairment (MCI) or cognitive composite scores in the WRAP cohort is well-taken. We agree that the selection criteria of the WRAP cohort may influence these findings, as it consists of individuals with a specific risk profile for Alzheimer's disease. This selection could indeed mitigate the observed association between these factors and cognitive outcomes, which we initially found surprising.

Regarding the environmental factors, we appreciate your clarification and understand the confusion. Our intention was to discuss the potential for selection bias and confounding factors in biobank datasets for the identified associations, which might not necessarily be direct environmental effects.

Overall, the work has merit, but I am left without a clear impression of the improvement in the approach over similar methods. Likewise, the results are interesting, but similar findings are described with the data that was used in the study, which are over 5 years old at the time of this review.

We thank the reviewer a lot for their endorsement of the BADGER framework. We believe that our method, BADGER, improves on existing approaches by effectively linking genetic data with the detailed phenotypic information in biobanks and large disease GWAS. This enhances our ability to detect associations without needing individual-level data, offering clearer insights while reducing issues like reverse causality and confounding factors.

Even though the IGAP dataset is over five years old, it remains one of the largest publicly available datasets for Alzheimer’s Disease. Likewise, the UK biobank is one of the largest publicly available human traits datasets, which researchers continue to use. These datasets' continued utility demonstrates their value in the research community. Additionally, the versatility of the BADGER framework makes it suitable for future research investigating the relationship between human traits and various diseases using different datasets.

Reviewer #2 (Public Review):

Summary:

Yan, Hu, and colleagues introduce BADGERS, a new method for biobank-wide scanning to find associations between a phenotype of interest, and the genetic component of a battery of candidate phenotypes. Briefly, BADGERS capitalizes on publicly available weights of genetic variants for a myriad of traits to estimate polygenic risk scores for each trait, and then identify associations with the trait of interest. Of note, the method works using summary statistics for the trait of interest, which is especially beneficial for running in population-based cohorts that are not enriched for any particular phenotype (ie. with few actual cases of the phenotype of interest).

Here, they apply BADGERS on Alzheimer's disease (AD) as the trait of interest, and a battery of circa 2,000 phenotypes with publicly available precalculated genome-wide summary statistics from the UK Biobank. They run it on two AD cohorts, to discover at least 14 significant associations between AD and traits. These include expected associations with dementia, cognition (educational attainment), and socioeconomic status-related phenotypes. Through multivariate modelling, they distinguish between (1) clearly independent components associated with AD, from (2) by-product associations that are inflated in the original bivariate analysis. Analyses stratified according to APOE inclusion show that this region does not seem to play a role in the association of some of the identified phenotypes. Of note, they observe overlap but significant differences in the associations identified with BADGERS and other Mendelian randomization (MR), hinting at BADGERS being more powerful than classical top variant-based MR approaches. They then extend BADGERS to other AD-related phenotypes, which serves to refine the hypotheses about the underlying mechanisms accounting for the genetic correlation patterns originally identified for AD. Finally, they run BADGERS on a pre-clinical cohort with mild cognitive impairment. They observe important differences in the association patterns, suggesting that this preclinical phenotype (at least in this cohort) has a different genetic architecture than general AD.

We thank the reviewer a lot for the conclusion and positive comments.

Strengths:

BADGERS is an interesting new addition to a stream of attempts to "squeeze" biobank data beyond pure association studies for diagnosis. Increasingly available biobank cohorts do not usually focus on specific diseases. However, they tend to be data-rich, opening for deep explorations that can be useful to refine our knowledge of the latent factors that lead to diagnosis. Indeed, the possibility of running genetic correlation studies in specific sub-settings of interest (e.g. preclinical cohorts) is arguably the most interesting aspect of BADGERS. Classical methods like LDSC or two-sample MR capitalize on publicly available summary statistics from large cohorts, or having access to individual genotype data of large cohorts to ensure statistical power. Seemingly, BADGERS provides a balanced opportunity to dissect the correlation between traits of interest in settings with small sample size in which other methods do not work well.

We thank the reviewer a lot for the conclusion and positive comments.

Weaknesses:

However, the increased statistical power is just hinted, and for instance, they do not explore if LDSC would have identified these associations. Although I suspect that is the case, this evidence is important to ensure that the abovementioned balance is right. Finally, as discussed by the authors, the reliance on polygenic risk scoring necessarily undermines the causality evidence gained through BADGERS. In this sense, BADGERS provides an alternative to strict instrumental-variable based analysis, which can be particularly useful to generate new mechanistic hypotheses.

We thank the reviewer a lot for the comments. We understand the importance of comparing BADGER to other methods. The comparison with LDSC, while not directly relevant to BADGER’s causal inference aims, is indeed an interesting aspect to consider for future studies. In this paper, we focused on comparing BADGER with Mendelian Randomization (MR), which shares its causal inference objective.

As a result, BADGERS identified a total of 48 traits that reached Bonferroni-corrected statistical significance. In contrast, MR-IVW only identified nine traits with Bonferroni-corrected statistical significance. Among these nine traits, seven were also identified by BADGERS. This demonstrates that BADGER holds higher power in detecting causal relationships.

Regarding the use of polygenic risk scoring, we agree that it holds challenges in directly inferring causality. While BADGERS offers an innovative way to explore genetic correlations and can help generate new hypotheses about disease mechanisms, it does not replace the causal inferences that can be drawn from instrumental-variable-based analyses. Instead, it should be viewed as a complementary tool that can illuminate potential genetic relationships and guide further causal investigations.

In summary, after 15 years of focus on diagnosis that would require having individual access to large patient cohorts, BADGERS can become an excellent tool to dig into trait heterogeneity, especially if it turns out to be more powerful than other available methodologies.

We thank the reviewer a lot for the conclusion and positive comments.

typo, also mention the name "Ordinary Least Squares"

typo, should be pyplot

nicht definiert

welchen?

Author response:

We thank the reviewers and editors for their time and effort reviewing and improving this manuscript. We also thank them for their support.

Following the guidelines received by eLife we submit here the preliminary author’s response to the Public review with our planned changes to the manuscript.

Reviewer 1.

Comment 1. Issue on cross-reactivities of MafB antibodies.

We are confident that our description of MafB V1 interneurons is correct despite some cross-reactivity with one of the antibodies used. We test all antibodies we use, and unfortunately, we found an inverse relationship between sensitivity and specificity with the two MafB antibodies used in this study. We chose for quantification the one with highest sensitivity, despite the presence of some cross-reactivity in interneurons other than the dorsal and ventral (Renshaw) V1 populations we focus on. The dorsal and ventral (Renshaw) V1 populations we describe here are also reactive with the more specific antibody (although with lower sensitivity) and both are neatly labeled in a MafB-GFP reporter mouse as described in Figure 3. We will add an image to the supplement with MafB-GFP V1 Interneurons at P5 showing the immunoreactivity of both MafB antibodies as suggested by the reviewer. We agree with the reviewer that this will give further support to the characterization of these populations by either immunocytochemical or genetic means at P5.

Unfortunately, we cannot show lack of immunoreactivity for MafB antibodies in MafB GFP/GFP knockout mice at P5 because MafB global KOs die at birth as a result of respiratory failure. This is due to removal of inhibitory interneurons in brainstem centers critical for respiration (Blanchi at al. 2003 MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth. Nat Neurosci. 6(10):1091-100. doi: 10.1038/nn1129. PMID: 14513037). This is why we used tissues from late embryos for testing antibody specificity in KO spinal cords. We will make this clearer in the text.

Comment 2. Overlap of V1 clades with lineage labeled Foxp2-V1s at P5.

We collected the data requested by the reviewer for P5 Foxp2-V1 interneurons and this will be added to an updated version of this figure. In comparison to the results with the OTP mouse, we only found marginal overlap at P5 with Renshaw cells, Pou6f2, and Sp8 V1s in our genetic intersection to label Foxp2-V1s. We apologize for not showing the data. We will make this clearer.

Reviewer 2.

Comment 1. Paper VERY hard to read.

We will make every effort to make the paper more readable by moving methodological discussions to supplementary materials. We strive to keep our methods as rigorous, clean, and replicable as possible, and that sometimes requires lengthy explanations of the details and reasoning behind our approaches. We will make sure this does not distract from the principal scientific messages we want to convey. We agree with the reviewer that these should be emphasized over methodological detail, and we will correct any mistakes in the text that lead to confusion. Thank you for pointing out this problem that we hope to correct in a new version. Why focus on Foxp2 V1s? We focus in the Foxp2 population for several reasons: 1) This is the largest population of V1s, and it is the one with a close spatial association to motoneurons, in particular limb motoneurons; 2) Given previous results (Benito-Gonzalez and Alvarez, 2012, cited in bibliography) it likely includes many reciprocal inhibitory interneurons; 3) We do not have the mice for studying the Pou6f2 (or Sp8) population, but similar studies are now being carried out in the Bikoff lab.

Comment 2. Lack of functional studies.

Functional studies are currently being carried out, both during development of limb function in postnatal mice as well as in adult animals. These studies required the creation of several new animal models and reagents. As with the present manuscript, we thoroughly characterize all animals and methods. This takes time and space. These studies are beyond the goals and length of the current manuscript, but we agree with the reviewer that these are the critical next experiments that need to be performed. We are now finalizing studies on the role of Foxp2-V1 interneurons in the postnatal development of limb coordination and validating approaches for silencing them in the adult while also optimizing behavioral assays and recordings. The data presented here on Foxp2-V1 interneuron heterogeneity and relations with limb motoneurons gives the necessary context for raising stronger hypotheses and aiding in the interpretation of future results in functional studies.

Synapse counts.

We respectfully disagree with the reviewer’s comments on our synapse density estimates. To fully explain the reasons and prevent any ambiguity, we need to focus on detailed methodological aspects. We apologize for the lengthy response. Two major issues were raised:

(1) Focus on the cell body.

The issue pointed by the reviewer of potential synapses in distal dendrites from V1 subgroups not projecting proximally was already discussed in the text. The reason we focus on the cell body is because 1) it is not feasible to study the full dendritic arbor of so many different types of motoneurons and 2) it allows us to identify V1 subpopulations that likely exert stronger modulation of motoneuron firing by targeting the proximal somatodendritic membrane. The fact that synaptic organization on motoneurons is similar on cell bodies and proximal dendrites (first 100 µm) suggests that inputs from V1 clades other than Renshaw cells are likely further away, and therefore there is limited benefit to include analyses of proximal dendrites in these data. Additionally, dendrites would be difficult to consistently follow in Chat immunostained tissue. We are currently using novel viral approaches to obtain labeling of single motoneurons and their full dendritic trees for more in depth dendritic analyses in the mouse. The classical method based on single cell in vivo intracellular labeling using micropipettes is presently very low yield in the adult mouse. We are experienced with detailed single motoneuron dendritic arbor analyses in cat and rat motoneurons (Alvarez et al. 1997 Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord. J Comp Neurol. 379(1):150-70; Alvarez et al., 1998 Distribution of 5-hydroxytryptamine-immunoreactive boutons on alpha-motoneurons in the lumbar spinal cord of adult cats. J Comp Neurol. 393(1):69-83; Rotterman et al., 2014. Normal distribution of VGLUT1 synapses on spinal motoneuron dendrites and their reorganization after nerve injury. J Neurosci. 34(10):3475-92. doi: 10.1523/JNEUROSCI.4768-13.2014). Based on this experience, we do not believe it is feasible to include similar analyses to compare all motor columns throughout 6 segments of the spinal cord in this study. We agree with the reviewer that these are important data sets that need to be collected and they are planned for future experiments. These analyses will address different questions than the ones posed and answered in our current manuscript.

(2) Number of motoneurons analyzed.

We disagree with the reviewer assessment that our conclusions might be biased because of the numbers of motoneurons analyzed. We sampled a total of 295 motoneurons in 5 different mice (117 LMC/HMC, 99 MMC, and 79 PGC motoneurons), and we used stringent methods for synapse detection. Due to a technical error, Mouse 3 lacked data in upper lumbar and Th13, but all other mice included data in almost all motor columns and segments. We disagree with the characterization that these are small samples. For full transparency, all motoneurons analyzed were identified in Figure 6D. Each of the nearly 300 motoneuron cell bodies was carefully reconstructed through several optical planes to obtain an accurate estimate of synapse density. More automatic methods in current use in the literature sometimes analyze larger samples, but our methods are designed to avoid methodological biases inherent to these automatic methods. We do not use image thresholding to extract synaptic contacts because they lack accuracy identifying single synapses. Thus, estimates using this technique frequently refer to coverage, not synapse density. In addition, it is hard to keep threshold criteria consistent across multiple optical planes to analyze enough section thickness to estimate a motoneuron surface. This is because tissue light diffraction alters thresholding levels continuously across optical planes. Thus, many authors present data as linear densities across a perimeter (in a single plane) measuring many cells in one field in one plane. We avoid cell body linear densities (or coverage) because they bias counts towards larger synapses that have higher probability of being present at any single confocal plane. Moreover, estimates along a surface reduces synapse sampling variability and better estimate synaptic coverage compared to estimates derived from analyzing single cross-sections. We also confirm each genetically labeled varicosity as a likely synapse by accumulation of VGAT. In this manner we restrict our counts to synaptic boutons and not axons or intervaricose regions. Previously, we used bassoon to show the accuracy of our methods (Wootz et al. 2013 Alterations in the motor neuron-Renshaw cell circuit in the Sod1(G93A) mouse model. J Comp Neurol. 521(7):1449-69. doi: 10.1002/cne.23266). That means that our densities are true synaptic densities, which are difficult to extract from automatic methods that estimate fluorescence coverage over larger samples of somatic profiles but fail to individualize synapses and frequently bias results. These bulk methods introduce significant confounds in data interpretation: Is higher coverage due to bigger synapses or more synapses? Do threshold structures represent true synapses or also include axons? To what extent does sub- or over-thresholding in different planes affect identification of structures in contact with the motoneuron surface? We avoid all these problems. Not surprisingly, a nested ANOVA demonstrated consistent significant differences among motor columns and segments.

In summary, while more automatic methods allow larger samples, they disregard true synaptic densities and are based on thresholding methods with high variability in different motoneurons, optical planes and histological sections, thereby they require much larger numbers of motoneurons to overcome their many biases and sources of error. This is not our case. Our sample size is large enough considering the accuracy of our methods and data quality. This is demonstrated by consistency in statistical results across motor columns in different segments and mice.

Comment 3. Possibility of anterograde transsynaptic labeling from primary afferents infected with rabies virus.

This is a fair question that we did not clearly explain. The reviewer compares our results with those of Pimpinella et al., 2022. The methods used are different. To obtain anterograde tracing, these authors used Cre lines to achieve high levels of expression of TVA and RV glycoprotein in specific subtypes of sensory neurons including proprioceptors. Then EnVa-coated Rabies virus was injected directly inside the spinal cord for cell-type specificity. This method transynaptically labeled in the anterograde direction interneurons receiving inputs from specific types of sensory afferents, but the method does not have the muscle specificity required in our analyses. In our case, we used intramuscular injections at P5 of AAV1-G for transcomplementation with Rabies virus delta G injected in the same muscles later, at P15. In previous studies in which we used the RV-delta G virus without AAV1G, we analyzed motoneuron and primary afferent infection rates and found both to be considerably reduced with injection age. In our hands, there is almost no RV infection of primary afferents when Rabies virus is injected i.m. at P15, but there is some limited motoneuron infection remaining (that we used to our advantage in this paper to avoid primary afferent and developmental confounds).

Unfortunately, these methodological studies are presently communicated only in abstract form (GomezPerez et al., 2015 and 2016; Program Nos. 242.08 and 366.06). Therefore, we will add to the supplementary information some images from serial sections to those illustrated in the paper and that will show a few “start” LG motoneurons that remained labeled at this survival time point and the lack of any dorsal horn primary afferent labeling. This is consistent with our yet unpublished data that is based on a larger number of animals and more extensive time courses.

Comment 4. Temporal resolution of birth-dating.

We agree with the reviewer, and that is the reason we explicitly discuss that temporal resolution is not perfect (we also add a few more caveats that affect temporal resolution beyond the reviewers’ comments). However, the method is good enough to differentiate temporal sequences of neurogenesis with close to 12-hour resolution, once enough animals are analyzed to compensate for methodological temporal overlaps. That is the reason for our Figure 1D.

Reviewer 3

Comment 1. Text is too long and main message buried in technical details.

We agree and similar to our response to the first comment of Reviewer 2, we will revise the writing to make it more straightforward while moving some of the information on methods and technical discussion to supplementary materials. As demonstrated by reviewer 2 comments, methodological discussions are still important to best interpret the data presented in this paper.

I love that you stated this so plainly! :)

delete this

Die G7 Staaten haben sich beim Treffen ihrer Energieminister in Turin darauf geeinigt, ab 2035 keinen Strom mehr aus Kohlekraftwerken zu produzieren – es sei denn, es werde Carbon Capture and Storage verwendet. Länder, die auf Kohlestrom angewiesen seien, werden nicht zum Verzicht auf Kohle aufgefordert. https://www.theguardian.com/world/2024/apr/30/g7-agree-to-end-use-of-unabated-coal-power-plants-by-2035

identify

spell out

bottom left

pro tip: number the vision board images so they are easier to refer to.

you can delete this and make your vision board larger

The girls' homes, despite being places of refuge, often become sites of alienation and isolation. As working-class immigrants, their parents are compelled to prioritize economic stability, often at the expense of quality family time. The absence of intimate family rituals, such as daily dinners and regular conversations, compounds their feelings of loneliness and longing for the sense of community they experienced in their countries of origin.It illustrates this longing through Chelle's melancholy recollection of being home alone in the US, contrasting it with the sense of belonging and support she felt surrounded by extended family members and neighbors in the Philippines. This juxtaposition highlights the stark difference in social structures and support systems between their home countries and the US.

The researcher acknowledges various aspects of her identity, including being a Japanese citizen, an Asian woman, a non-native English speaker, and a doctoral student. She recognizes that these identities shape her understanding of phenomena and influence how she interacts with and builds relationships with the girls in her study. She demonstrates an awareness of the privilege afforded by her middle-class background and Japanese citizenship relative to the girls, many of whom come from working-class backgrounds and families from developing countries. This recognition of power differentials informs her approach to the research, as she navigates the 'ambiguous insider/outsider position' with sensitivity and reflexivity.

Drawing on scholarly works that delve into the sites of belonging and power constructed by Asian American young women, the passage exemplifies how they actively engage in processes of negotiation, resistance, and adaptation. For instance, Maira's study on Indian American young women demonstrates how they use Bhangra remix music and hybrid fashion to navigate between multiple cultural identities. Similarly, Ngo's research on Hmong American young women challenges the negative connotations of early marriage, revealing how they employ this practice as a means of resisting structural constraints.

The excerpt adeptly articulates the internal and external pressures these girls confront, including the expectations of their parents to uphold their homeland traditions and values, as well as the societal norms and media representations that often impose narrow and idealized standards of identity and beauty. It underscores the pervasive nature of these messages, which permeate various facets of their lives, from family dynamics to educational institutions and popular culture.Furthermore, it poignantly depicts the struggle of Asian American immigrant girls to carve out a sense of belonging and agency within this liminal space. Despite feeling displaced and alienated, they actively engage in the process of identity negotiation, creating their own community—the Basement Group—as a sanctuary where they can embrace their cultural hybridity, challenge dominant narratives, and envision alternative futures.

This introduction paints a vivid picture of the lunchtime scene at Maple High, where a diverse group of students congregates in the school basement to form what they call the "Basement Group." The description captures the bustling atmosphere, with students engaging in various activities such as sharing food, conversing in multiple languages, listening to music, and dancing together.What stands out is the sense of community and belonging that the Basement Group provides for these students, particularly Asian American immigrant high school girls. Despite the challenges and tensions they may face, this space serves as a sanctuary where they feel comfortable, empowered, and affirmed.The introduction effectively sets the stage for further exploration into the dynamics of this community and the experiences of its members. By focusing specifically on the perspectives and voices of Asian American girls who are founders and core members, the article promises to provide valuable insights into their lived realities and the ways in which they navigate identity, culture, and belonging within the context of their high school environment.

I think step-by-step studies that control variables are important. It is precisely because of the various details of the research objects that we pay attention to that determine the rigor and objectivity of our research. We can also count them on a large scale in the future. thereby completing the objectivity of the entire study

I think it is precisely because of the different environments that we can more objectively observe the changes in the children of a family in different periods. This way we can see whether the child's own changes have a lot to do with the family. It is precisely because of these differences that they lead different lives.

Stereotypes and body image also weigh heavily on them. Regardless of gender, they are required to have a body that they think looks good, otherwise they will make jokes at home that make you sad, intentionally or unintentionally, as if just because you gain a little weight, the whole world will leave you and you will be The whole world is abandoned. They ask you to do your best in everything, and your children never stop for a moment. They don't have time to look at the world around them, and they don't have time to enjoy campus life. Everything should be the same as a robot. What they think is good is good, but when they ask for it, it becomes wrong. They will only tell you with what they have given, and you are not qualified to refute what they have given so much.

Strict gender roles and expectations, their own daughter has very strict requirements and does not feel that her daughter should succeed. Rather than success, they think that success is when their daughter marries a good man. This is actually very abnormal. After working hard for more than 20 years to get close to the best university and get the best job, they are required to be successful only if they are married. This is really unreasonable but true.

I think family stress really affects a child throughout his or her life. When their parents ask them to get into a good university without considering the setbacks in the process, a child can only face it alone. Because they only see what they have paid but not what their children have borne under their pressure and effort. They often lose their best childhood years. When they still fail to get a good result after working hard, they will be swept away by the previous pressure again and press them harder on the beach.

They want to build a community where people in their own circle can speak freely and develop as they please. The original intention is good, but I think it will only get harder and harder in the future. Because this method of not communicating with the outside world is not feasible. From an early age, this is why we have to go to school instead of being educated at home. Generally speaking, if a country closes itself off, it will only lead to the country's regression. I wonder if we need a hundred flowers to bloom internally, but we also need to learn knowledge from the outside that is in line with everyone's thoughts and progress.

physician could be lower case throughout your reflection

delete this.

explain what these acronyms mean.

replace with: In addition to my academic interests,

delete comma

add years to your activities

LEAD

work or employment

eLife assessment

This fundamental study for the first time defines genetically the role of the Clock gene in basal metazoa, using the cnidarian Nematostella vectensis. With convincing evidence, the study provides insight into the early evolution of circadian clocks. Clock in this species is necessary for daily rhythms under constant conditions, but not under a rhythmic light/dark cycle, suggesting that the major role of the circadian oscillator in this species could be a stabilizing function under non-rhythmic environmental conditions.

Kilden synes ikke at omhande unge men forskning om unge. Sørg derfor for at dobbeltchecke at det rent faktisk er kilden (fremfor een af kildens kilder) der pointerer hvad I refererer.

Det er lidt diffust hvad jeres substantielle kapitel handler om.

Undertitlerne indikerer at kapitlet omhandler jeres valg af teori og metode, men hvor jeg i et sådant kapitel ville forvente en fremadrettet præsentation af planer og intentioner med jeres valg af tilgang til projektet, synes teksten indholdsmæssigt i højere grad at rumme en reflektion af erfaringer med anvendelsen af teori og metoder. Hvis kapitlet er ment som præsentation fremadrettet så virker det distraherende at det er skrevet i datid, og bør nok begrænses til at omhandle planer (ikke også erfaringer).

Omvendt, hvis det er et evaluerende kapitel, så vil det nok være en god idé at indikere det tydeligere i dets overskrifter, og tydeligere knytte jeres vurderinger om succes (eksempelvis det direkte citerede om at det var "særligt værdifuldt") direkte på jeres teorier og metoder og planer.

Hvordan har det været gavnligt? Vi savner empiriske eksempler.

Der er mange henvisninger til metoden og hvordan I har brugt den, men der er ingen koblinger mellem metoden og teorien og empiriske eksempler fra jeres interviews. Overvej at skrive kapitlet med større fokus på empiriske eksempler.

Det lader tid at designprocessen er central for jeres projekt. Overvej om D&K kunne være mere relevant end STS

TRIN-modellen er ikke en metode

Something something.

Die heftigen Regenfälle im Südosten Frankreichs, ein sogenannter "épisode méditerranéen", werden nicht ausreichen, um die Grundwasservorräte im französischen Department Pyrénées Orientales wieder aufzufüllen, die sich durch eine zweijährige Trockenheit extrem verringert haben. https://www.liberation.fr/environnement/episode-mediterraneen-un-phenomene-precoce-insuffisant-pour-les-nappes-phreatiques-des-pyrenees-orientales-20240430_BQACSFW2ZNAMLE2WYVH5DNNEYY/

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In den Ländern, die sich in Paris 2015 einer Initiative gegen das Verbrennen von nicht genutztem Erdgas (flaring) angeschlossen hatten, wird das Verbrennen mit offener Flamme oft nur durch Verbrennung in geschlossenen Anlagen ersetzt, wie eine investigative journalistische Recherche ergab. Die Menge der Emissionen sinkt dadurch nicht wesentlich, aber diese Anlagen sind für Satelliten nicht äußerlich erkennbar. https://www.theguardian.com/environment/2024/may/02/methane-emissions-gas-flaring-hidden-satellite-monitors-oil-gas

Ressourcen für die Recherche zu Methan-Emissionen: https://gijn.org/resource/new-tools-investigate-methane-emissions/

This not necessarily points out that The specific examples of a polished slab are figure A and D. At first I read it as those figures were examples of meteorites, in general.. You may add a sentence to say something like "Examples of a polished slides are shown in figures A and D.

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for - SRG complexity mapping - possible candidate

severless ml systems categories

how interactive ML systems operate

how analytical ML systems operate

Author response:

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

eLife assessment

This study presents a valuable contribution to cardiac arrhythmia research by demonstrating long noncoding RNA Dachshund homolog 1 (lncDACH1) tunes sodium channel functional expression and affects cardiac action potential conduction and rhythms. Whereas the evidence for functional impact of lncDACH1 expression on cardiac sodium currents and rhythms is convincing, biochemical experiments addressing the mechanism of changes in sodium channel expression and subcellular localization are incomplete.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this study, the authors show that a long-non coding RNA lncDACH1 inhibits sodium currents in cardiomyocytes by binding to and altering the localization of dystrophin. The authors use a number of methodologies to demonstrate that lncDACH1 binds to dystrophin and disrupts its localization to the membrane, which in turn downregulates NaV1.5 currents. Knockdown of lncDACH1 upregulates NaV1.5 currents. Furthermore, in heart failure, lncDACH1 is shown to be upregulated which suggests that this mechanism may have pathophysiolgoical relevance.

Strengths:

(1) This study presents a novel mechanism of Na channel regulation which may be pathophysiologically important.

(2) The experiments are comprehensive and systematically evaluate the physiological importance of lncDACH1.

Weaknesses:

(1). What is indicated by the cytoplasmic level of NaV1.5, a transmembrane protein? The methods do not provide details regarding how this was determined. Do you authors means NaV1.5 retained in various intracellular organelles?

Thank you for the good suggestion. Our study showed that Nav1.5 was transferred to the cell membrane by the scaffold protein Dystropin in response to the regulation of LncDACH1, but not all Nav1.5 in the cytoplasm was transferred to the cell membrane. Therefore, the cytoplasmic level of Nav1.5 represents the Nav1.5 protein that is not transferred to the cell membrane but stays in the cytoplasm and various organelles within the cytoplasm when Nav1.5 is regulated by LncDACH1

(2) What is the negative control in Fig. 2b, Fig. 4b, Fig. 6e, Fig. 7c? The maximum current amplitude in these seem quite different. -40 pA/pF in some, -30 pA/pF in others and this value seems to be different than in CMs from WT mice (<-20 pA/pF). Is there an explanation for what causes this variability between experiments and/or increase with transfection of the negative control? This is important since the effect of lncDACH1 is less than 50% reduction and these could fall in the range depending on the amplitude of the negative control.

Thank you for the insightful comment. The negative control in Fig. 2b, Fig. 4b, Fig. 6e are primary cardiomyocytes transfected with empty plasmids. The negative control in Fig.7c are cardiomyocytes of wild-type mice injected with control virus. When we prepare cells before the patch-clamp experiments, the transfection efficiency of the transfection reagent used in different batches of cells, as well as the different cell sizes, ultimately lead to differences in CMS.

(3) NaV1.5 staining in Fig. 1E is difficult to visualize and to separate from lncDACH1. Is it possible to pseudocolor differently so that all three channels can be visualized/distinguished more robustly?

Thank you for the good suggestion. We have re-added color to the original image to distinguish between the three channels.

Author response image 1.

(4) The authors use shRNA to knockdown lncDACH1 levels. It would be helpful to have a scrambled ShRNA control.

Thank you for the insightful comment. The control group we used was actually the scrambled shRNA, but we labeled the control group as NC in the article, maybe this has caused you to misunderstand.

(5) Is there any measurement on the baseline levels of LncDACH1 in wild-type mice? It seems quite low and yet is a substantial increase in NaV1.5 currents upon knocking down LncDACH1. By comparison, the level of LncDACH1 seems to be massively upregulated in TAC models. Have the authors measured NaV1.5 currents in these cells? Furthermore, does LncDACH1 knockdown evoke a larger increase in NaV1.5 currents?

Thank you for the insightful comment.

(1).The baseline protein levels of LncDACH1 in wild-type mice and LncDACH1-CKO mice has been verified in a previously published article(Figure 3).(Hypertension. 2019;74:00-00. DOI: 10.1161/HYPERTENSIONAHA.119.12998.)

Author response image 2.

(2). We did not measure the Nav1.5 currents in cardiomyocytes of the TAC model mice in this artical, but in another published paper, we found that the Nav1.5 current in the TAC model mice was remarkably reduced than that in wild-type mice(Figure 4).(Gene Ther. 2023 Feb;30(1-2):142-149. DOI: 10.1038/s41434-022-00348-z)

Author response image 3.

This is consistent with our results in this artical, and our results show that LncDACH1 levels are significantly upregulated in the TAC model, then in the LncDACH1-TG group, the Nav1.5 current is significantly reduced after the LncDACH1 upregulation(Figure 3).

Author response image 4.

(6) What do error bars denote in all bar graphs, and also in the current voltage relationships?

Thank you for the good comment. All the error bars represent the mean ± SEM. They represent the fluctuation of all individuals of a set of data based on the average value of this set of data, that is, the dispersion of a set of data.

Reviewer #2 (Public Review):

This manuscript by Xue et al. describes the effects of a long noncoding RNA, lncDACH1, on the localization of Nav channel expression, the magnitude of INa, and arrhythmia susceptibility in the mouse heart. Because lncDACH1 was previously reported to bind and disrupt membrane expression of dystrophin, which in turn is required for proper Nav1.5 localization, much of the findings are inferred through the lens of dystrophin alterations.

The results report that cardiomyocyte-specific transgenic overexpression of lncDACH1 reduces INa in isolated cardiomyocytes; measurements in whole heart show a corresponding reduction in conduction velocity and enhanced susceptibility to arrhythmia. The effect on INa was confirmed in isolated WT mouse cardiomyocytes infected with a lncDACH1 adenoviral construct. Importantly, reducing lncDACH1 expression via either a cardiomyocyte-specific knockout or using shRNA had the opposite effect: INa was increased in isolated cells, as was conduction velocity in heart. Experiments were also conducted with a fragment of lnDACH1 identified by its conservation with other mammalian species. Overexpression of this fragment resulted in reduced INa and greater proarrhythmic behavior. Alteration of expression was confirmed by qPCR.

The mechanism by which lnDACH1 exerts its effects on INa was explored by measuring protein levels from cell fractions and immunofluorescence localization in cells. In general, overexpression was reported to reduce Nav1.5 and dystrophin levels and knockout or knockdown increased them.

Thank you for summarizing our work and thank you very much for your appreciation on our work.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors report the first evidence of Nav1.5 regulation by a long noncoding RNA, LncRNA-DACH1, and suggest its implication in the reduction in sodium current observed in heart failure. Since no direct interaction is observed between Nav1.5 and the LncRNA, they propose that the regulation is via dystrophin and targeting of Nav1.5 to the plasma membrane.

Strengths:

(1) First evidence of Nav1.5 regulation by a long noncoding RNA.

(2) Implication of LncRNA-DACH1 in heart failure and mechanisms of arrhythmias.

(3) Demonstration of LncRNA-DACH1 binding to dystrophin.

(4) Potential rescuing of dystrophin and Nav1.5 strategy.

Thank you very much for your appreciation on our work.

Weaknesses:

(1) Main concern is that the authors do not provide evidence of how LncRNA-DACH1 regulates Nav1.5 protein level. The decrease in total Nav1.5 protein by about 50% seems to be the main consequence of the LncRNA on Nav1.5, but no mechanistic information is provided as to how this occurs.

Thank you for the insightful comment.

(1) The mechanism of the whole article is as mentioned in the discussion at the end of the article: LncDACH1 binds to dystrophin and thus inhibits membrane trafficking of Nav1.5, Dystrophin is a well-characterized Nav1.5 partner protein. It indirectly interacts with Nav1.5 via syntrophin, which binds with the C-terminus of dystrophin and with the SIV motif on the C-terminus of Nav1.5(Circ Res. 2006;99:407-414. doi: 10.1161/01.RES.0000237466.13252.5e)(Circulation.2014;130:147-160.doi:10.1161/CIRCULATIONAHA.113.007852).

And we performed pulldown and RNA immunoprecipitation experiments to verify it (Figure 1).

Author response image 5.

2) Then we found that overexpression of lncDACH1 increased the ubiquitination of Nav1.5, which explains the downregulation of total Nav1.5 protein (Online Supplementary Figure 12).

Author response image 6.

3). Lastly,we found that lncDACH1 failed to pulldown Nav1.5 and anti-Nav1.5 did not precipitate lncDACH1( Supplementary Fig. 1).

Author response image 7.

These data indicated that lncDACH does not interact with Nav1.5 directly. It participates in the regulation of Nav1.5 by binding to dystrophin.Cytoplasmic Nav1.5 that failed to target on plasma membrane may be quickly distinguished and then degraded by these ubiquitination enzymes.

(2) The fact that the total Nav1.5 protein is reduced by 50% which is similar to the reduction in the membrane reduction questions the main conclusion of the authors implicating dystrophin in the reduced Nav1.5 targeting. The reduction in membrane Nav1.5 could simply be due to the reduction in total protein.

Thank you for the insightful comment. We do not rule out the possibility that the reduction in membrane Nav1.5 maybe be due to the reduction in total protein, but we don't think this is the main mechanism. Our data indicates that the membrane and total protein levels of Nav1.5 were reduced by 50%. However, the cytoplasmic Nav1.5 increased in the hearts of lncDACH1-TG mice than WT controls rather than reduced like membrane and total protein(Figure 1).

Author response image 8.

Therefore, we think the mian mechanism of the whole article is as mentioned in the discussion at the end of the article: LncDACH1 binds to dystrophin and thus inhibits membrane trafficking of Nav1.5.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) In Fig. 6E the error bars are only in one direction for cF-lncDACH1. It seems that this error overlaps for NC and cF-lncDACH1 at several voltages, yet it is marked as statistically significant. Also in Fig. 7C, what statistical test was used? Do the authors account for multiple comparisons?

Thank you for the insightful comment.

(1) We have recalculated the two sets of data and confirmed that there are indeed statistically significant between the two sets of data for NC and cF-lncDACH1 at In Fig. 6E, The overlaps in the picture may only be visually apparent.

(2) The data in Fig. 7C are expressed as mean ± SEM. Statistical analysis was performed using unpaired Student’s t test or One-Way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc analysis.

(2) line 57, "The Western blot" remove "The"

Sorry for the mistake. We have corrected it.

(3) line 61, "The opposite data were collected" It is unclear what is meant by opposite.

Sorry for the mistake. We have corrected it.

(4) Lines 137-140. This sentence is complex, I would simplify as two sentences.

Sorry for the mistake. We have corrected it.

(5) Line 150, "We firstly validated" should be "we first validated"

Sorry for the mistake. We have corrected it.

(6) Line 181, "Consistently, the membrane" Is this statement meant to indicate that the experiments yielded a consistent results or that this statement is consistent with the previous one? In either case, this sentence should be reworded for clarification.

Sorry for the mistake. We have corrected it.

(7) Line 223, "In consistent, the ex vivo" I am not sure what In consistent means here.

Thank you for the good suggestion. We mean that the results of ex vivo is consistent with the results of in vivo. We have corrected it to make it clearer.

(8) Line 285. "a bunch of studies" could be rephrased as "multiple studies"

Sorry for the mistake. We have corrected it.

(9) Line 299 "produced no influence" Do you mean produced no change?

Thank you for the good suggestion.As you put it,we mean it produced no change.

(10) Line 325 "is to interact with the molecules" no need for "the molecules

Sorry for the mistake. We have corrected it.

(11) lines 332-335. This sentence is very confusing.

Thank you for the insightful comment. We have corrected it.

(12) Lines 341-342. It is unnecessary to claim primacy here.

Thank you for the good suggestion. We have removed this sentence.

(13) Line 373. "Sodium channel remodeling is commonly occured in" perhaps rephrase as occurs commonly

Thank you for the insightful comment. We have corrected it.

Reviewer #2 (Recommendations For The Authors):

Critique

(1) Aside from some issues with presentation noted below, these data provide convincing evidence of a link between lncDACH1 and Na channel function. The identification of a lncDACH1 segment conserved among mammalian species is compelling. The observation that lncDACH1 is increased in a heart failure model and provides a plausible hypothesis for disease mechanism.

Thank you very much for your appreciation on our work.

(2) Has a causal link between dystrophin and Na channel surface expression has been made, or is it an argument based on correlation? Is it possible to rule out a direct effect of lncDACH1 on Na channel expression? A bit more discussion of the limitations of the study would help here.

Thank you for the insightful comment.

(1). Dystrophin is a well-characterized Nav1.5 partner protein. It indirectly interacts with Nav1.5 via syntrophin, which binds with the C-terminus of dystrophin and with the SIV motif on the C-terminus of Nav1.5(Circ Res. 2006;99:407-414. doi: 10.1161/01.RES.0000237466.13252.5e)(Circulation.2014;130:147-160.doi:10.1161/CIRCULATIONAHA.113.007852).

Author response image 9.

(2).we performed pulldown and RNA immunoprecipitation experiments. The data showed that lncDACH1 failed to pulldown Nav1.5 and anti-Nav1.5 did not precipitate lncDACH1 (Online Supplementary Figure 11). These data indicated that lncDACH does not interact with Nav1.5 directly. ( Supplementary Fig. 1)

Author response image 10.

(3) What normalization procedures were used for qPCR quantification? I could not find these.

Thank you for the good suggestion.The expression levels of mRNA were calculated using the comparative cycle threshold (Ct) method (2−ΔΔCt). Each data point was then normalized to ACTIN as an internal control in each sample. The final results are expressed as fold changes by normalizing the data to the values from control subjects. We have added the normalization procedures in the methods section of the article.

(4) In general, I found the IF to be unconvincing - first, because the reported effects were not very apparent to me, but more importantly, because only exemplars were shown without quantification of a larger sample size.

Thank you for the good suggestion. Accordingly, we quantified the immunostaining data. The data have been included in Supplementary Figure 2- 16.The sample size is labeled in the caption.

Author response image 11.

Fluorescence intensity of lncDACH1, dystrophin and Nav1.5 in isolated cardiomyocytes of lncDACH1-TG mice. a,b, Membrane levels of dystrophin (dys) and Nav1.5. N=9 for dys. N=8 for Nav1.5. P<0.05 versus WT group. c,d, Cytoplasm levels of dystrophin and Nav1.5. N=9. P<0.05 versus WT group. e, Fluorescence in situ hybridization (FISH) images of LncDACH1. N=10. *P<0.05 versus WT group. P-values were determined by unpaired t test.

Author response image 12.

Fluorescence intensity of dystrophin and Nav1.5 in cultured neonatal cardiomyocyte overexpressing lncDACH1. a,b, Membrane levels of dystrophin and Nav1.5. N=9. P<0.05 versus NC group. c,d, Cytoplasm levels of dystrophin and Nav1.5. N=9 for dys. N=12 for Nav1.5. P<0.05 versus NC group. P-values were determined by unpaired t test.

Author response image 13.

Fluorescence intensity of lncDACH1, dystrophin and Nav1.5 in isolated cardiomyocytes of lncDACH1-cKO mice. a,b, Membrane levels of dystrophin (dys) and Nav1.5. N=12 for dys. N=8 for Nav1.5. P<0.05 versus WT group. c,d, Distribution of cytoplasm levels of dystrophin and Nav1.5. N=12. P<0.05 versus WT group. e, Fluorescence in situ hybridization (FISH) images of LncDACH1 expression. N=8. *P<0.05 versus WT group. P-values were determined by unpaired t test.

Author response image 14.

Fluorescence intensity of dystrophin and Nav1.5 in cultured neonatal cardiomyocytes after knocking down of lncDACH1. a,b, Distribution of membrane levels of dystrophin and Nav1.5. N=11 for dys. N=8 for Nav1.5.P<0.05 versus NC group. c,d, Distribution of cytoplasm levels of dystrophin and Nav1.5. N=12 for dys. N=9 for Nav1.5.P<0.05 versus NC group. P-values were determined by unpaired t test.

Author response image 15.

Fluorescence intensity of dystrophin and Nav1.5 in isolated cardiomyocytes overexpressing cF-lncDACH1. a,b, Membrane levels of dystrophin (dys) and Nav1.5. N=9 for dys. N=7 for Nav1.5. P<0.05 versus NC group. c,d, Cytoplasm levels of dystrophin and Nav1.5. N=6 for dys. N=7 for Nav1.5. P<0.05 versus NC group. P-values were determined by unpaired t test.

Author response image 16.

Fluorescence intensity of dystrophin and Nav1.5 in cultured neonatal cardiomyocytes overexpressing cF-lncDACH1. a,b, Membrane levels of dystrophin and Nav1.5. N=10 for dys. N=11 for Nav1.5. P<0.05 versus NC group. c,d, Cytoplasm levels of dystrophin and Nav1.5. N=7 for dys. N=6 for Nav1.5.P<0.05 versus NC group. P-values were determined by unpaired t test.

Author response image 17.

Fluorescence intensity of Nav1.5 in human iPS differentiated cardiomyocytes overexpressing cF-lncDACH1. a, Membrane levels of Nav1.5. N=8 for Nav1.5. P<0.05 versus NC group. b, Cytoplasm levels of Nav1.5. N=10 for Nav1.5.P<0.05 versus NC group. P-values were determined by unpaired t test.

(5) More information on how the fractionation kit works would be helpful. How are membrane v. cytoplasm fractions identified?

a. I presume the ER is part of the membrane fraction? When Nav1.5 is found in the cytoplasmic fraction, what subcompartment is it in - the proteasome?

b. In the middle panel of A - is the dystrophin signal visible on the WB for WT? I assume the selected exemplar is the best of the blots and so this raises concerns. Much is riding on the confidence with which the fractions report "membrane" v "cytoplasm."

Thank you for the insightful comment.

(1). How the fractionation kit works:

The kit utilizes centrifuge column technology to obtain plasma membrane structures with native activity and minimal cross-contamination with organelles without the need for an ultracentrifuge and can be used for a variety of downstream assays. Separation principle: cells/tissues are sensitized by Buffer A, the cells pass through the centrifuge column under the action of 16000Xg centrifugation, the cell membrane is cut to make the cell rupture, and then the four components of nucleus, cytoplasm, organelle and plasma membrane will be obtained sequentially through differential centrifugation and density centrifugation, which can be used for downstream detection.

Author response image 18.

(2). How are membrane v. cytoplasm fractions identified:

The membrane proteins and cytosolic proteins isolated by the kit, and then the internal controls we chose when performing the western blot experiment were :membrane protein---N-cadherin cytosolic protein---β-Actin

Most importantly, when we incubate either the primary antibody of N-cadherin with the PVDF membrane of the cytosolic protein, or the primary antibody of the cytosolic control β-Actin with the PVDF membrane of the membrane protein, the protein bands cannot be obtained in the scan results

Author response image 19.

(6) More detail in Results, figures, and figure legends will assist the reader.

a. In Fig. 5, it would be helpful to label sinus rhythm vs. arrhythmia segments.

Thank you for the good suggestion. We've marked Sinus Rhythm and Arrhythmia segments with arrows

Author response image 20.

b. Please explain in the figure legend what the red bars in 5A are

Thank you for the insightful comment. We've added the explanation to the figure legend .The red lines in the ECG traces indicate VT duration.

c. In 5C, what the durations pertain to.

Thank you for the good suggestion. 720ms-760ms refers to the duration of one action potential, with 720ms being the peak of one action potential and 760ms being the peak of another action potential.The interval duration is not fixed, in this artical, we use 10ms as an interval to count the phase singularities from the Consecutive phase maps. Because the shorter the interval duration, the larger the sample size and the more convincing the data.

d. In the text, please define "breaking points" and explain what the physiological underpinning is. Define "phase singularity."

Thank you for the insightful comment. Cardiac excitation can be viewed as an electrical wave, with a wavefront corresponding to the action potential upstroke (phase 0) and a waveback corresponding to rapid repolarization (phase 3). Normally, Under normal circumstances, cardiac conduction is composed of a sequence of well-ordered action potentials, and in the results of optical mapping experiments, different colors represent different phases.when a wave propagates through cardiac tissue, wavefront and waveback never touch.when arrhythmias occur in the heart, due to factors such as reenfrant phenomenon, the activation contour will meet the refractory contour and waves will break up, initiating a newly spiral reentry. Corresponding to the optical mapping result graph, different colors representing different time phases (including depolarization and repolarization) come together to form a vortex, and the center of the vortex is defined as the phase singularity.

(7) In reflecting on why enhanced INa is not proarrhythmic, it is noted that the kinetics are not altered. I agree that is key, but perhaps the consequence could be better articulated. Because lncDACH1 does not alter Nav1.5 gating, the late Na current may not be enhanced to the same effect as observed with LQT gain-of-function Nav1.5 mutations, in which APD prolongation is attributed to gating defects that increase late Na current.

Thank you for the good suggestion. Your explanation is very brilliant and important for this article. We have revised the discussion section of the article and added these explanations to it.

Reviewer #3 (Recommendations For The Authors):

(1) Experiments to specifically address the reduction in total Nav1.5 protein should be included.

Thank you for the insightful comment. We examined the ubiquitination of Nav1.5. We found that overexpression of lncDACH1 increased the ubiquitination of Nav1.5, which explains the downregulation of total Nav1.5 protein (Online Supplementary Figure 12).

Author response image 21.

(2) Experiments to convincingly demonstrate that LncRNA-DACH1 regulates Nav1.5 targeting via dystrophin are missing. As it is, total reduction in Nav1.5 seems to be the explanation as to why there is a decrease in membrane Nav1.5.

Thank you for the insightful comment. we performed pulldown and RNA immunoprecipitation experiments. The data showed that lncDACH1 can pulldown dystrophin(Figure 1),but failed to pulldown Nav1.5 and anti-Nav1.5 did not precipitate lncDACH1( Supplementary Fig. 1). These data indicated that lncDACH does not interact with Nav1.5 directly. It participates in the regulation of Nav1.5 by binding to dystrophin.

Author response image 22.

for - nondual coach

Boa tarde,

este é um aspeto a meu ver importantíssimo. As atividades deverão ter sempre em conta que, acima de tudo, têm como objetivo ajudar os alunos a aprender, e reter informação, e deverão ser sempre desenvolvidas com este propósito, e de o fazerem da forma mais eficaz possível.

Saudações, David Vinagreiro