Heinz bodies

TMP-SMX, fava bean, dapsone, primaquine, moth balls

heme is source of bilirubin, which is probably source of elevated unconjugated bilirubin.

high AST and normal ALT: RBCs have AST but not ALT inside them, so this represents more RBC breakdown as opposed to liver damage (both would be elevated).

higher reticulocyte=hemolysis

need LDH, bilirubin, haptoglobin, blood smear, Coombs test (looking for antibodies stuck to RBC), DAT (direct= RBCs have antibiotics already attached), IAT (indirect=Ab in plasma)

summary: this case is a 62-year old male with a history of urinary tract trouble and anemic episode, presenting with dysuria, fever, CVA tenderness, with concern for urinary tract infection.

look for stones or prostatic obstruction

mechanical obstruction can lead to increased infection risk

Africa is in the American subconscious despite limited knowledge. It appears in media, creating stereotypes, like tribes, famine, and wild animals, shaping perceptions based on assumptions, not facts.

This sentence is an example specifically of the typical media stereotype of Africa. It lines up with the most common movies that integrate Africa into them. The "hot, dangerous, and tribal" part of it.

This part of the text shows that many Americans have Africa as a subconscious idea in their head. This is mainly because of the things they've been told through media but also because of school not exactly teaching them completely. I believe that if schools would talk about the development of Africa, people wouldn't categorize Africa with the words used in the text. This is an example of many different popular shortcomings made about Africa. The only reason these terms are associated with Africa still is because people in America haven't took the time to learn about actually understanding Africa so how can it be taught? That's why it still persists even today.

What society/the news shows and tells us.

I did not know that Africa was this big. That is a very interesting fact.

This part of the text is a media stereotype because people get put an image of Africa into their mind where the only things that happen in Africa are bad, like how newspapers are about aids, genocide, Ebola, or civil war.

Can make up if absent, in-class/group work

because we can do anytime, no absence leinency

Given in lab sections, 3 midterms, no final

Can do any time

great

great

十分な

dreaming hypothesis: dreams are like being mad because what is thought to be perceived is not real

(4 946)

The spacing should be the same as above/below (it is currently larger)

At point i (Immobilisation) we did a link to note 13. Therefore, either remove this reference in point i (Immobilisation corporelles) or a a reference to note 6 in paragraph N Incertitude relative à la mesure to be consistant…. I don't have a preference. i let you choose. Please apply same approach to english version

Est-ce possible de mettre le titre "Valeur comptable nette" en haut (au-dessus des années) pour qu'il soit là une fois et de laisser les années en bas comme dans le PDF?

Maison-Laurier with a dash between Maison and Laurier

Descriptive and prescriptive grammar often clash instead of collaborating. Descriptive grammar explains how people actually speak/write; whereas, prescriptive grammar dictates how people should speak/write.

I use descriptive grammar in my everyday writing but i also try to be prescriptive as well.

Add link to note 6

The spacing is larger than it should be (refer to note n)

The spacing is larger than it should be (refer to note n)

The spacing is larger than it should be (refer to note n)

The spacing is larger than it should be (refer to note n)

The spacing is larger than it should be (refer to note n)

The spacing is larger than it should be (refer to note n)

The spacing is larger than it should be (refer to note n)

Shouldn't be in bold

1,374,290

Note to Hugues: Revoir à la fin s'assurer que le lien apporte à la note 13 (Actuellement la note sur les immo. indique note 14)

Silly Things To Laugh About

Things We Dreamt Of Seeing As Kids

Simple, Wholesome Things To Be Happy About

Unusual Facts That Aren't True But Are Worth Being Happy For

Small Characters To Be Happy About

Cartoon Worlds We Would Love To Live In

Things To Celebrate Every Day

Everyday Moments To Be Happy About

Board & Card Games To Play On A Rainy Day

The statement in this paragraph about commercials remains the same today. The ads distributed during the shows are often related to the type of program being broadcast. For example, if the show is for an adult audience, the ads will be aimed at promoting products for adults.

In this initial paragraph, Williams explains that while the categories of broadcasting shows are essential, developers of entertainment need to understand that the flow is most relevant in the distribution of the programs.

A claim of policy is the easiest claim to spot,

I didn't know this. I will definitely try to gamify my resume now.

Ideas are useful to share.

Colloquial means it is informal, but in ordinary conversation.

Everybody creates creative work based on people's work. Nothing is really new.

Using a template is not plagiarism as long as the details are added my own words and proper credit has been given.

A person who criticize, objects to, or opposes something.

A text not yet published. When I am starting to write something, it helps to use template.

A picture of exaggeration. Audiences don't like when I insult the other side of the argument.

A person who blamed for wrongdoing, mistakes, or fault for others.

Regard or represent as being a little worth and insulting.

If no one disagrees, then I am stating the obvious. That is boring.

The common belief is arguing against everybody.

The page says that I don't have to argue against famous person. It can be anyone including myself.

The page says that I don't have to argue against famous person. It can be anyone including myself.

It is not necessarily disagreeing, but it is building upon argument.

Contrast makes easier to see including topic for argument.

Giving a basis for opinion.

It's important to listen and acknowledge others.

The templates will help practice, but not automatically making a good writer.

For practice, I am using templates to create muscle memory.

Template is a tool for easily replicating writing.

Writing is like muscle memory.

How do you show that the shape of E is the same in the past? Do you just look at it, or is there some algorithm? Slide 17.

Clarify?

Why monthly? Is monthly variability low for each snow station?

Clarify Rossby waves.

Like, r = sqrt(x^2 + y^2) distance?

Confused on this terminology.

Large-scale climate pattern that connects distant places on Earth.

Celebrity Luxury Treatment To Be Happy About

Moments As Sweet As Candy

Red Carpet Luxuries To Be Happy About

Cosy Things To Do On A Snow Day

Cheesecake Recipes To Be Happy About

Cosy Comforts To Be Happy About

Ways To Bring The Outside In

Ways To Make Bedtime More Soothing & Relaxing

Imaginative Fantasies To Journal About

Feel Good Movies To Be Happy About

Creative Ways To Banish Boredom

Ways To Recover From A Long, Hard Sleep

Luxuries To Experience When You're In The Bath

Ways To Make Life Comfy

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Reply to the reviewers

I have already provided a document with a point-by-point response. I do not wish to re-format all of the text again in this HTML box. The document I provided can be published as it is.

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Referee #2

Evidence, reproducibility and clarity

Summary:

This study demonstrates an improved integral gene drive (IGD) for use in Anopheles gambiae. Inserting the coding sequence for Cas9 in-frame with a germline-specific gene (nanos) improved the performance of this IGD relative to previously reported systems while reducing fitness costs. Integration of the gRNA cassette within a synthetic intron is an elegant solution to constraining the minimal elements of the IGD within a single insertion. The results of this study found that while the IGD can be used to propagate anti-malarial effectors (MM-CP) within a population, fitness costs and resistance alleles were higher than anticipated, potentially limiting the application of this particular IGD design without further optimisation.

The results comprehensively demonstrate the effective transmission and stability of the IGD over several generations, while also characterising the limitations of the system. Although I don't think the authors make any claims which are not supported by their results. It might be good to provide more of an explanation for how the performance of this IGD compares to the zpg IGD reported in Ellis et al 2022 for readers less familiar with the IGD literature.

The manuscript is overall very well written with clear results and methods. However, I found the descriptions referring to the effects of the maternal, paternal, and even grandmaternal inheritance hard to follow. The statistical analysis and replications are adequate as well.

Referee cross-commenting

I agree with the other reviewer's comments regarding the need to clarify a few points made in the overall well written manuscript.

Significance

Gene drives are the most promising genetic biocontrol method for controlling the spread of malaria. However, there are many technical challenges that have made the development of gene drives quite difficult. This study works to address one such challenge - constraining the expression of Cas9 to the germline by integrating it within an endogenous loci rather than using semi-synthetic promoters. While IGD have been demonstrated before, this study further improves on their performance while reducing off-target effects.

The manuscript is written for a highly specialized audience that is very familiar with the genetic biocontrol, and especially the gene-drive field of research.

My fields of research include genetic biocontrol and insect synthetic biology.

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Referee #1

Evidence, reproducibility and clarity

In this study, the authors develop a complete integral drive system in Anopheles gambiae malaria mosquitoes. This type of gene drive is interesting, with special advantages and disadvantages compared to more common designs. Here, the authors develop the Cas9 element and combine it with a previously developed antimalaria effector element. The new element performs very well in terms of drive efficiency, but it has unintended fitness costs, and a higher than desirable rate of functional resistance allele formation. Nevertheless, this study represents a very good step forward toward developing effective gene drives and is thus of high impact.

The format of the manuscript is a bit suboptimal for review. Please add line numbers next time for easy reference. It would also help to have spaces between paragraphs and to have figures (with legends) added to the text where they first appear.

It might be useful to add subsections to the results, just like in the methods section. It could even be expanded a bit with some specific parts from the discussion, through this is optional.

Abstract: The text says: "As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations." However, it does seem to change things based on Figure 3c.

Page 2: "drive technologies for public health and pest control applications" needs a period afterward.

Page 2: "The fitness costs, homing efficiency, and resistance rate of the gene drive is" should be "The fitness costs, homing efficiency, and resistance rate of the gene drive are".

Page 2: "When they target important mosquito genes, gene drives are designed to ensure that the nuclease activity window (germline) does not overlap with that of the target gene (somatic)." is note quite correct. This is, of course, sensible for suppression drives, but it's not a necessary requirement for modification drives with rescue elements in many situations.

Page 2: "recessive somatic fitness cost phenotypes" is unclear. I think that you are trying to avoid the recessive fitness cost of null alleles becoming a dominant fitness cost from a gene drive allele (in drive-wild-type heterozygotes).

Page 2: "This optimization approach has had only limited success, and suboptimal performance is commonly attributed to not capturing all the regulatory elements specific to the germline gene's expression9,12". I don't think this is correct. There are several examples where a new promoter helped a lot. The zpg promoter in Anopheles gambiae allowed success at the dsx site in suppression cage studies (Kyrou et al 2018), and nanos gave big improvement to modification drives at the cardinal locus (Carballer et al 2020). In flies, several promoters were tested, and one allowed success in cage experiments (Du et al 2024). In Aedes, the shu promoter allowed for high drive performance (Anderson et al 2023), though this last one hasn't been tested in more difficult situations. I think you could certainly argue in the general case that not all promoters will work the way their transcriptome says, but there are many examples where they seem to be pretty good.

Page 2: "make it more likely that mutations that disrupt the drive components are selected against though loss of function of the host gene." I think that this needs a bit more explanation. You are referring to mutations in regulatory elements or frameshift mutations. This will make it more resistant to mutation. Also, these mutations would tend to have a minor effect expect perhaps in the cargo gene of a modification drive. By using a cargo gene in an integral drive, you could still keep it somewhat safer, but whether this is 1.2x or 10x safer is unclear.

Page 3: "they can incur severe unintended fitness costs". This is central to integral drives and this manuscript. It's worth elaborating on.

Page 3: "Regulatory elements from germline genes that have worked sub-optimally in traditional gene drive designs for the reasons outlined above may work well in an IDG design20." This is setting up the integral drive with nanos, but nanos DOES work well in traditional Anopheles gambiae gene drive designs. It is possible that you might end up with less somatic expression than Hammond et al 2020 (though the comparison is unclear due to batch effects in that study), but there is no direct comparison in this manuscript to that.

Page 3: "This suggests an impact of maternal deposition on drive efficiency only in female drive carriers." This is quite strange. Usually, I would expect to see an equal reduction in efficiency between male and female progeny. Could this be due to limited sample size? Random idea: It's also possible that almost all maternal deposition was mosaic and wouldn't be enough to direct affect drive conversion. However, it could cause enough of a fitness cost TOGETHER with new drive expression in females that perhaps only tissues with randomly low expression rates properly developed and led to progeny, reducing drive inheritance? Another possibility: Could the drive/resistance males have impaired fertility, so these ones are underrepresented in the batch cross? If nanos is needed in males and a single drive copy is not quite enough for good fertility or mating competitiveness, they may be underrepresented in your crosses. They might have worse fertility than drive homozygous males, which at least have two partially working copies of nanos rather than just one (in many cells, at least). Maybe check the testis for abnormal phenotypes?

Overall, it would be favorable if the drive allele was somewhere more fit than a nonfunctional resistance allele. This could already be achieved in this drive, but it doesn't get much mention.

Page 3: There should be a comma after, "Interestingly, while many of the observed mutations were predicted to abolish nanos expression" and "This could indicate that in these experiments".

Page 3 last sentence: Please improve the clarity.

Removing the EGFP is supposed to restore the fitness, and this was helpful in some previous integral drive constructs. This could get a bit more mention (it is possible that I missed this somewhere in the manuscript).

Page 4: The MM-CP line and it's association with the integral drive strategy could get a little more introduction. Maybe even a supplemental figure showing the general idea.

Page 5: "cassette is predicted to disrupt the CP function entirely (Fig. 5d)" also lacks a period.

Page 5: "The subsequent stabilization of the nanosd frequency and the lack of rapid loss suggests that any associated fitness cost is primarily recessive." This is not quite correct because by this point, drive/wild-type heterozygotes are rare, and this is where you'd find a potential dominant fitness cost. It should be correct in the end stages where it is a mix of drive and functional/nonfunctional resistance alleles (though the nonfunctional resistance alleles may cause greater fitness costs when together with a drive - see above).

Page 6: "Maternal deposition of Cas9, or Cas9;gRNA, into the zygote can lead to cutting at stages when homing is not favoured, and has been commonly observed for canonical Anopheles nanos drives9,10,35." Reference 35 (which is more suitable for referencing an example of nanos in other Anopheles) found some resistance alleles by deep sequencing, but the timing that they formed was unclear (it's not certain if it was maternal deposition). This study may be a more suitable reference: Carballar-Lejarazú R, Tushar T, Pham TB, James AA. Cas9-mediated maternal-effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae. Genetics, 2022.

Page 8: "could further reduce the likelihood of resistance allele formation by increasing the frequency of HDR events." Multiple gRNAs would mostly help by reducing functional resistance allele formation, especially since drive conversion is already very high in Anopheles.

Page 8, last paragraph: This conclusion is perhaps a little optimistic considering the functional resistance alleles, which should get a little more attention in the summary or elsewhere in the discussion section.

Figure 1d: The vertical text saying "Non-WT" is confusing. The circles themselves show + and -. Also, "-" isn't necessarily a knockout allele, so I'm not sure if - is the best symbol for resistance.

Figure 2e: The vertical scale is not the most intuitive. Consider rearranging it to "Transition from larvae to pupae" starting at zero and going to 1 when all the larvae become pupae.

Figure 2e-f: For both of these, there are clear differences between males and females. Thus, when comparing drive homozygotes to wild-type, it would probably be better to have separate statistical comparisons for males and females.

Figure 3: Can any of these transcription results in individual genes potentially explain the observed fitness cost?

Figure 3b: The scale here also doesn't quite make sense. It's the fraction of underdeveloped ovaries, but the graph is also perhaps trying to show whether just 1-2 ovaries are present, or maybe how many ovaries are undeveloped, but then it would say "zero"? This should be clarified. Number of ovaries and how well-developed they are is separate (it can be put on the same graph, but needs to be more clear).

Figure 4f: The vertical axis should say "ONNV."

Figure 5c-d: These should be labeled as the most common resistance allele. Also, I'm not sure how relevant it is, but we also found an alternate start codon here: Hou S, Chen J, Feng R, Xu X, Liang N, Champer J. A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance. J Genet Genomics, 2024. Maybe this is a more common problem than one would expect?

Figure 5cd,S4,S5: They have a bit of a weird plot. Why not make four line graphs for each? Also, some alleles use the  symbol. + is wild-type, which is well understood, but - as resistance is not always clear, and seeing them together may confuse readers. Additionally, the fact that you have the most common resistance allele in Figure 5cd might mean that you know more about the genotype? If so, it would be best to separate wild-type and resistance alleles in whatever the final figure looks like.

Some supplemental raw data files would be useful if they were available, but the figures are through enough that this isn't essential.

Review by:

Jackson Champer, with major assistance from Ruobing Feng (essentially section B) and Jie Du

Referee cross-commenting

We don't have any cross-comments, other than supporting the idea of slightly more comparisons to the authors' previous construct.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

A key innovation of the nanosd gene drive is its integral gene drive (IGD) design, which inserts the drive cassette directly into the A. gambiae nanos gene, incorporating only the minimal components necessary for drive function. The drive achieves high transmission rates, without causing widespread disruption of gene expression or increasing susceptibility to malaria parasites, and imposes an acceptable fitness cost-primarily a reduction in female fecundity when homozygous. The strong performance of nanosd can be attributed to its design: Cas9 is expressed in the correct cells and timing to induce efficient homing, effectively hijacking the nanos gene's natural expression profile. However, despite the careful design aimed at preserving nanos function, the rescue was incomplete: homozygous female drive carriers exhibited a clear reduction in ovarian function.

In caged population trials, both the drive and a co-introduced anti-malaria effector gene reached high frequencies, even in the presence of emerging resistance alleles. Because the drive is inserted into an essential gene, nonfunctional resistance alleles are selected against and tend to be purged over time. Nonetheless, functional resistance remains a concern. The use of a single, though precisely positioned gRNA targeting the native nanos gene ATG site increases the likelihood of generating functional resistance alleles. Over the long term, if the drive imposes fitness costs, it may be outcompeted by such functional resistance alleles, potentially undermining the goal of sustained population modification.

Overall, this study represent a notable advance in Anopheles mosquito gene drive development and can be considered as high impact. - Place the work in the context of the existing literature (provide references, where appropriate).

Previous IGD efforts in Drosophila, mice and mosquitoes have demonstrated nearly super‐Mendelian inheritance but often at the expense of host fitness. For example, Nash et al. built an intronic‐gRNA Cas9 drive at the D. melanogaster rcd-1r locus that propagated efficiently through cage populations (Nash et al., 2022), and Gonzalez et al. reported that a Cas9 drive inserted at the germline zpg locus in Anopheles stephensi biased inheritance by ~99.8% (Gonzalez et al., 2025). However, these strong drives disrupted essential genes: in A. gambiae, inserting Cas9 into zpg produced efficient homing but rendered females largely sterile (Ellis et al., 2022). A similar germline Cas9 knock-in in Mus musculus enabled gene conversion in both sexes, albeit with only modest efficiency and potential fitness trade-offs (Weitzel et al., 2021). The current nanosd IGD is explicitly designed to overcome this limitation by selecting a more permissive gene target and using a minimal drive cassette, so as to preserve mosquito viability while maintaining robust drive efficiency, although still with reduced female drive homozygotes fertility.

This nanosd gene drive like previous homing drives in Anopheles, is capable of achieving a high level of inheritance bias. Although it uses the endogenous nanos regulatory elements, which have less leaky somatic expression compared to using vasa (Gantz et al., 2015; Hammond et al., 2016; Hammond et al., 2017) or zpg promoters(Hammond et al., 2021; Kyrou et al., 2018), to drive Cas9 expression and thereby reduces somatic expression-induced female sterility, the incomplete rescue of nanos function still leads to reduced female fertility in drive homozygotes. - State what audience might be interested in and influenced by the reported findings.

It's worth noting the broad audience that will find this work relevant. Gene drive developers and molecular geneticists will be impressed by the good drive performance and directly influenced by the design principles showcased here. The study's integral gene drive architecture that leverages the endogenous nanos regulatory elements, in-frame E2A peptide linkage for co-expression, and intronic insertion of gRNA and selectable markers addresses long-standing challenges in promoter leakage, somatic fitness costs, and resistance allele evolution. What's more, vector biologists and malaria researchers will be interested in the successful deployment of a gene drive in A. gambiae that actually carries a disease-blocking trait. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

We have worked on CRISPR gene drive development in both fruit flies and Anopheles mosquitoes and have experience with modeling their spread.

References

Ellis, D.A., Avraam, G., Hoermann, A., Wyer, C.A.S., Ong, Y.X., Christophides, G.K., and Windbichler, N. (2022). Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector Anopheles gambiae. PLOS Genetics 18, e1010244-e1010244.

Gantz, V.M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V.M., Bier, E., and James, A.A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc Natl Acad Sci U S A 112, E6736-E6743.

Gonzalez, E., Anderson, M.A.E., Ang, J.X.D., Nevard, K., Shackleford, L., Larrosa-Godall, M., Leftwich, P.T., and Alphey, L. (2025). Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi. Scientific Reports 15, 13408.

Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., Gribble, M., Baker, D., Marois, E., Russell, S., et al. (2016). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34, 78-83.

Hammond, A., Karlsson, X., Morianou, I., Kyrou, K., Beaghton, A., Gribble, M., Kranjc, N., Galizi, R., Burt, A., Crisanti, A., et al. (2021). Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance. PLOS Genetics 17, e1009321-e1009321.

Hammond, A.M., Kyrou, K., Bruttini, M., North, A., Galizi, R., Karlsson, X., Kranjc, N., Carpi, F.M., D'Aurizio, R., Crisanti, A., et al. (2017). The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLOS Genetics 13, e1007039-e1007039.

Kyrou, K., Hammond, A.M., Galizi, R., Kranjc, N., Burt, A., Beaghton, A.K., Nolan, T., and Crisanti, A. (2018). A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature Biotechnology 36, 1062-1066.

Nash, A., Capriotti, P., Hoermann, A., Papathanos, P.A., and Windbichler, N. (2022). Intronic gRNAs for the construction of minimal gene drive systems. Frontiers in Bioengineering and Biotechnology 0, 570-570. Weitzel, A.J., Grunwald, H.A., Ceri, W., Levina, R., Gantz, V.M., Hedrick, S.M., Bier, E., and Cooper, K.L. (2021). Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline. Plos Biol 19, e3001478-e3001478.

What are Goodwill's competitive advantages?

Ways To Make Your Living Lots Of Fun

Luxuries To Experience In A Hotel

Exciting Things To Do On Fun National Days

Ways To Feel Happy About Life

Thunderbirds Fantasies To Be Happy About

you should take a trip to lake chargoggagoggmanchauggagoggchaubunagungamaugg

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Magnificent Rooms To Be Happy About

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Fun Things To Do On An Australian Road Trip

eLife Assessment

This work characterizes the function and localization of SLC4A1 variants associated with distal renal tubular acidosis in human patients. Cell culture and limited animal studies provide partial but incomplete support to the authors' claim that the variants disrupt normal protein degradative flux by alkalinizing the intracellular pH. The study is valuable in providing preliminary evidence for future exploration of the link between intracellular pH regulation by SLC4A1 and kidney cell function in vivo.

Reviewer #1 (Public review):

Summary:

This study is an evaluation of patient variants in the kidney isoform of AE1 linked to distal renal tubular acidosis. Drawing on observations in the mouse kidney, this study extends findings to autophagy pathways in a kidney epithelial cell line.

Strengths:

Experimental data are convincing and nicely done.

Weaknesses:

Some data are lacking or not explained clearly. Mutations are not consistently evaluated throughout the study, which makes it difficult to draw meaningful conclusions.

Reviewer #2 (Public review):

Context and significance:

Distal renal tubular acidosis (dRTA) can be caused by mutations in a Cl-/HCO3- exchanger (kAE1) encoded by the SLC4A1 gene. The precise mechanisms underlying the pathogenesis of the disease due to these mutations are unclear, but it is thought that loss of the renal intercalated cells (ICs) that express kAE1 and/or aberrant autophagy pathway function in the remaining ICs may contribute to the disease. Understanding how mutations in SLC4A1 affect cell physiology and cells within the kidney, a major goal of this study, is an important first step to unraveling the pathophysiology of this complex heritable kidney disease.

Summary:

The authors identify a number of new mutations in the SLC4A1 gene in patients with diagnosed dRTA that they use for heterologous experiments in vitro. They also use a dRTA mouse model with a different SLC4A1 mutation for experiments in mouse kidneys. Contrary to previous work that speculated dRTA was caused mainly by trafficking defects of kAE1, the authors observe that their new mutants (with the exception of Y413H, which they only use in Figure 1) traffic and localize at least partly to the basolateral membrane of polarized heterologous mIMCD3 cells, an immortalized murine collecting duct cell line. They go on to show that the remaining mutants induce abnormalities in the expression of autophagy markers and increased numbers of autophagosomes, along with an alkalinized intracellular pH. They also reported that cells expressing the mutated kAE1 had increased mitochondrial content coupled with lower rates of ATP synthesis. The authors also observed a partial rescue of the effects of kAE1 variants through artificially acidifying the intracellular pH. Taken together, this suggests a mechanism for dRTA independent of impaired kAE1 trafficking and dependent on intracellular pH changes that future studies should explore.

Strengths:

The authors corroborate their findings in cell culture with a well-characterized dRTA KI mouse and provide convincing quantification of their images from the in vitro and mouse experiments.

Weaknesses:

The data largely support the claims as stated, with some minor suggestions for improving the clarity of the work. Some of the mutants induce different strengths of effects on autophagy and the various assays than others, and it is not clear why this is from the present manuscript, given that they propose pHi and the unifying mechanism.

Reviewer #3 (Public review):

Summary:

The authors have identified novel dRTA causing SLC4A1 mutations and studied the resulting kAE1 proteins to determine how they cause dRTA. Based on a previous study on mice expressing the dRTA kAE1 R607H variant, the authors hypothesize that kAE1 variants cause an increase in intracellular pH, which disrupts autophagic and degradative flux pathways. The authors clone these new kAE1 variants and study their transport function and subcellular localization in mIMCD cells. The authors show increased abundance of LC3B II in mIMCD cells expressing some of the kAE1 variants, as well as reduced autophagic flux using eGFP-RFP-LC3. These data, as well as the abundance of autophagosomes, serve as the key evidence that these kAE1 mutants disrupt autophagy. Furthermore, the authors demonstrate that decreasing the intracellular pH abrogates the expression of LC3B II in mIMCD cells expressing mutant SLC4A1. Lastly, the authors argue that mitochondrial function, and specifically ATP synthesis, is suppressed in mIMCD cells expressing dRTA variants and that mitochondria are less abundant in AICs from the kidney of R607H kAE1 mice. While the manuscript does reveal some interesting new results about novel dRTA causing kAE1 mutations, the quality of the data to support the hypothesis that these mutations cause a reduction in autophagic flux can be improved. In particular, the precise method of how the western blots and the immunofluorescence data were quantified, with included controls, would enhance the quality of the data and offer more supportive evidence of the authors' conclusions.

Strengths:

The authors cloned novel dRTA causing kAE1 mutants into expression vectors to study the subcellular localization and transport properties of the variants. The immunofluorescence images are generally of high quality, and the authors do well to include multiple samples for all of their western blots.

Weaknesses:

Inconsistent results are reported for some of the variants. For example, R295H causes intracellular alkalinization but also has no effect on intracellular pH when measured by BCECF. The authors also appear to have performed these in vitro studies on mIMCD cells that were not polarized, and therefore, the localization of kAE1 to the basolateral membrane seems unlikely, based upon images included in the manuscript. Additionally, there is no in vivo work to demonstrate that these kAE1 variants alter intracellular pH, including the R607H mouse, which is available to the authors. The western blots are of varying quality, and it is often unclear which of the bands are being quantified. For example, LAMP1 is reported at 100kDa, the authors show three bands, and it is unclear which one(s) are used to quantify protein abundance. Strikingly, the authors report a nonsensical value for their quantification of LCRB II in Figure 2, where the ratio of LCRB II to total LCRB (I + II) is greater than one. The control experiments with starvation and bafilomyocin are not supportive and significantly reduce enthusiasm for the authors' findings regarding autophagy. There are labeling errors between the manuscript and the figures, which suggest a lack of vigilance in the drafting process.

When going through the principles of clear directions we can see a list of things that seem useful. I believe that in the video we can see examples that would seem to translate in real life quite well.

When looking at the section labeled "register" it is interesting to see what truly goes into it. The difference between casual and urgent it quite clear. I would like to see the difference in urgent for a first year teacher and a highly expericned teacher.

Substance

Halloween Things To Be Happy About

Fun Ways To Keep Fit & Healthy On Halloween

'80s Cartoons To Be Happy About

Kids' Action Show Characters That Might Become Your Friends

Things To Do Indoors When It Rains On Halloween Night

'80s Toys To Be Nostalgic About

Soothing Camping Activities To Be Happy About

Books For Kids To Read & Be Inspired By

Soothing Things To Do At A Slumber Party

Fun Things You Can Do To Stay Up Late

Lonely Places Where You Can Think

Echoes Of The Past To Make You Nostalgic

MeTV Toons Characters To Be Happy About

Cosy Camping Moments To Make You Happy

Slumber Party Moments To Be Happy About

Friendliest TV & Book Characters To Be Happy About

Teatime Treats To Be Happy About

Specials & Shows From Your Childhood To Be Happy About

Cheesy Dishes To Be Happy About

This shows how some people take technology and blames it for things that happens in society. I feel like the definition itself doesn't really target if it could be a good thing or bad thing. some people might feel like technological determinism is good because it helped Society evolve, while others might say its ruining society because its making humans more dependent on technology. Just like anything I feel like it has its pros and cons. Technogly comes from humans and evolution to make modern day things more easy to do.

A type of grape

supprimer

maybe we can just plug in models of various scales and see how the performance project?

I guess this is needed when generator-verifier gap exists?

hard domain-specific constraints

important

important

• This is a reference to Harriet Beecher Stowe's Uncle Tom's Cabin, which was a prominent abolitionist text. Here, Harris is suggesting that it was actually a kind portrait of slavery.

Maybe: https://www.fltimes.com/lifestyle/pompey-smash-a-freed-slave-in-waterloo/article_a2f9cca0-cb6b-11e5-b708-ff1e1eb6e597.html

Cartoon Dogs To Be Happy About

Alcohol Free Substitutes To Think About

Cereals We Have Yet To Have

Moments Of Softness Around Animals

Ways To Spend Time With Your Favourite Friends

Things To Do When You Feel Sick

Classic Characters To Be Happy About

Things You Can Make Out Of Food

Characters You'd Wish To Sleep With

Calm Things To Take Your Mind Off Noisy Sports Games

Things To Think About When You're Hungry

Most Unlikely Friendships

Ways To Be Like Sonic

يتكم الجزء اني بقدر اعمل متغيرات داخل Class تكون public واستدعيها في blade مثل ما يوضح في الصورة

لما تعمل صفحة جديدة بكون في ملفات افتراضية يتم انشائها اذا بدي اخصص الملف اول ما اعملو شو يكون داخلو بنفذ الكوماند المرفق وبعدل ببساطة الملفات الافتراضية لانشاء الملفات

اذا حذفت render function تلقائيا livewire رح تبحث علي اول view يتطابق اسمه مع اسم Control وتبعثو للواجهة

لما تعمل Component بضمن html داخل نفس Class يعني بنشاء ملف واحد في App/Livewire وما بعمل ملف داخل view

نستطيع من خلالها تحديد اي layout نريد تمرير البيانات له

return view('livewire.posts.index') ->layout('components.layouts.app', ['title' => 'Latest Posts']); // تمُرِّر بيانات للّياوت أيضًا

كوماند مخصص لانشاء Component ينتج عن تنفيد هذا الكوماند ملفين

CLASS: app/Livewire/Counter.php المنطق VIEW: resources/views/livewire/counter.blade.php الواجهة

اذا بدي اعمل الملفات داخل مجلد

Ways To Feel Warm & Cosy

Luxurious Things That Feel Like Spa Treatment

Things That Feel Like A Gentle, Loving Touch

Moments Of Gentle, Loving Softness

Moments Of Cosy Softness

It's interesting how these criticisms of mass society emerged right before postmodernism, which had a major focus on challenging universal truths and messages. I wonder if this is the research community's response to modernism. Does the film industry ever respond to what comes out of related research fields?

Things to Think About When You Listen To Peaceful Music

Places To Visit Around Town

Most Unlikely Sources Of Inspiration

Fun Things To Do At Easter

It's so difficult to talk to someone with differing views on fundamental topics like gender views or political topics because people tend to be very stubborn about their personal beliefs.

Good definition of critical reading, writing, and thinking. Critical thinking is often mentioned, but rarely defined.

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eLife Assessment

This study presents the important finding that lysosomal damage triggers inflammatory signaling through ubiquitination and the TAB-TAK1-IKK-NF-kB axis. The data obtained from the unbiased transcriptomic and proteomic analyses are convincing and provide invaluable information to the field. Although further experiments will be required to clarify how TAB2/3 are recruited after various types of lysosome damage, this work will be of interest to researchers in the fields of organelle biology and inflammation.

Reviewer #1 (Public review):

Summary:

Lysosomal damage is commonly found in many diseases including normal aging and age-related disease. However, the transcriptional programs activated by lysosomal damage has not been thoroughly characterized. This study aims to investigate lysosome damage-induced major transcriptional responses and the underlying signaling basis. The authors have convincingly shown that lysosomal damage activates a ubiquitination-dependent signaling axis involving TAB, TAK1, and IKK, which culminate in the activation of NF-kB and subsequent transcriptional upregulation of pro-inflammatory genes and pro-survival genes. Overall, the major aims of this study are successfully achieved.

Strengths:

This study is well-conceived and strictly executed, leading to clear and well-supported conclusions. Through unbiased transcriptomics and proteomics screens, the authors identifies NF-kB as a major transcriptional program activated upon lysosome damage. TAK1 activation by lysosome damage-induced ubiquitination is found to be essential for NF-kB activation and MAP kinase signaling. The transcriptional and proteomic changes are shown to be largely driven by TAK1 signaling. Finally, the TAK1-IKK signaling is shown to provide resistance to apoptosis during lysosomal damage response. The main signaling axis of this pathway has been convincingly demonstrated.

Overall, this study identifies major transcriptional responses following lysosomal damage through unbiased approaches. It is important to consider the impact of these pathways in disease settings where lysosomal integrity is compromised.

Comments on revisions:

The authors have adequately addressed all previous comments. I have no further recommendations.

Reviewer #2 (Public review):

Summary:

Endo et al. investigate the novel role of ubiquitin response upon lysosomal damage in activating cellular signaling for cell survival. The authors provide a comprehensive transcriptome and proteome analysis of aging-related cells experiencing lysosomal damage, identifying transcription factors involved in transcriptome and proteome remodeling with a focus on the NF-κB signaling pathway. They further characterized the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis in controlling gene expression, inflammatory responses, and apoptotic processes.

Strengths:

In the aging-related model, the authors provide a comprehensive transcriptome and characterize the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis. Through compelling experiments and advanced tools, they elucidate its critical role in controlling gene expression, inflammatory responses, and apoptotic processes.

Weaknesses:

The study lacks deeper connections with previous research, particularly:

• The established role of TAB-TAK1 in AMPK activation during lysosomal damage

• The potential significance of TBK1 in NF-κB signaling pathways

Comments on revisions:

The authors have successfully addressed all the raised questions and the manuscript is now significantly improved.

Reviewer #3 (Public review):

Summary:

The response to lysosomal damage is a fast-moving and timely field. Besides repair and degradation pathways, increasing interest has been focusing on damaged-induced signaling. The authors conducted both transcriptomics and proteomics to characterize the cellular response to lysosomal damage. They identify a signaling pathway leading to activation of NFkappaB. Based on this and supported by Western blot and microscopy data, the authors nicely show that TAB2/3 and TAK1 are activated at damaged lysosomes and kick off the pathway to alter gene expression, which induces cytokines and protect from cell death. TAB2/3 activation is proposed to occur through K63 ubiquitin chain formation. Generally, this is a careful and well conducted study that nicely delineates the pathway under lysosomal stress. The "omics" data serves a valuable resource for the field. More work should be invested into how TAB2/3 are activated at the damaged lysosomes, also to increase novelty in light of previous reports.

Strengths:

Generally, this is a careful and well-conducted study that nicely delineates how the NFkB pathway is activated under lysosomal stress and modulates cell behavior. The "omics" data serves as a valuable resource for the field.

Weaknesses:

While activation of TAB2/3 by K63-linked Ub chains is convincing, more work needs to be done on how they are recruited by distinct damage types to probe relevance for different pathophysiological conditions."

Comments on revisions:

The authors have addressed much of my criticism. Specifically, they have put (with new experiments) the data on the TAB2/3-TAK1 pathway in perspective to the previously reported LUBAC-mediated activation of NFkB. They also addressed the question about the significance of K63-linked chains for TAB2/3 activation with new complementation experiments (a K63-specific NZF mutant failed to rescue).

The third point (types of damage as triggers) raises more questions, though. The authors find that, in contrast to LLOMe, GPN or DC661-induced damage does not activate TAK1 (consistent with lower damage levels). However, the authors still observe K63 ubiquitylation. This goes along with their finding that TAB2 is recruited in the absence of any ubiquitylation (blocked by TAK-243). It argues that TAB2 is recruited by an unknown cue (that may be damage-specific) and then activated by K63. The authors need to clarify whether TAB2 is or is not recruited in the GPN/DC661 conditions (in which K63 occurs, but TAK1 is not activated). The point about the effects of other damage types was also raised by reviewer #1 and should be solved. The fact that TAB2 is recruited independently of K63 should also be visualized in the model. The manuscript will then be an important contribution to the field.

Author response:

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

Reviewer #1 (Public review):

Summary:

Lysosomal damage is commonly found in many diseases including normal aging and age-related disease. However, the transcriptional programs activated by lysosomal damage have not been thoroughly characterized. This study aimed to investigate lysosome damage-induced major transcriptional responses and the underlying signaling basis. The authors have convincingly shown that lysosomal damage activates a ubiquitination-dependent signaling axis involving TAB, TAK1, and IKK, which culminates in the activation of NF-kB and subsequent transcriptional upregulation of pro-inflammatory genes and pro-survival genes. Overall, the major aims of this study were successfully achieved.

Strengths:

This study is well-conceived and strictly executed, leading to clear and well-supported conclusions. Through unbiased transcriptomics and proteomics screens, the authors identified NF-kB as a major transcriptional program activated upon lysosome damage. TAK1 activation by lysosome damage-induced ubiquitination was found to be essential for NF-kB activation and MAP kinase signaling. The transcriptional and proteomic changes were shown to be largely driven by TAK1 signaling. Finally, the TAK1-IKK signaling was shown to provide resistance to apoptosis during lysosomal damage response. The main signaling axis of this pathway was convincingly demonstrated.

Weaknesses:

One weakness was the claim of K63-linked ubiquitination in lysosomal damage-induced NF-kB activation. While it was clear that K63 ubiquitin chains were present on damaged lysosomes, no evidence was shown in the current study to demonstrate the specific requirement of K63 ubiquitin chains in the signaling axis being studied. Clarifying the roles of K63-linked versus other types of ubiquitin chains in lysosomal damage-induced NF-kB activation may improve the mechanistic insights and overall impact of this study.

Another weakness was that the main conclusions of this study were all dependent on an artificial lysosomal damage agent. It will be beneficial to confirm key findings in other contexts involving lysosomal damage.

We would like to thank Reviewer #1 for the positive and constructive comments on our study. For a main concern regarding the molecular mechanism by which TAB proteins are activated in response to lysosomal damage, we have added the experimental results to support that the lysosomal accumulation of K63 ubiquitin chains serves as a trigger to activate the TAB-TAK1 pathway. We also investigated and discussed the role of LUBAC-mediated M1 ubiquitin chains in NF-kB activation and the effects of other lysosomal-damaging compounds. Please see the response to “Reviewer #3 (Public review): Suggestions:”.

Reviewer #2 (Public review):

Summary:

Endo et al. investigate the novel role of ubiquitin response upon lysosomal damage in activating cellular signaling for cell survival. The authors provide a comprehensive transcriptome and proteome analysis of aging-related cells experiencing lysosomal damage, identifying transcription factors involved in transcriptome and proteome remodeling with a focus on the NF-κB signaling pathway. They further characterized the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis in controlling gene expression, inflammatory responses, and apoptotic processes.

Strengths:

In the aging-related model, the authors provide a comprehensive transcriptome and characterize the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis. Through compelling experiments and advanced tools, they elucidate its critical role in controlling gene expression, inflammatory responses, and apoptotic processes.

Weaknesses:

The study lacks deeper connections with previous research, particularly:

• The established role of TAB-TAK1 in AMPK activation during lysosomal damage

• The potential significance of TBK1 in NF-κB signaling pathways

We would like to thank Reviewer #2 for the helpful comments on our study. To achieve a more comprehensive understanding of the signaling pathways involved in the lysosomal damage response, we investigated additional related signal mediators, such as TBK1 and LUBAC. The citations related to AMPK have been incorporated.

Reviewer #3 (Public review):

Summary:

The response to lysosomal damage is a fast-moving and timely field. Besides repair and degradation pathways, increasing interest has been focusing on damaged-induced signaling. The authors conducted both transcriptomics and proteomics to characterize the cellular response to lysosomal damage. They identify a signaling pathway leading to activation of NFkappaB. Based on this and supported by Western blot and microscopy data, the authors nicely show that TAB2/3 and TAK1 are activated at damaged lysosomes and kick off the pathway to alter gene expression, which induces cytokines and protect from cell death. TAB2/3 activation is proposed to occur through K63 ubiquitin chain formation. Generally, this is a careful and well conducted study that nicely delineates the pathway under lysosomal stress. The "omics" data serves as a valuable resource for the field. More work should be invested into how TAB2/3 are activated at the damaged lysosomes, also to increase novelty in light of previous reports.

Strengths:

Generally, this is a careful and well-conducted study that nicely delineates the pathway under lysosomal stress. The "omics" data serves as a valuable resource for the field.

Weaknesses:

More work should be invested into how TAB2/3 are activated at the damaged lysosomes, also to increase novelty in light of previous reports. Moreover, different damage types should be tested to probe relevance for different pathophysiological conditions.

We would like to thank Reviewer #3 for the valuable comments on our study. We have added the experimental results to address two concerns raised by Reviewer #3. Please see the response to “Reviewer #3 (Public review): Suggestions:”.

Suggestions:

(1) A recent paper claims that NFkappaB is activated by Otulin/M1 chains upon lysosome damage through TBK1 (PMID: 39744815). In contrast, Endo et al. nicely show that ubiquitylation is needed (shown by TAK-243) for NFkB activation but only have correlative data to link it specifically to K63 chains. On page 15, line 11, the authors even argue a "potential" involvement of K63. This point should be better dealt with. Can the authors specifically block K63 formation? K63R overexpression or swapping would be one way. Is the K63 ligase ITCH involved (PMID: 38503285) or any other NEDD4-like ligase? This could be compared to LUBAC inhibition. Also, the point needs to be dealt with more controversially in the discussion as these are alternative claims (M1 vs K63, TAB vs TBK1).

It is well-characterized that the NZF domain of TAB proteins preferentially associates with K63-linked ubiquitin chains. Therefore, we performed the add-back experiment using siRNA-resistant TAB2 WT and mutants incapable of binding to K63-linked ubiquitin chains, dNZF and E685A, to elucidate the requirement of K63 ubiquitin chains for TAK1 activation. We investigated whether the add-back of TAB2 mutants rescues the activation of TAK1 in TAB2-depleted cells (Fig. 2E). TAB2 WT, but not dNZF and E685A, rescued TAK1 activation in response to LLOMe, suggesting that the specific interaction of TAB proteins and K63 ubiquitin chains is a key mechanism to activate TAK1. We also found that the treatment of an E1 inhibitor TAK-243 effectively prevented the lysosomal accumulation of K63 ubiquitin chains, but TAB2 was recruited to damaged lysosomes (Fig. S2B). This suggests that the recruitment of TAB proteins to damaged lysosomes is independent of the association with K63 ubiquitin chains. Collectively, it is postulated that TAB proteins require interaction with K63 ubiquitin chains for TAK1 activation, but not for recruitment to damaged lysosomes. We have added the sentences (p9, lines 7-20, and p10, lines 8-10).

Next, we confirmed that LUBAC functions are essential for NF-kB activation in the lysosomal damage response. RNF31/HOIP is a component of LUBAC that catalyzes M1 ubiquitination. The depletion of RNF31 showed no significant effects on TAK1 activation, but abolished IKK activation (Fig. S4G). It is well-characterized that LUBAC-mediated M1 ubiquitin chains recruit IKK subunits and transduce the signaling to downstream in the canonical pathway. We assume that K63 ubiquitin chains in damaged lysosomes initially activate TAB-TAK1 and trigger LUBAC-mediated M1 ubiquitination, and subsequently, M1 ubiquitination functions to recruit the IKK complex. Consequently, activated TAK1 phosphorylates IKK subunits in damaged lysosomes, leading to NF-kB activation. We also examined whether TBK1 is involved in the activation of NF-kB. TBK1 was phosphorylated upon LLOMe, and depletion of TAB and TAK1 resulted in a slight reduction of TBK1 phosphorylation (Fig. S4D, E). The treatment of a TBK1 inhibitor BX-795 exhibited no or little effects on TAK1 activation, but abolished phosphorylation of IKK and IkBa (Fig. S4F). These suggest that TBK1 is required for the activation of NF-kB. We have added the sentences (p13, line 13-p14, line 10).

As mentioned by Reviewer #3, it is important to identify the E3 ligase responsible for K63 ubiquitination in the lysosomal damage response. We have been aiming to identify such E3 ligase(s). However, depletions of ITCH and other E3 ligases that have been tested exhibited no or little effects on K63 ubiquitination and TAK1 activation.  We would like to explore E3 ligase(s) in future study.

(2) It would be interesting to know what the trigger is that induces the pathway. Lipid perturbation by LLOMe is a good model, but does activation also occur with GPN (osmotic swelling) or lipid peroxidation (oxidative stress) that may be more broadly relevant in a pathophysiological way? Moreover, what damage threshold is needed? Does loss of protons suffice? Can activation be induced with a Ca2+ agonist in the absence of damage?

To further clarify the initial trigger that induces TAB-TAK1 activation coupled with lysosomal damage, we examined other damage sources, GPN and DC661, which induce hyperosmotic stress and lipid peroxidation in lysosomes, respectively, thereby resulting in lysosomal membrane damage. Under our experimental conditions, the treatment of these compounds did not result in significant accumulation of Gal-3, indicating a reduced level of lysosomal membrane permeabilization compared with LLOMe (Fig. S2C, D), and no or little TAK1 activation was observed (Fig. S2E). TAB proteins require their association with K63 ubiquitin chains for TAK1 activation. It is therefore postulated that the severe lysosomal membrane permeabilization that triggers the formation and cytosolic exposure of K63 ubiquitin chains may be a determinant of TAB-TAK1 activation. In our future work, we would like to examine broad stimulation of lysosomal damage and further elucidate the initial mechanism of TAB-TAK1 activation. We have added the sentences (p9, line 21-p10, line 7).

(3) The authors nicely define JNK and p38 activation. This should be emphasized more, possibly also in the abstract, as it may contribute to the claim of increased survival fitness.

We further tested whether the inhibition of JNK affects the anti-apoptotic effect (Fig. S5B). The inhibition of JNK resulted in an increase in the cleaved caspase-3. This suggests that the anti-apoptotic action in the lysosomal damage response requires JNK as well as IKK. We have added the sentences in results to emphasize the pivotal role of stress-induced MAPKs (p15, lines 7-11).

Reviewer #1 (Recommendations for the authors):

(1) Although the ubiquitination-TAB-TAK1-IKK axis was previously characterized in other contexts, specific evidence supporting lysosomal recruitment of these components by ubiquitination during lysosome damage would be beneficial.

We found that the treatment of an E1 inhibitor TAK-243 abolished the lysosomal accumulation of K63 ubiquitin chains, but TAB2 and TAK1 were recruited to damaged lysosomes (Fig. S2B). This suggests that the recruitment of TAB proteins to damaged lysosomes is independent of the association with K63-linked ubiquitin chains. Next, we investigated whether the add-back of TAB2 mutants incapable of binding K63 ubiquitin chains rescues the activation of TAK1 in TAB2-depleted cells (Fig. 2E). K63 ubiquitin binding of TAB2 was essential for TAK1 activation in response to LLOMe. Taken together, it is suggested that TAB proteins require their interaction with K63 ubiquitin chains for TAK1 activation, but not for recruitment to damaged lysosomes. We have added the sentences (p9, lines 7-20, and p10, lines 8-10). Please also see the response to “Reviewer #3 (Public review): Suggestions:”.

(2) The activation of p38 and JNK by lysosomal damage does not fit well into the main conclusions of the paper, since IKK knockdown was sufficient to block cellular resistance to apoptosis (caspase cleavage in Fig. 5f). Are p38 and JNK also important for cell survival during lysosomal damage?

We found that the inhibition of JNK resulted in an increase in the cleaved caspase-3, suggesting that the anti-apoptotic action in the lysosomal damage response requires both IKK and JNK (Fig. S5B). We have added the sentences (p15, lines 7-11).

(3) Cell death tests are recommended to support the conclusions related to apoptosis.

As suggested by Reviewer #1, we performed the cell death assay using propidium iodide (PI) and confirmed that HeLa cells co-treated with LLOMe and TAK-243 or HS-276 exhibited increased cell death (Fig. 5E). This indicates a direct correlation between the degree of caspase-3 cleavage and cell death, possibly apoptosis.

(4) Page 8, line 19-21, gal3 is not exposed upon lysosomal damage. It is recruited from the cytosol by the exposed beta-galactoside-containing glycans on lysosomal membrane proteins.

We have corrected the corresponding sentence (p7, lines 17-20).

(5) Carefully checking grammar throughout the text is recommended. Below are a few examples:

a) Page 4, line 10, remove "that".

b) "K63 ubiquitin" shall be replaced with "K63 ubiquitination" or "K63 ubiquitin chains".

c) Page 8, line 9, "remain" should be "remains".

We have carefully checked the revised manuscript.

Reviewer #2 (Recommendations for the authors):

Despite the novelty and significance of these findings in advancing the field, several technical and experimental limitations require further clarification:

We have responded to each comment. Please see below.

The manuscript should introduce or discuss previous research showing that TAB-TAK1 facilitates AMPK activation during lysosomal damage and TAK1's increased association with damaged lysosomes (PMID: 31995728).

We have added the reference (PMID: 31995728) and the sentences (p17, lines 15-20).

Figure 2A: The differential LAMP1 staining intensity between control and LLOMe-treated cells needs explanation. The weaker LAMP1 signal in control and puncta changes, especially during 5-minute LLOMe treatment, require detailed clarification

We have added the explanation (p8, lines 17-21).

Recent literature (PMID: 34585663) reports TBK1 activation during lysosomal damage. The authors should investigate or discuss whether TBK1 potentially contributes to NF-κB signaling in this context.

We experimentally investigated whether TBK1 is involved in the TAB-TAK1 pathway. We confirmed that TBK1 was activated upon LLOMe (Fig. S4D). Depletions of TAB and TAK1 exhibited a modest decrease in TBK1 phosphorylation (Fig. S4E). The inhibition of TBK1 by BX-795 did not affect TAK1 activation, but abolished phosphorylation of IKK and IkBa (Fig. S4F). This suggests that TBK1 is required for NF-kB activation. We have added the reference (PMID: 34585663) and the sentences (p13, lines 13-21, p14, lines 8-10, and p18, lines 15-20).

The introduction of lysosomal damage response lacks comprehensive mechanistic information. For example, while ESCRT is discussed, other critical mechanisms such as lipid transfer and stress granule formation in lysosomal repair should be incorporated. Moreover, mTOR and AMPK signaling pathways undergo significant changes upon lysosomal damage.

We have added the sentences (p3, lines 16-18, and p3, line 21-p4, line 1).

The statement "lysosomal permeabilization causes the dissociation of mTORC1 from lysosomes" should explicitly reference PMID: 29625033.

We have added the suggested reference (PMID: 29625033, p4, line 19).

The claim that "The elimination of damaged lysosomes through lysophagy requires a period of more than half a day" needs a specific publication citation.

We have added the reference (PMID: 23921551) to claim the time-scale of lysosomal clearance (p4, line 21).

Figure 1G: The label "WO after 2h" lacks explanation in the figure legend and requires detailed interpretation.

To simplify the figures, we have deleted the label “WO after 2 h” (Fig. 1G, 3F, 5D, F-J, S4G, S5A). Instead, we have added the explanation in the figure legends (Fig. 1G).

Reviewer #3 (Recommendations for the authors):

(1) page 8, line 13: it is recommended to phrase colocalisation "at" damaged lysosomes rather than "in" damaged lysosomes as the resolution does not allow the claim of influx into lysosomes.

We have corrected the word (p8, line 17).

(2) page 11, line 22: why is "whereas" used to link two events driven by the same mechanism.

We have corrected the word (p13, line 8).

تصمیم

eLife Assessment

This important work describes the adaptation and evaluation of two red-shifted anion channelrhodopsins (RubyACRs) for optogenetic inhibition in Drosophila. The study provides convincing evidence for the effectiveness of RubyACRs in fly neurons, including electrophysiology, calcium imaging, and behavioral analysis. With minor revisions to address potential toxicity and compatibility with 2-photon imaging, this paper and the publicly available fly lines it describes will be resources that are of value to the neuroscience community.

Reviewer #1 (Public review):

Summary:

This study by Bushey et al., focuses on two newly released red-shifted anion-Channelrhodopsins (A1ACR and HfACR, referred as Ruby-ACRs) in Drosophila. Here, the authors use a combination of electrophysiology, calcium imaging, and behavioral analyses to demonstrate the advantages of Ruby-ACRs over previous optogenetic silencers like the green-shifted GtACR1 and the blue-shifted GtACR2: higher photocurrent, faster kinetics, and operating at a light spectrum range that prevents unwanted behavioral effects in the fly. The availability of these new red-shifted silencers constitutes a great addition to the Drosophila genetic toolkit.

Strengths:

(1) The authors generate both UAS and LexAop RubyACR reagents and test them in a variety of preparations (electrophysiological recordings, calcium imaging, different behavioral paradigms) that cover the breadth of the fly research environment.

(2) The optical stimulation parameters are carefully measured and characterized. Especially impressive is that they managed to titrate over both wavelength and intensity across their various assays. This provides a comprehensive dataset to the community.

(3) Tools are made available to the community through the stock center.

Weaknesses:

(1) The authors could better describe their construct and choice of parameters for the chosen construct. I am specifically wondering about the following points:

a) Why use that particular backbone (not the most commonly used one across recent literature (pJFRC7 is more common).

b) Why do the CsChrimson and GTACR1 have a Kir sequence in it, and why did the authors not put this in the RubyACRs? I would also prefer if authors don't refer to GtACR1 as GTACR-Kir in text (e.g., in line 72); instead, they should either refer to it as GtACR1 or GtACR1-kir-mVenus (based on the full genotype mentioned in their table at the end). Same for CsChrimson-kir. From what I understand, this is just a Kir trafficking sequence and not the entire Kir sequence, which can confuse the readers.

c) Finally, I would also encourage authors to deposit plasmids on Addgene.

(2) Figure 2 is interesting, but it is a bit unfortunate that there is a YFP baseline in most of the samples here (except Chrimson88; this should also be mentioned). I wonder how the YFP baseline impacts this data. Could the high intensity stimulation (red light) lead to bleaching of YFP or tdTomato that reduces the baseline in the green channel? All this also makes me wonder if authors tried tagging the RubyACRs with other fluorophores or non-fluorescent tags and how that impacted their functioning. Non-YFP-tagged versions would be more useful for applications involving GCaMP imaging.

(3) Another point for Figure 2: Since RubyACRs seem to have such a broad activation range, I wonder how much the imaging light (920nm) impacts the baseline in these experiments. If there were plots without the red light stimulation and just varying imaging light intensity, that could be useful to the research community.

(4) Also, for Figures 2C - D, in the methods authors indicate that the stimulation light intensities were progressively increased. Could this lead to desensitization of opsin? Wouldn't randomized intensities be a better way to do this? Perhaps it should be mentioned as a caveat.

(5) In Figure 3E the bottom middle panel Vglut-Gal4,GtACR1 shows a major increase in walking at light onset. This seems very different than all other conditions, and I could not find any discussion of this. It would help if some explanation were provided for this.

Reviewer #2 (Public review):

Summary:

Bushey et al. investigate the feasibility of using RubyACRs, specifically A1ACR1 and HfACR1 (described previously in (Govorunova et al., 2020)) as red-shifted inhibitory opsins in Drosophila melanogaster. The study employs a wide range of techniques to demonstrate successful neuronal inhibition. Electrophysiology experiments established that HfACR1 was most effective at hyperpolarizing cells, compared to A1ACR1 and GtACR1; both RubyACRs also appeared to be more effective than GtACR1 when the latter was actuated by green light. The authors further demonstrate successful neuronal inhibition using calcium imaging. RubyACRs were also shown to be useful in in vivo behavioral setups, specifically in spontaneous locomotion, associative learning, and courtship paradigms. In the courtship assay, in particular, the authors test multiple wavelengths of light at various light intensities, thus providing a rigorous analysis of the RubyACRs' efficacy under different light conditions.

Strengths:

The work provides the Drosophila field with a promising new tool. Red-shifted opsins are particularly advantageous in behavioral assays as red light penetrates the cuticle better than green or blue light, and provides less visual stimulation to the fly. It is also ideal for imaging as it allows for simultaneous optogenetic stimulation and GCamp imaging. A particular strength of the paper is the direct demonstration of RubyACR's capacity to inhibit neurons via electrophysiology and calcium imaging. Furthermore, inhibition effects in the three behavioral assays are strong and convincing. Given the apparent efficacy of RubyACRs and the advantages of a red-sensitive anion channelrhodopsin, this tool has great potential.

Weaknesses:

This work convincingly demonstrates the efficacy and potential utility of RubyACRs in Drosophila for imaging and behavior. However, the lethality/toxicity of RubyACRs is a relevant concern that should be addressed in-depth rather than glossed over, as it may pose a major obstacle to use. Discussing this issue in the present study will also help guide potential users and will set the stage for potential future efforts to ameliorate RubyACRs as optogenetic inhibitors.

Major concerns:

(1) Table 1 demonstrates high lethality in the RubyACRs compared to GtACR1. For example, in the MI04979-VGlut driver, GtACR1 expression resulted in 32.9% lethality, while HfACR1 expression resulted in 98.7% lethality. This lethality presents an obstacle to the potential adoption of this tool, and should be discussed in detail, rather than in passing. The authors might like to present "% lethality" rather than "% survived", as the former is more relevant when discussing the relative yield and health of flies that can be used in experiments.

(2) In Figure 3D, driver>opsin flies have lower locomotion during the baseline (i.e., dark) phase, compared to opsin-only controls or GtACR1 flies. For some comparisons, flies are walking around 10-fold slower. For example, in the case of VGlut-GAL4>HfACR1, test flies are walking at <1 mm/s, while "Empty" test flies are walking at ~10 mm/s. This suggests that, for these drivers, neuronal and/or network function is affected. It opens the possibility that the lethality and locomotor defects could be due to cell-autonomous toxicity. We ask the authors to provide a description of this effect in the Results and to discuss it in the Discussion. Relatedly, VGlut-GAL4>GtACR1 flies in red light exhibit a locomotion increase, but this data is not mentioned in the text. The use of differing scales for the Y-axes in these panels can be confusing when the reader is expected to compare velocity across different panels. It would be best if the y-axes were set to a single range, e.g., 0 to 12 mm/s.

(3) Lethality in broad drivers could result from cell-autonomous toxicity or neuronal dysfunction resulting from RubyACR expression. Ideally, the authors would address or even investigate the possible mechanisms of toxicity of the RubyACRs. Do cells and/or synapses expressing RubyACRs have normal morphology and function? For example, the authors could compare cell survival between flies with RubyACR expression and flies with a fluorescent protein with no opsin. The authors may also want to present lethality data for other, less broad drivers (such as MB320C, which was used for the associative memory assay) in order to demonstrate whether this problem is confined to broad drivers such as VGlut-GAL4, or if this is a problem with narrow drivers as well. If new experiments are not possible, these issues should at least be mentioned in the Discussion.

Minor concerns

(1) The specific method used for quantifying lethality is mentioned briefly in Table 1 but is not detailed in the Methods. The authors derive lethality by comparing to a sibling control group with either the opsin or the driver alone, but the opsin alone or driver alone may cause some lethality by themselves. We suggest the use of a viability assay, e.g. (Rockwell et al., 2019), which would give potential users a clearer picture of which developmental stage is most affected by opsin expression, as well as allow opsin-only, driver-only and experimental groups to be assessed separately (lethality would then be reported as the % of embryos that reach each stage of development, and eventually enclosure).

(2) For the calcium imaging analysis in Figure 2, the U-shaped curve observed for mean ΔF/F0 for A1ACR1 and HfACR1 may not be due to actual desensitization for the channels, as the authors suggest (lines 143-145), but may be due simply to a shifting baseline. The authors use the 5-s period preceding stimulation onset as F0, but in some cases (e.g., HfACR1 at 250 uW/mm2), calcium fluorescence rises above baseline and remains high post-stimulation (ΔF/F0 of +0.5, which we observe is the same magnitude as the ΔF/F0 of -0.5 observed during inhibition), thus affecting the ΔF/F0 for subsequent trials. The authors should discuss this incomplete recovery in the text, or (if available) use a static channel instead to provide a stable F0 for calculating ΔF/F0. Alternatively, if the authors wish to rigorously test the hypothesis that high light intensity indeed results in desensitization of these channels, they may consider using different flies for each light intensity or longer inter-stimulus intervals.

(3) For Figure 3C (Flybowl assay), the authors mention that "simply expressing the opsins decreased baseline locomotor activity compared to empty driver lines". However, the "Empty" controls in 3C appear to refer to opsin-only controls, not driver-only controls. The driver-only controls are not presented in the figure. The use of "empty" differs between the text and the figure, as the text refers to "empty" driver lines, while the figure uses "empty" to apparently refer to opsin-only controls. We recommend changing the terminology across all figures to be unambiguous, e.g., by using "opsin-only" or "driver-only" as opposed to the ambiguous "empty". In addition, the fact that opsin-only controls move less than driver-only controls may suggest some toxicity as a result of the opsin-only construct; this should be discussed further.

(4) Figures 4 and 5 lack the reporting of driver-only controls.

(5) Figures 3 and 4 lack positive controls; that is, the benchmarking of the efficacy of RubyACRs in their respective behavioral paradigms against a known inhibitor, e.g., GtACR1 with green light. To confirm that this GtACR1 transgene is functional, the authors could include GtACR1 with green light as a positive control for these two figures, as they have done for Figure 5-supplement 2 and 3.

(6) Several citations are missing. In their discussion, the authors highlight that shorter wavelengths of light are more attenuated by tissue (lines 278-281); this should be accompanied by the relevant citations (Inagaki et al., 2014). Similarly, the claim that behavioral experiments exhibit greater sensitivity to shorter wavelengths should be substantiated (lines 281-283).

References:

Govorunova EG, Sineshchekov OA, Li H, Wang Y, Brown LS, Spudich JL. 2020. RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption. Proc Natl Acad Sci U S A 117:22833-22840.

Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY, Tsien RY, Anderson DJ. 2014. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat Methods 11:325-332.

Rockwell AL, Beaver I, Hongay CF. 2019. A direct and simple method to assess Drosophila melanogaster's viability from embryo to adult. J Vis Exp e59996.

Reviewer #3 (Public review):

Summary:

This study by Bushey et al. adapts and evaluates two newly developed red-shifted optogenetic inhibitors, A1ACR1 and HfACR1, collectively referred to as RubyACRs, for neuronal silencing in Drosophila melanogaster. Traditional optogenetic inhibitors such as GtACR1 and GtACR2 are activated by green (~515 nm) and blue (~470 nm) light, respectively, which poses several limitations in Drosophila. Specifically, shorter-wavelength light suffers from reduced tissue penetration and increased absorption, and is visible to flies, potentially confounding behavioral assays, particularly those involving visual processing. In contrast, RubyACRs are activated by red light (~610-660 nm), which penetrates the cuticle more effectively and thus can be more potent in manipulating fly behavior. In the current manuscript, the authors first demonstrate that both A1ACR1 and HfACR1 can be robustly expressed in fly neurons and are properly trafficked to the plasma membrane. Upon red-light stimulation, both opsins produce strong and sustained hyperpolarization in larval motor neurons, outperforming GtACR1 in both magnitude and temporal dynamics. Next, using two-photon calcium imaging in the visual system, the authors further demonstrate that activation of RubyACRs significantly reduces GCaMP6s signal, indicating that they can reliably inhibit neuronal activity. Importantly, unlike reported in some mammalian studies, RubyACRs do not appear to trigger paradoxical depolarization at axon terminals in the fly visual system, as no evidence of aberrant depolarization is observed in motion-detecting Mi1 neurons.

In the second part of the manuscript, the authors characterize the effects of RubyACRs on fly behavior (walking, learning, and courtship song). Using the inhibition of genetically labelled neurons that regulate these behaviors, the authors demonstrate that stimulation of RubyACRs leads to potent suppression of locomotion, courtship song, or dopamine-dependent associative learning.

Strengths:

Altogether, the experiments conducted in this manuscript demonstrate that RubyACRs are powerful tools for optogenetic inhibition in Drosophila, with advantages in spectral compatibility, behavioral specificity, and potential applications in vivo two-photon calcium imaging.

Weaknesses:

The manuscript is strong, but it can be further improved with a few additional analyses and minor revisions. Especially, a more detailed evaluation of RubyACRs with two-photon excitation will help clarify to what extent these opsins can be simultaneously used together with green GECIs, such as GCaMPs.

Author response:

We thank the reviewers for their thoughtful and thorough consideration of the work. We appreciate the positive reception they give the work, and plan to address several of the comments with further experiments. To outline that work (and ensure that we are on the right track to addressing those concerns), we summarize the core concerns that prompt new experiments:

(1) Does the YFP tag on the ACRs interfere with simultaneous GCaMP imaging of RubyACR-expressing cells and could bleaching of the YFP complicate interpretation of the experiments here?

We will test whether 920 nm (2p) and 650 nm (1p) excitation cause YFP bleaching that interferes with interpretation of inhibitory calcium (i.e. GCaMP) signals. Because the YFP tag enhances opsin sensitivity, we prioritized these tagged RubyACRs for initial characterization. FLAG-tagged ACRs are in progress, but will take time to fully characterize. Considering that the RubyACR-EYFP versions work very well, and in many cases people will want the YFP tag, either for visualizing expression or to maximize sensitivity, we feel the current work is a valuable contribution on its own. Indeed several labs have already requested these lines.

(2) Are the ACRs activated by two-photon illumination?

We will examine GCaMP signals at increasing 2p intensities to determine whether imaging unintentionally activates RubyACRs, as well as whether 2p illumination could be used for intentional opsin activation.

(3) How toxic is the expression of these opsins?

We will update the quantification of toxicity in Table 1 to include all the drivers we used in this study. In fact the toxicity we observed was primarily with the vGlut driver, which was why that was the only information in the table. The other drivers we used did not appreciably reduce survival rate, but showing the one case where it did have a big effect left a strong and understandably inaccurate impression that toxicity was a big pitfall. We note that the widely used CSChrimson has similar % survival to the RubyACRs when expressed with these vGlut drivers.

We also plan to examine whether ACR expression leads to cell-autonomous perturbations. We will determine whether expression leads to some frequency of neuronal cell death, and we will evaluate whether any morphological effects occur.

We will also clarify in the Discussion that potential toxicity may be driver-specific (as it is here) and should be evaluated case-by-case by investigators using the tool.

(4) Use functional imaging to confirm inhibition of the neurons used only for behavioral experiments (pIP10 & PPL1-γ1pedc)

We will perform these imaging experiments. One caveat is that inhibition may not be readily detectable with GCaMP, as the resting calcium levels in pIP10 and PPL1-γ1pedc neurons may already be quite low. This differs from the non-spiking Mi1 neurons, where inhibition was clearly observed with GCaMP. For this reason, we consider the behavioral results stronger evidence of efficacy, but we agree that imaging could provide useful supporting evidence, recognizing that a negative result would be difficult to interpret.

(5) Confirm that the GtACR1 will inhibit locomotion in the flybowl when activated with green light, its spectral peak.

We will perform this benchmark experiment. Please note that our intention with this study was to find an effective red-light activated opto-inhibitor because these wavelengths are much less perturbing to behavior. In that respect, regardless of GtACR1’s performance with green light, the RubyACRs clearly provide important new tools for Drosophila behavioral neuroscience.

Always keep this in my mind.

to have someone's attention completely so that they cannot think of anything else. (verb)

بيقوم بتحميل الصفحة اول ما تعمل تمرر الماوس على الرابط

**يمنع اعادة التحميل الكاملة للصفحة يجلب الصفحة من خلال ajax بيبدل المحتوى القديم بالجديد بشكل يشبه Single Page Application **

• Informativo nº 806
• CORTE ESPECIAL
• Processo: EAREsp 1.766.665-RS, Rel. Ministro Francisco Falcão, Rel. para acórdão Ministro Ricardo Villas Bôas Cueva, Corte Especial, por maioria, julgado em 3/4/2024.

Ramo do Direito DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Multa cominatória. Valor exorbitante. Desproporcionalidade. Valor acumulado. Possiblidade de revisão. Exigência de postura ativa do devedor. Sucessivas revisões. Impossibilidade. Preclusão consumativa.

DESTAQUE - Incide a preclusão consumativa sobre o montante acumulado da multa cominatória, de forma que, já tendo havido modificação, não é possível nova alteração, preservando-se as situações já consolidadas.

INFORMAÇÕES DO INTEIRO TEOR - A controvérsia diz respeito à ocorrência de preclusão sobre decisão que revisa o valor de astreintes. Sobre tema, a Corte Especial, no julgamento do EAREsp n. 650.536-RJ, firmou o entendimento de ser possível a redução quando o valor for exorbitante, levando-se em conta a razoabilidade e a proporcionalidade, e a fim de evitar o enriquecimento sem causa do credor.

• No entanto, a questão demanda reflexões mais aprofundadas, especialmente porque essa decisão, muito embora tenha sido proferida sob a égide do CPC atual, baseou-se especialmente em jurisprudência majoritária construída à época em que vigia o CPC/1973, com destaque para o Tema Repetitivo n. 706: "A decisão que comina astreintes não preclui, não fazendo tampouco coisa julgada" (REsp n. 1.333.988/SP, Segunda Seção, Rel. Ministro Paulo de Tarso Sanseverino, DJe 11/4/2014).

• Além disso, não se levou em consideração que o CPC/2015 alterou substancial e expressamente o regime jurídico das astreintes no tocante à possibilidade de modificação. Com efeito, de acordo com a premissa estabelecida no julgamento do EAREsp n. 650.536-RJ, a regra que permite ao magistrado alterar a multa cominatória estaria prevista no art. 461, § 6°, do CPC/1973 e no seu correspondente, art. 537, § 1°, do CPC/2015. Todavia, há uma diferença substancial entre essas duas regras, em particular no que diz respeito a quais valores podem ser modificados.

• A partir da análise dessas regras supracitadas, percebe-se a nítida intenção do legislador de autorizar a revisão ou a exclusão apenas da "multa <u>vincenda</u>", ou seja, a decisão não pode ter eficácia retroativa para atingir o montante acumulado da multa. Por outro lado, há quem sustente a possibilidade de decisão com efeitos retroativos no caso de redução do montante da multa que já incidiu, pois a expressão "vincendas" diria respeito apenas à multa que está incidindo.

• Contudo, não há motivo para submeter a modificação e a exclusão a regimes jurídicos diversos. A regra do art. 537, § 1°, do CPC deixa claro que o legislador optou por preservar as situações já consolidadas, independentemente de se tratar da multa que está incidindo ou do montante oriundo da sua incidência. Analisando a questão com mais profundidade, tem-se que a pendência de discussão acerca do montante da multa não guarda relação com o seu vencimento, mas, sim, com a sua definitividade.

• Dessa forma, se a incidência da multa durante o período de inadimplência alcança valores exorbitantes, seja porque o devedor permaneceu inerte e não requereu a revisão ou exclusão, seja porque o magistrado não agiu de ofício, qualquer decisão que venha a ser proferida somente poderia provocar, em regra, efeitos <u>prospectivos</u>.

• Percebe-se que o legislador do CPC/2015 optou por levar em consideração a postura do devedor, a fim de premiar aquele que, muito embora inadimplente num primeiro momento, acaba por cumprir a obrigação, ainda que parcialmente, ou que demonstra a impossibilidade de cumprimento. Significa dizer que somente tem direito à redução da multa aquele que abandona a recalcitrância.

• Desse modo, a partir da regra expressa do art. 537, §1°, do CPC, somente seria possível alterar o valor acumulado das multas vincendas e, consoante disposto no inciso II, a redução exige postura <u>ativa</u> do devedor, consubstanciada no cumprimento parcial da obrigação ou na demonstração de sua impossibilidade.

• De qualquer sorte, na hipótese, há outro óbice para a revisão pretendida, qual seja a preclusão pro judicato consumativa, pois já havia sido revisado o valor da multa diária.

• O STJ sedimentou, por meio de recurso especial julgado na sistemática dos repetitivos, que "a decisão que comina astreintes não preclui, não fazendo tampouco coisa julgada" (Tema 706), conforme já anotado. Trata-se, no entanto, de não incidência de preclusão <u>temporal</u>, de forma que o valor da multa pode ser modificado a qualquer tempo. Não se trata de ausência de preclusão consumativa, sob pena de grave violação da segurança jurídica.

• Dessa forma, uma vez fixada a multa, é possível alterá-la ou excluí-la a qualquer momento. No entanto, uma vez reduzido o valor, não serão lícitas sucessivas revisões, a bel prazer do inadimplente recalcitrante, sob pena de estimular e premiar a renitência sem justa causa. <u>Em outras palavras, é possível modificar a decisão que comina a multa, mas não é lícito modificar o que já foi modificado</u>.

• Considerando que a multa cominatória é um importantíssimo instrumento para garantir a efetividade das decisões judiciais e pode ser fixada de ofício, trata-se de matéria de ordem pública. No caso, a multa fixada em sentença transitada em julgado pode ser alterada na fase de execução porque tem natureza de técnica processual, de modo que não é acobertada pela coisa julgada material. Uma vez fixada ou alterada no início da execução, mantém tal natureza e, portanto, pode ser modificada a qualquer momento, inclusive de ofício.

• Todavia, o valor acumulado da multa deixa de ser técnica processual e passa a integrar o patrimônio do exequente como crédito de valor, perdendo a natureza de matéria de ordem pública. Com efeito, nos termos do art. 537, § 2°, do CPC, "o valor [acumulado] da multa será devido ao exequente".

• Além disso, mesmo se considerada também a multa acumulada como matéria de ordem pública, deve incidir a preclusão pro judicato consumativa, de forma que, tendo havido modificação, não é possível nova alteração, preservando-se as situações já consolidadas, como deixa claro o art. 537, § 1°, do CPC ao se referir a "multa vincenda". Isso porque há preclusão consumativa em relação às questões de ordem pública, inclusive àquelas que estão fora da esfera de disponibilidade das partes, tais como os pressupostos processuais e as condições da ação, conforme entendimento sedimentado no STJ.

• Assim sendo, e com maior razão, há preclusão consumativa no tocante ao montante acumulado da multa cominatória, pois ostenta natureza patrimonial e disponível.

eLife Assessment

This valuable study employs a formalized computational model of learning to assess memory deficits in Alzheimer's Disease with the goal of developing an early diagnosis tool. Using an established mouse model of the disease, the authors studied multiple behavioral tasks and ages with the goal of showing similarities in behavioral deficits across tasks. Using the model, the authors indicate specific deficits in memory (overgeneralization and over differentiation) in mice with the transgene for the disease. The evidence presented is solid, yet certain concerns remain regarding the interpretation of the results of the modeling.

Reviewer #1 (Public review):

I applaud the authors' for providing a thorough response to my comments from the first round of review. The authors' have addressed the points I raised on the interpretation of the behavioral results as well as the validation of the model (fit to the data) by conducting new analyses, acknowledging the limitations where required and providing important counterpoints. As a result of this process, the manuscript has considerably improved. I have no further comments and recommend this manuscript for publication.

Reviewer #2 (Public review):

Summary:

This manuscript proposes that the use of a latent cause model for assessment of memory-based tasks may provide improved early detection in Alzheimer's Disease as well as more differentiated mapping of behavior to underlying causes. To test the validity of this model, the authors use a previously described knock-in mouse model of AD and subject the mice to several behaviors to determine whether the latent cause model may provide informative predictions regarding changes in the observed behaviors. They include a well-established fear learning paradigm in which distinct memories are believed to compete for control of behavior. More specifically, it's been observed that animals undergoing fear learning and subsequent fear extinction develop two separate memories for the acquisition phase and the extinction phase, such that the extinction does not simply 'erase' the previously acquired memory. Many models of learning require the addition of a separate context or state to be added during the extinction phase and are typically modeled by assuming the existence of a new state at the time of extinction. The Niv research group, Gershman et al. 2017, have shown that the use of a latent cause model applied to this behavior can elegantly predict the formation of latent states based on a Bayesian approach, and that these latent states can facilitate the persistence of the acquisition and extinction memory independently. The authors of this manuscript leverage this approach to test whether deficits in production of the internal states, or the inference and learning of those states, may be disrupted in knock-in mice that show both a build-up of amyloid-beta plaques and a deterioration in memory as the mice age.

Strengths:

I think the authors' proposal to leverage the latent cause model and test whether it can lead to improved assessments in an animal model of AD is a promising approach for bridging the gap between clinical and basic research. The authors use a promising mouse model and apply this to a paradigm in which the behavior and neurobiology are relatively well understood - an ideal situation for assessing how a disease state may impact both the neurobiology and behavior. The latent cause model has the potential to better connect observed behavior to underlying causes and may pave a road for improved mapping of changes in behavior to neurobiological mechanisms in diseases such as AD.<br /> The authors also compare the latent cause model to the Rescorla-Wagner model and a latent state model allowing for better assessment of the latent cause model as a strong model for assessing reinstatement.

Weaknesses:

I have several substantial concerns which I've detailed below. These include important details on how the behavior was analyzed, how the model was used to assess the behavior, and the interpretations that have been made based on the model.<br /> (1) There is substantial data to suggest that during fear learning in mice separate memories develop for the acquisition and extinction phases, with the acquisition memory becoming more strongly retrieved during spontaneous recovery and reinstatement. The Gershman paper, cited by the authors, shows how the latent causal model can predict this shift in latent causes by allowing for the priors to decay over time, thereby increasing the posterior of the acquisition memory at the time of spontaneous recovery. In this manuscript, the authors suggest a similar mechanism of action for reinstatement, yet the model does not appear to return to the acquisition memory after reinstatement, at least based on the simulation and examples shown in figures 1 and 3. More specifically, in figure 1, the authors indicate that the posterior probability of the latent cause, zA (the putative acquisition memory), increases, partially leading to reinstatement. This does not appear to be the case as test 3 (day 36) appears to have similar posterior probabilities for zA as well as similar weights for the CS as compared to the last days of extinction. Rather, the model appears to mainly modify the weights in the most recent latent cause, zB - the putative the 'extinction state', during reinstatement. The authors suggest that previous experimental data have indicated that spontaneous recovery or reinstatement effects are due to an interaction of the acquisition and extinction memory. These studies have shown that conditioned responding at a later time point after extinction is likely due to a balance between the acquisition memory and the extinction memory, and that this balance can shift towards the acquisition memory naturally during spontaneous recovery, or through artificial activation of the acquisition memory or inhibition of the extinction memory (see Lacagnina et al. for example). Here the authors show that the same latent cause learned during extinction, zB, appears to dominate during the learning phase of reinstatement, with rapid learning to the context - the weight for the context goes up substantially on day 35 - in zB. This latent cause, zB, dominates at the reinstatement test, and due to the increased associative strength between the context and shock, there is a strong CR. For the simulation shown in figure 1, it's not clear why a latent cause model is necessary for this behavior. This leads to the next point.

(2) The authors compared the latent cause model to the Rescorla-Wagner model. This is very commendable, particularly since the latent cause model builds upon the RW model, so it can serve as an ideal test for whether a more simplified model can adequately predict the behavior. The authors show that the RW model cannot successfully predict the increased CR during reinstatement (Appendix figure 1). Yet there are some issues with the way the authors have implemented this comparison:<br /> (2A) The RW model is a simplified version of the latent cause model and so should be treated as a nested model when testing, or at a minimum, the number of parameters should be taken into account when comparing the models using a method such as the Bayesian Information Criterion, BIC.<br /> (2B) The RW model provides the associative strength between stimuli and does not necessarily require a linear relationship between V and the CR. This is the case in the original RW model as well as in the LCM. To allow for better comparison between the models, the authors should be modeling the CR in the same manner (using the same probit function) in both models. In fact, there are many instances in which a sigmoid has been applied to RW associative strengths to predict CRs. I would recommend modeling CRs in the RW as if there is just one latent cause. Or perhaps run the analysis for the LCM with just one latent cause - this would effectively reduce the LCM to RW and keep any other assumptions identical across the models.<br /> (2C) In the paper, the model fits for the alphas in the RW model are the same across the groups. Were the alphas for the two models kept as free variables? This is an important question as it gets back to the first point raised. Because the modeling of the reinstatement behavior with the LCM appears to be mainly driven by latent cause zB, the extinction memory, it may be possible to replicate the pattern of results without requiring a latent cause model. For example, the 12-month-old App NL-G-F mice behavior may have a deficit in learning about the context. Within the RW model, if the alpha for context is set to zero for those mice, but kept higher for the other groups, say alpha_context = 0.8, the authors could potentially observe the same pattern of discrimination indices in figure 2G and 2H at test. Because the authors don't explicitly state which parameters might be driving the change in the DI, the authors should show in some way that their results cannot simply be due to poor contextual learning in the 12 month old App NL-G-F mice, as this can presumably be predicted by the RW model. The authors' model fits using RW don't show this, but this is because they don't consider this possibility that the alpha for context might be disrupted in the 12-month-old App NL-G-F mice. Of course, using the RW model with these alphas won't lead to as nice of fits of the behavior across acquisition, extinction, and reinstatement as the authors' LCM, the number of parameters are substantially reduced in the RW model. Yet the important pattern of the DI would be replicated with the RW model (if I'm not mistaken), which is the important test for assessment of reinstatement.

(3) As stated by the authors in the introduction, the advantage of the fear learning approach is that the memory is modified across the acquisition-extinction-reinstatement phases. Although perhaps not explicitly stated by the authors, the post-reinstatement test (test 3) is the crucial test for whether there is reactivation of a previously stored memory, with the general argument being that the reinvigorated response to the CS can't simply be explained by relearning the CS-US pairing, because re-exposure the US alone leads to increase response to the CS at test. Of course there are several explanations for why this may occur, particularly when also considering the context as a stimulus. This is what I understood to be the justification for the use of a model, such as the latent cause model, that may better capture and compare these possibilities within a single framework. As such, it is critical to look at the level of responding to both the context alone and to the CS. It appears that the authors only look at the percent freezing during the CS, and it is not clear whether this is due to the contextual-US learning during the US re-exposure or to increased responding to the CS - presumably caused by reactivation of the acquisition memory. The authors do perform a comparison between the preCS and CS period, but it is not clear whether this is taken into account in the LCM. For example, the instance of the model shown in figure 1 indicates that the 'extinction cause', or cause z6, develops a strong weight for the context during the reinstatement phase of presenting the shock alone. This state then leads to increased freezing during the final CS probe test as shown in the figure. If they haven't already, I think the authors must somehow incorporate these different phases (CS vs ITI) into their model, particularly since this type of memory retrieval that depends on assessing latent states is specifically why the authors justified using the latent causal model. In more precise terms, it's not clear whether the authors incorporate a preCS/ITI period each day the cue is presented as a vector of just the context in addition to the CS period in which the vector contains both the context and the CS. Based on the description, it seemed to me that they only model the CRs during the CS period on days when the CS is presented, and thereby the context is only ever modeled on its own (as just the context by itself in the vector) on extinction days when the CS is not presented. If they are modeling both timepoints each day that the CS I presented, then I would recommend explicitly stating this in the methods section.

(4) The authors fit the model using all data points across acquisition and learning. As one of the other reviewers has highlighted, it appears that there is a high chance for overfitting the data with the LCM. Of course, this would result in much better fits than models with substantially fewer free parameters, such as the RW model. As mentioned above, the authors should use a method that takes into account the number of parameters, such as the BIC.

(5) The authors have stated that they do not think the Barnes maze task can be modeled with the LCM. Whether or not this is the case, if the authors do not model this data with the LCM, the Barnes maze data doesn't appear valuable to the main hypothesis. The authors suggest that more sophisticated models such as the LCM may be beneficial for early detection of diseases such as Alzheimer's, so the Barnes maze data is not valuable for providing evidence of this hypothesis. Rather, the authors make an argument that the memory deficits in the Barnes maze mimic the reinstatement effects providing support that memory is disrupted similarly in these mice. Although, the authors state that the deficits in memory retrieval are similar across the two tasks, the authors are not explicit as to the precise deficits in memory retrieval in the reinstatement task - it's a combination of overgeneralizing latent causes during acquisition, poor learning rate, over differentiation of the stimuli.

Reviewer #3 (Public review):

Summary:

This paper seeks to identify underlying mechanisms contributing to memory deficits observed in Alzheimer's disease (AD) mouse models. By understanding these mechanisms, they hope to uncover insights into subtle cognitive changes early in AD to inform interventions for early-stage decline.

Strengths:

The paper provides a comprehensive exploration of memory deficits in an AD mouse model, covering early and late stages of the disease. The experimental design was robust, confirming age-dependent increases in Aβ plaque accumulation in the AD model mice and using multiple behavior tasks that collectively highlighted difficulties in maintaining multiple competing memory cues, with deficits most pronounced in older mice.

In the fear acquisition, extinction, and reinstatement task, AD model mice exhibited a significantly higher fear response after acquisition compared to controls, as well as a greater drop in fear response during reinstatement. These findings suggest that AD mice struggle to retain the fear memory associated with the conditioned stimulus, with the group differences being more pronounced in the older mice.

In the reversal Barnes maze task, the AD model mice displayed a tendency to explore the maze perimeter rather than the two potential target holes, indicating a failure to integrate multiple memory cues into their strategy. This contrasted with the control mice, which used the more confirmatory strategy of focusing on the two target holes. Despite this, the AD mice were quicker to reach the target hole, suggesting that their impairments were specific to memory retrieval rather than basic task performance.

The authors strengthened their findings by analyzing their data with a leading computational model, which describes how animals balance competing memories. They found that AD mice showed somewhat of a contradiction: a tendency to both treat trials as more alike than they are (lower α) and similar stimuli as more distinct than they are (lower σx) compared to controls.

Weaknesses:

While conceptually solid, the model struggles to fit the data and to support the key hypothesis about AD mice's inability to retain competing memories. These issues are evident in Figure 3:

(1) The model misses trends in the data, including the gradual learning of fear in all groups during acquisition, the absence of a fear response at the start of the experiment, and the faster return of fear during reinstatement compared to the gradual learning of fear during acquisition. It also underestimates the increase in fear at the start of day 2 of extinction, particularly in controls.

(2) The model explains the higher fear response in controls during reinstatement largely through a stronger association to the context formed during the unsignaled shock phase, rather than to any memory of the conditioned stimulus from acquisition (as seen in Figure 3C). In the experiment, however, this memory does seem to be important for explaining the higher fear response in controls during reinstatement (as seen in Author Response Figure 3). The model does show a necessary condition for memory retrieval, which is that controls rely more on the latent causes from acquisition. But this alone is not sufficient, since the associations within that cause may have been overwritten during extinction. The Rescorla-Wagner model illustrates this point: it too uses the latent cause from acquisition (as it only ever uses a single cause across phases) but does not retain the original stimulus-shock memory, updating and overwriting it continuously. Similarly, the latent cause model may reuse a cause from acquisition without preserving its original stimulus-shock association.

These issues lead to potential overinterpretation of the model parameters. The differences in α and σx are being used to make claims about cognitive processes (e.g., overgeneralization vs. over differentiation), but the model itself does not appear to capture these processes accurately.

The authors could benefit from a model that better matches the data and captures the retention and retrieval of fear memories across phases. While they explored alternatives, including the Rescorla-Wagner model and a latent state model, these showed no meaningful improvement in fit. This highlights a broader issue: these models are well-motivated but may not fully capture observed behavior.

Conclusion:

Overall, the data support the authors' hypothesis that AD model mice struggle to retain competing memories, with the effect becoming more pronounced with age. While I believe the right computational model could highlight these differences, the current models fall short in doing so.